G2Cdb::Gene report

Gene id
Gene symbol
Homo sapiens
low density lipoprotein receptor-related protein 1
G00000110 (Mus musculus)

Databases (7)

ENSG00000123384 (Ensembl human gene)
4035 (Entrez Gene)
448 (G2Cdb plasticity & disease)
LRP1 (GeneCards)
107770 (OMIM)
Marker Symbol
HGNC:6692 (HGNC)
Protein Sequence
Q07954 (UniProt)

Synonyms (2)

  • CD91
  • LRP

Literature (208)

Pubmed - other

  • Low-density lipoprotein receptor-related protein 1 is an essential receptor for trichosanthin in 2 choriocarcinoma cell lines.

    Jiao Y and Liu W

    Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, The Chinese Academy of Sciences, 320 Yue Yang Road, Shanghai 200031, China.

    Type-I ribosome-inactivating protein-trichosanthin (TCS) exhibits selective cytotoxicity toward different types of cells. It is believed that the cytotoxicity results from the inhibition of ribosomes to decrease protein synthesis, thereby indicating that there are specific mechanisms for TCS entry into target cells to reach the ribosomes. Low-density lipoprotein (LDL) receptor-related protein 1 (LRP1) is a large scavenger receptor that is responsible for the binding and endocytosis of diverse biological ligands on the cell surface. In this study, we demonstrated that 2 choriocarcinoma cell lines can significantly bind and internalize TCS. In contrast, Hela cell line displayed no obvious TCS binding and endocytosis. Furthermore LRP1 gene silencing in JAR and BeWo cell lines blocked TCS binding; TCS could also interact with LRP1.The results of our study established that LRP1 was a major receptor for phagocytosis of TCS in JAR and BeWo cell lines and might be the molecular basis of TCS abortificient and anti-choriocarcinoma activity.

    Biochemical and biophysical research communications 2010;391;4;1579-84

  • Alpha2-macroglobulin inhibits the malignant properties of astrocytoma cells by impeding beta-catenin signaling.

    Lindner I, Hemdan NY, Buchold M, Huse K, Bigl M, Oerlecke I, Ricken A, Gaunitz F, Sack U, Naumann A, Hollborn M, Thal D, Gebhardt R and Birkenmeier G

    Institute of Biochemistry, Department of Ophthalmology, University of Leipzig, Leipzig, Germany.

    Targets that could improve the treatment of brain tumors remain important to define. This study of a transformation-associated isoform of alpha2-macroglobulin (A2M*) and its interaction with the low-density lipoprotein receptor-related protein-1 (LRP1) suggests a new mechanism for abrogating the malignant potential of astrocytoma cells. LRP1 bound A2M* found to be associated with an inhibition of tumor cell proliferation, migration, invasion, spheroid formation, and anchorage-independent growth. Transcriptional studies implicated effects on the Wnt/beta-catenin signaling pathway. Notably, LRP1 antibodies could phenocopy the effects of A2M*. Our findings suggest a pathway of tumor suppression in astrocytoma that might be tractable to therapeutic exploitation.

    Cancer research 2010;70;1;277-87

  • Role of the low-density lipoprotein receptor-related protein-1 in regulation of chondrocyte differentiation.

    Kawata K, Kubota S, Eguchi T, Moritani NH, Shimo T, Kondo S, Nishida T, Minagi S and Takigawa M

    Department of Biochemistry and Molecular Dentistry, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama, Japan.

    The low-density lipoprotein receptor-related protein 1 (LRP1) is known as an endocytic and signal transmission receptor. We formerly reported the gene expression and the localization of LRP1 in cartilage tissue and chondrocytes, but its roles in the differentiation of chondrocytes remained to be investigated. Here, in order to address this issue, we employed RNAi strategy to knockdown lrp1 in chondrocytic cells and obtained findings indicating a critical role therein. As a result of lrp1 knockdown, aggrecan and col2a1 mRNA levels were decreased. However, that of col10a1 or mmp13 mRNA was rather increased. Under this condition, we performed a promoter assay for Axin2, which is known to be induced by activation of the WNT/beta-catenin (betacat) signaling pathway. Thereby, we found that Axin2 promoter activity was enhanced in the lrp1 knockdown cells. Furthermore, when the WNT/beta-catenin pathway was activated in chondrocytic cells by WNT3a or SB216763, which inhibits the phosphorylation of GSK3beta, the mRNA levels of aggrecan and col2a1 were decreased, whereas that of mmp13 was increased. Additionally, the level of phosphorylated protein kinase C (PKC) zeta was also decreased in the lrp1 knockdown cells. When the phosphorylation of PKCzeta was selectively inhibited, aggrecan and col2a1 mRNA levels decreased, whereas the mmp13 mRNA level increased. These data demonstrate that LRP1 exerts remarkable effects to retain the mature phenotype of chondrocytes as a critical mediator of cell signaling. Our findings also indicate that the onset of hypertrophy during endochondral ossification appears to be particularly dependent on the WNT and PKC signaling initiated by LRP1.

    Journal of cellular physiology 2010;222;1;138-48

  • Low density lipoprotein receptor-related protein 1 promotes anti-apoptotic signaling in neurons by activating Akt survival pathway.

    Fuentealba RA, Liu Q, Kanekiyo T, Zhang J and Bu G

    Department of Pediatrics, Hope Center for Neurological Disorders, Washington University School of Medicine, St. Louis, Missouri 63110, USA.

    The low density lipoprotein receptor-related protein 1 (LRP1) is a multi-ligand receptor abundantly expressed in neurons. Previous work has shown that brain LRP1 levels are decreased during aging and in Alzheimer disease. Although mounting evidence has demonstrated a role for LRP1 in the metabolism of apolipoprotein E/lipoprotein and amyloid-beta peptide, whether LRP1 also plays a direct role in neuronal survival is not clear. Here, we show that LRP1 expression is critical for the survival of primary neurons under stress conditions including trophic withdrawal, the presence of apoptosis inducers, or amyloid-beta-induced neurotoxicity. Using lentiviral short hairpin RNA to knock down endogenous LRP1 expression, we showed that a depletion of LRP1 leads to an activation of caspase-3 and increased neuronal apoptosis, an effect that was rescued by a caspase-3 inhibitor. A correlation between decreased Akt phosphorylation and the activation of caspase-3 was demonstrated in LRP1 knocked down neurons. Notably, LRP1 knockdown decreased insulin receptor levels in primary neurons, suggesting that decreased neuronal survival might be a consequence of an impaired insulin receptor signaling pathway. Correspondingly, both insulin receptor and phospho-Akt levels were decreased in LRP1 forebrain knock-out mice. These results demonstrate that LRP1 mediates anti-apoptotic function in neurons by regulating insulin receptor and the Akt survival pathway and suggest that restoring LRP1 expression in Alzheimer disease brain might be beneficial to inhibiting neurodegeneration.

    Funded by: NIA NIH HHS: P01 AG030128, R01 AG027924, R01 AG031784, R01 AG035355

    The Journal of biological chemistry 2009;284;49;34045-53

  • A candidate gene approach to genetic prognostic factors of IgA nephropathy--a result of Polymorphism REsearch to DIstinguish genetic factors Contributing To progression of IgA Nephropathy (PREDICT-IgAN).

    Yamamoto R, Nagasawa Y, Shoji T, Inoue K, Uehata T, Kaneko T, Okada T, Yamauchi A, Tsubakihara Y, Imai E, Isaka Y and Rakugi H

    Department of Geriatric Medicine and Nephrology, Osaka University Graduate School of Medicine, Osaka, Japan.

    Background: Renal prognosis of IgA nephropathy (IgAN) is affected by environmental and genetic factors. Other studies demonstrated that some atherosclerotic disease-related genes were significantly associated with renal prognosis.

    Methods: The Polymorphism REsearch to DIstinguish genetic factors Contributing To progression of IgAN (PREDICT-IgAN) was a multicentre retrospective observational study to investigate associations between progression of IgAN (a 50% increase of serum creatinine level and slope of eGFR) and a hundred atherosclerotic disease-related gene polymorphisms, mainly single nucleotide polymorphisms (SNPs) in 320 IgAN patients who had more than a normal range of urinary protein (> or =0.25 g/day) at diagnosis.

    Results: During 8.3 +/- 4.2 years of a follow-up period, 83 patients (25.9%) developed progression. In log-rank tests, glycoprotein Ia GPIa C807T and G873A and intercellular adhesion molecule-1 ICAM-1 A1548G (K469E) were found to be significantly associated with progression even after adjustment for multiple comparisons by the method of Bonferroni (adjusted P = 0.0174, 0.0176 and 0.0430, respectively). In a multivariate Cox proportional-hazards model, GPIa 807TT (873CC) [versus 807TT, adjusted hazard ratio 2.05 (95% confidence interval 1.13-3.71)] and ICAM-1 1548GG [versus 1548AA, 2.55 (1.40-4.65)] were identified as independent genetic predictors of progression, along with conventional clinical prognostic factors such as eGFR, urinary protein and use of antihypertensives at diagnosis.

    Conclusions: PREDICT-IgAN distinguished GPIa C807T/ G873A and ICAM-1 A1548G from multiple athero- sclerotic disease-related gene polymorphisms by their predictive indicator for progression of IgAN.

    Nephrology, dialysis, transplantation : official publication of the European Dialysis and Transplant Association - European Renal Association 2009;24;12;3686-94

  • Association of the LRP1 gene and cognitive performance with amnestic mild cognitive impairment in elderly Chinese.

    Shi YM, Zhou H, Zhang ZJ, Yu H, Bai F, Yuan YG, Deng LL and Jia JP

    School of Clinical Medicine, Southeast University, Nanjing, China.

    Background: The genetic region coding for low-density lipoprotein receptor-related protein1 (LRP1) is considered an intriguing susceptibility locus for Alzheimer's disease (AD). Amnestic mild cognitive impairment (aMCI) is characterized by episodic memory impairment and represents the prodromal stage of AD. Our aim in this study is to investigate the relationship between LRP1 genetic variation and aMCI, and the influence of LRP1 on cognitive performance.

    Methods: We performed a case-control association study analyzing five polymorphisms in LRP1 gene by TaqMan Assays-on-Demand SNP Genotyping. All samples were derived from Chinese subjects (109 cases, 104 healthy controls) and assessed using multi-dimension neuropsychological instruments.

    Results: We identified haplotypes within the region containing the LRP1 gene. Of these, haplotype TAA (T: rs1800194; A: rs11837145; A: rs10876967) was significantly associated with aMCI, being over-represented in aMCI versus healthy controls. Haplotype TAA was associated with poor performance on episodic memory in all subjects.

    Conclusions: This study confirms the association between genetic variants in LRP1 and aMCI. Moreover, we have identified a relationship between LRP1 genetic variation and specific aspects of neurocognitive function. Our convergent results suggest that LRP1 plays an important role in cognitive function and possibly in the pathogenesis of aMCI.

    International psychogeriatrics 2009;21;6;1072-80

  • hSSTR2 expression and octreotide treatment reverses multidrug resistance of BxPC-3 human pancreatic cancer cells.

    Sui C, Ma Q, Nan K, Xiao J, Suo A, Sha H and Zhao L

    Department of Oncology, First Affiliated Hospital of Medical College, Xi'an Jiaotong University, Xi'an, PR China.

    Pancreatic cancer is generally refractory to most chemotherapeutic agents. We investigated whether hSSTR2 expression and octreotide treatment reverse multidrug resistance of human pancreatic cancer cells. We used pancreatic cancer cells that were transfected by using a lentivirus expression system, which allowed stable expression of the hSSTR2 gene in the pancreatic cancer cells. BxPC-3 cells were transfected with hSSTR2 through a lentivirus vector pWP XL-MOD-SSTR2 in order to enable the expression of hSSTR2. The transfected cells were treated with different concentrations of octreotide and with the chemotherapeutic agents cisplatin, epirubicin, fluorouracil and gemcitabine. The changes in IC50 following treatment with chemotherapeutic agents were determined, and the expression of different MDR indicating marker genes, multidrug resistance gene-1 (MDR1), multidrug resistance-associated protein 2 (MRP2), and lung resistance-related protein (LRP), were evaluated. Octreotide treatment of the transfected cells significantly decreased the IC50 of chemotherapeutic agents in a dose-dependent manner. hSSTR2 gene transfection decreased MDR1, MRP2 and LRP expression by 57, 47 and 56%, respectively (P<0.01), and octreotide treatment (1.6 microg/ml) for 48 h, decreased it further by 88, 73 and 87<, respectively (P<0.01). These data suggested that the down-regulation of MDR genes is responsible for the improvement in the chemotherapeutic sensitivity of hSSTR2-expressing pancreatic cancer cells, when these cells are subjected to octreotide treatment.

    Oncology reports 2009;22;6;1391-6

  • MicroRNA-205 inhibits tumor cell migration through down-regulating the expression of the LDL receptor-related protein 1.

    Song H and Bu G

    Department of Pediatrics, Washington University School of Medicine, St. Louis, MO 63110, USA. almamater70@gmail.com

    Low-density lipoprotein receptor-related protein 1 (LRP1) is a multifunctional endocytic receptor that plays critical roles in the pathogenesis of several human diseases including tumor metastasis and Alzheimer's disease. However, mechanisms that regulate LRP1 expression under physiological and pathophysiological conditions are not unclear. In human cell lines, we found that miR-205 down-regulates the expression of LRP1 by targeting sequences in the 3'UTR of LRP1 mRNA. This effect was abolished by deleting the miR-205 seed site in the 3'UTR of LRP1. The ectopic expression of miR-205 also significantly mitigated migration of both U87 and SK-LU-1 cells. These results, for the first time, demonstrate that expression of human LRP1 is regulated in part by a specific miRNA, leading to decreased tumor cell migration.

    Funded by: NIA NIH HHS: R01 AG027924, R01 AG027924-04, R01 AG031784, R01 AG031784-02, R01-AG027924, R01-AG031784

    Biochemical and biophysical research communications 2009;388;2;400-5

  • LRP1 receptor controls adipogenesis and is up-regulated in human and mouse obese adipose tissue.

    Masson O, Chavey C, Dray C, Meulle A, Daviaud D, Quilliot D, Muller C, Valet P and Liaudet-Coopman E

    IRCM, Institut de Recherche en Cancérologie de Montpellier, INSERM, U896, Université Montpellier1, CRLC Val d'Aurelle Paul Lamarque, Montpellier, France.

    The cell surface low-density lipoprotein receptor-related protein 1, LRP1, plays a major role in lipid metabolism. The question that remains open concerns the function of LRP1 in adipogenesis. Here, we show that LRP1 is highly expressed in murine preadipocytes as well as in primary culture of human adipocytes. Moreover, LRP1 remains abundantly synthesised during mouse and human adipocyte differentiation. We demonstrate that LRP1 silencing in 3T3F442A murine preadipocytes significantly inhibits the expression of PPARgamma, HSL and aP2 adipocyte differentiation markers after adipogenesis induction, and leads to lipid-depleted cells. We further show that the absence of lipids in LRP1-silenced preadipocytes is not caused by lipolysis induction. In addition, we provide the first evidences that LRP1 is significantly up-regulated in obese C57BI6/J mouse adipocytes and obese human adipose tissues. Interestingly, silencing of LRP1 in fully-differentiated adipocytes also reduces cellular lipid level and is associated with an increase of basal lipolysis. However, the ability of mature adipocytes to induce lipolysis is independent of LRP1 expression. Altogether, our findings highlight the dual role of LRP1 in the control of adipogenesis and lipid homeostasis, and suggest that LRP1 may be an important therapeutic target in obesity.

    PloS one 2009;4;10;e7422

  • Functional expression of the alpha 2-macroglobulin receptor CD91 in salivary gland epithelial cells.

    Bourazopoulou E, Kapsogeorgou EK, Routsias JG, Manoussakis MN, Moutsopoulos HM and Tzioufas AG

    Department of Pathophysiology, School of Medicine, National University of Athens, Athens, Greece.

    CD91 molecule is a multifunctional receptor of alpha 2-macroglobulin, heat-shock proteins and calreticulin. CD91 has been implicated in cross-presentation of peptides chaperoned by these proteins to MHC molecules, thus eliciting antigen-specific immune responses. Hence, CD91 is considered as a major regulator of innate and acquired immune responses. Herein, we show that CD91 molecules are expressed by human salivary gland epithelial cells (SGEC), as indicated by immunohistochemical studies in minor salivary gland biopsy tissues (n = 21) as well as by the analyses of human long-term cultured non-neoplastic SGEC lines (n = 11) and the neoplastic HSG cell line. In these cell lines CD91 expression was evaluated by RT-PCR, flow cytometry and confocal microscopy. Standard internalization assays revealed that HSG and SGECs are capable to bind and internalize the CD91 ligand alpha 2-macroglobulin. This internalization is specific, as attested by inhibition studies using unlabeled alpha 2-macroglobulin and a blocking antibody against human CD91 receptor. Conclusively, our findings indicate that SGEC functionally express CD91 receptor, suggesting that this pathway might be involved in the presentation of exogenous antigens in SGEC.

    Journal of autoimmunity 2009;33;2;141-6

  • The type of LDLR gene mutation predicts cardiovascular risk in children with familial hypercholesterolemia.

    Guardamagna O, Restagno G, Rolfo E, Pederiva C, Martini S, Abello F, Baracco V, Pisciotta L, Pino E, Calandra S and Bertolini S

    Department of Pediatrics, University of Turin, Turin, Italy. ornella.guardamagna@unito.it

    Objective: To ascertain whether the molecular characterization of a defect in the low-density lipoprotein (LDL) receptor gene (LDLR) in children with heterozygous familial hypercholesterolemia (heFH) identifies subjects at greater risk of developing premature coronary artery disease (pCAD) later in life.

    We investigated 264 children with heFH from 201 families, along with 148 affected parents and 100 unaffected siblings. The lipid profile was assessed before any treatment was provided, and genotype analysis was performed to characterize LDLR defects. In a subgroup of children with heFH and controls, we measured aorta and carotid intima-media thickness (aIMT and cIMT). The prevalence of pCAD in parents and/or grandparents with heFH was recorded.

    Results: The children with heFH with a family history of pCAD had higher LDL cholesterol and apolipoprotein B levels and greater aIMT and cIMT than those with negative family history. Compared with carriers of LDLR-defective mutations, carriers of LDLR-negative mutations had a more severe phenotype, in terms of plasma lipid levels and IMT, and a higher prevalence of pCAD in first-degree relatives (36% vs 6.7%; P < .001).

    Conclusions: The study of heFH in children, in which other risk factors for CAD play a minor role, allows early identification of those at increased risk for developing pCAD, who merit more stringent clinical control and early pharmacologic treatment.

    The Journal of pediatrics 2009;155;2;199-204.e2

  • Defining the human deubiquitinating enzyme interaction landscape.

    Sowa ME, Bennett EJ, Gygi SP and Harper JW

    Department of Pathology, Harvard Medical School, Boston, MA 02115, USA.

    Deubiquitinating enzymes (Dubs) function to remove covalently attached ubiquitin from proteins, thereby controlling substrate activity and/or abundance. For most Dubs, their functions, targets, and regulation are poorly understood. To systematically investigate Dub function, we initiated a global proteomic analysis of Dubs and their associated protein complexes. This was accomplished through the development of a software platform called CompPASS, which uses unbiased metrics to assign confidence measurements to interactions from parallel nonreciprocal proteomic data sets. We identified 774 candidate interacting proteins associated with 75 Dubs. Using Gene Ontology, interactome topology classification, subcellular localization, and functional studies, we link Dubs to diverse processes, including protein turnover, transcription, RNA processing, DNA damage, and endoplasmic reticulum-associated degradation. This work provides the first glimpse into the Dub interaction landscape, places previously unstudied Dubs within putative biological pathways, and identifies previously unknown interactions and protein complexes involved in this increasingly important arm of the ubiquitin-proteasome pathway.

    Funded by: NIA NIH HHS: AG085011, R01 AG011085, R01 AG011085-16; NIDDK NIH HHS: K01 DK098285; NIGMS NIH HHS: GM054137, GM67945, R01 GM054137, R01 GM054137-14, R01 GM067945

    Cell 2009;138;2;389-403

  • An association analysis of Alzheimer disease candidate genes detects an ancestral risk haplotype clade in ACE and putative multilocus association between ACE, A2M, and LRRTM3.

    Edwards TL, Pericak-Vance M, Gilbert JR, Haines JL, Martin ER and Ritchie MD

    Department of Molecular Physiology and Biophysics and Center for Human Genetics Research, Vanderbilt University, Nashville, Tennessee 37232, USA.

    Alzheimer's disease (AD) is the most common form of progressive dementia in the elderly. It is a neurodegenerative disorder characterized by the neuropathologic findings of neurofibrillary tangles and amyloid plaques that accumulate in vulnerable brain regions. AD etiology has been studied by many groups, but since the discovery of the APOE epsilon4 allele, no further genes have been mapped conclusively to late-onset AD (LOAD). In this study, we examined genetic association with LOAD susceptibility in 738 Caucasian families (4,704 individuals) and an independent case-control dataset with 296 cases and 566 controls exploring 11 candidate genes (47 SNPs common to both samples). In addition to tests for main effects and haplotypes, the MDR-PDT was used to search for gene-gene interactions in the family data. We observed significant haplotype effects in ACE in family and case-control samples using standard and cladistic haplotype models. ACE was also part of significant 2 and 3-locus MDR-PDT joint effects models with Alpha-2-Macroglobulin (A2M), which mediates the clearance of Abeta, and Leucine-Rich Repeat Transmembrane-3 (LRRTM3), a nested gene in Alpha-3 Catenin (CTNNA3) which binds Presenilin-1. This result did not replicate in the case-control sample, and may not be a true positive. These genes are related to Abeta clearance; thus this constellation of effects might constitute an axis of susceptibility for LOAD. The consistent ACE haplotype result between independent family-based and unrelated case-control datasets is strong evidence in favor of ACE as a susceptibility locus for AD, and replicates results from several other studies in a large sample.

    Funded by: NIA NIH HHS: AG20135, R01 AG019757, R01 AG019757-06, R01 AG020135, R01 AG027944, R01 AG027944-01A2

    American journal of medical genetics. Part B, Neuropsychiatric genetics : the official publication of the International Society of Psychiatric Genetics 2009;150B;5;721-35

  • Specificity of binding of the low density lipoprotein receptor-related protein to different conformational states of the clade E serpins plasminogen activator inhibitor-1 and proteinase nexin-1.

    Jensen JK, Dolmer K and Gettins PG

    Department of Biochemistry and Molecular Genetics, University of Illinois at Chicago, Chicago, Illinois 60607, USA.

    The low density lipoprotein receptor-related protein (LRP) is the principal clearance receptor for serpins and serpin-proteinase complexes. The ligand binding regions of LRP consist of clusters of cysteine-rich approximately 40-residue complement-like repeats (CR), with cluster II being the principal ligand-binding region. To better understand the specificity of binding at different sites within the cluster and the ability of LRP to discriminate in vivo between uncomplexed and proteinase-complexed serpins, we have systematically examined the affinities of plasminogen activator inhibitor-1 (PAI-1) and proteinase nexin-1 (PN-1) in their native, cleaved, and proteinase-complexed states to (CR)(2) and (CR)(3) fragments of LRP cluster II. A consistent blue shift of the CR domain tryptophan fluorescence suggested a common mode of serpin binding, involving lysines on the serpin engaging the acidic region around the calcium binding site of the CR domain. High affinity binding of non-proteinase-complexed PAI-1 and PN-1 occurred to all fragments containing three CR domains (3-59 nm) and most that contain only two CR domains, although binding energies to different (CR)(3) fragments differed by up to 18% for PAI-1 and 9% for PN-1. No detectable difference in affinity was seen between native and cleaved serpin. However, the presence of proteinase in complex with the serpin enhanced affinity modestly and presumably nonspecifically. This may be sufficient to give preferential binding of such complexes in vivo at the relevant physiological concentrations.

    Funded by: NHLBI NIH HHS: R01 HL079430, R01HL79430; NIGMS NIH HHS: R01 GM054414, R01 GM54414

    The Journal of biological chemistry 2009;284;27;17989-97

  • Integrated associations of genotypes with multiple blood biomarkers linked to coronary heart disease risk.

    Drenos F, Talmud PJ, Casas JP, Smeeth L, Palmen J, Humphries SE and Hingorani AD

    Division of Cardiovascular Genetics, Department of Medicine, Royal Free and University College Medical School, 5 University St, London WC1E 6JF, UK.

    Individuals at risk of coronary heart disease (CHD) show multiple correlations across blood biomarkers. Single nucleotide polymorphisms (SNPs) indexing biomarker differences could help distinguish causal from confounded associations because of their random allocation prior to disease. We examined the association of 948 SNPs in 122 candidate genes with 12 CHD-associated phenotypes in 2775 middle aged men (a genic scan). Of these, 140 SNPs indexed differences in HDL- and LDL-cholesterol, triglycerides, C-reactive protein, fibrinogen, factor VII, apolipoproteins AI and B, lipoprotein-associated phospholipase A2, homocysteine or folate, some with large effect sizes and highly significant P-values (e.g. 2.15 standard deviations at P = 9.2 x 10(-140) for F7 rs6046 and FVII levels). Top ranking SNPs were then tested for association with additional biomarkers correlated with the index phenotype (phenome scan). Several SNPs (e.g. in APOE, CETP, LPL, APOB and LDLR) influenced multiple phenotypes, while others (e.g. in F7, CRP and FBB) showed restricted association to the index marker. SNPs influencing six blood proteins were used to evaluate the nature of the associations between correlated blood proteins utilizing Mendelian randomization. Multiple SNPs were associated with CHD-related quantitative traits, with some associations restricted to a single marker and others exerting a wider genetic 'footprint'. SNPs indexing biomarkers provide new tools for investigating biological relationships and causal links with disease. Broader and deeper integrated analyses, linking genomic with transcriptomic, proteomic and metabolomic analysis, as well as clinical events could, in principle, better delineate CHD causing pathways amenable to treatment.

    Funded by: British Heart Foundation: FS2005/125, PG2005/014, RG/08/008/25291; Wellcome Trust: 082178

    Human molecular genetics 2009;18;12;2305-16

  • Participation of the lipoprotein receptor LRP1 in hypoxia-HSP90alpha autocrine signaling to promote keratinocyte migration.

    Woodley DT, Fan J, Cheng CF, Li Y, Chen M, Bu G and Li W

    Department of Dermatology and the USC-Norris Comprehensive Cancer Center, the University of Southern California Keck School of Medicine, Los Angeles, CA 90033, USA.

    Hypoxia is a microenvironmental stress in many pathological conditions, including wound healing and tumor invasion. Under hypoxia, the cells are forced to adapt alternative and self-supporting mechanisms. Understanding these mechanisms may lead to new insights into human disorders. We report here a novel autocrine signaling mechanism by which hypoxia promotes human keratinocyte (HK) migration. First, hypoxia triggers HKs to secrete heat shock protein 90-alpha (HSP90alpha) via a HIF1-dependent pathway. The secreted HSP90alpha in turn promotes migration, but not proliferation, of the cells. Disruption of the secretion or extracellular function of HSP90alpha blocked hypoxia-stimulated HK migration. The ubiquitously expressed surface receptor, LRP1 (LDL-receptor-related protein 1), mediates the HSP90alpha signaling. Inhibition of LRP1 binding to extracellular HSP90alpha by neutralizing antibodies or genetic silencing of the LRP1 receptor by RNAi completely nullified hypoxia-driven HK migration. Finally, re-introducing a RNAi-resistant LRP1 cDNA into LRP1-downregulated HKs rescued the motogenic response of the cells to hypoxia. We propose that the hypoxia-HSP90alpha-LRP1 autocrine loop provides previously unrecognized therapeutic targets for human disorders such as chronic wounds and cancer invasion.

    Funded by: NIAMS NIH HHS: AR46538, GM/AR67100-01

    Journal of cell science 2009;122;Pt 10;1495-8

  • Novel role of RanBP9 in BACE1 processing of amyloid precursor protein and amyloid beta peptide generation.

    Lakshmana MK, Yoon IS, Chen E, Bianchi E, Koo EH and Kang DE

    Department of Neurosciences, University of California, San Diego, La Jolla, California 92093, USA.

    Accumulation of the amyloid beta (Abeta) peptide derived from the proteolytic processing of amyloid precursor protein (APP) is the defining pathological hallmark of Alzheimer disease. We previously demonstrated that the C-terminal 37 amino acids of lipoprotein receptor-related protein (LRP) robustly promoted Abeta generation independent of FE65 and specifically interacted with Ran-binding protein 9 (RanBP9). In this study we found that RanBP9 strongly increased BACE1 cleavage of APP and Abeta generation. This pro-amyloidogenic activity of RanBP9 did not depend on the KPI domain or the Swedish APP mutation. In cells expressing wild type APP, RanBP9 reduced cell surface APP and accelerated APP internalization, consistent with enhanced beta-secretase processing in the endocytic pathway. The N-terminal half of RanBP9 containing SPRY-LisH domains not only interacted with LRP but also with APP and BACE1. Overexpression of RanBP9 resulted in the enhancement of APP interactions with LRP and BACE1 and increased lipid raft association of APP. Importantly, knockdown of endogenous RanBP9 significantly reduced Abeta generation in Chinese hamster ovary cells and in primary neurons, demonstrating its physiological role in BACE1 cleavage of APP. These findings not only implicate RanBP9 as a novel and potent regulator of APP processing but also as a potential therapeutic target for Alzheimer disease.

    Funded by: NIA NIH HHS: AG 005131-24S1

    The Journal of biological chemistry 2009;284;18;11863-72

  • Insulin stimulates hepatic low density lipoprotein receptor-related protein 1 (LRP1) to increase postprandial lipoprotein clearance.

    Laatsch A, Merkel M, Talmud PJ, Grewal T, Beisiegel U and Heeren J

    Department of Biochemistry and Molecular Biology II: Molecular Cell Biology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany. laatsch@uke.uni-hamburg.de

    Background: While the role of insulin in glucose uptake and its aberration in diabetes are well established, the effect of insulin on lipoprotein clearance in the postprandial phase is not yet fully understood. The dietary lipids are carried in chylomicron remnants (CR) which are taken up into the liver mainly via LDLR-related protein 1 (LRP1). In this study, the effect of insulin on LRP1-mediated hepatic CR uptake was investigated.

    Methods: The study was based on determining the subcellular localisation of LRP1 by subcellular fractionation and immunofluorescence microscopy and correlating those findings with the hepatic uptake of fluorescently or radioactively labelled LRP1-specific ligands and CR in hepatoma cells, primary hepatocytes and mouse models.

    In vitro and in vivo, insulin stimulated the translocation of hepatic LRP1 from intracellular vesicles to the plasma membrane, which correlates with an increased uptake of LRP1-specific ligands. In wild-type mice, a glucose-induced insulin response increased the hepatic uptake of LRP1 ligands while in leptin-deficient obese mice (ob/ob), which are characterised by hepatic insulin resistance, insulin-inducible LRP1 ligand uptake was abolished. Finally, upon hepatic LRP1 knockdown, insulin no longer significantly enhanced CR uptake into the liver. The insulin-induced LRP1-mediated CR uptake, as demonstrated here, suggests that impaired hepatic LRP1 translocation can contribute to the postprandial lipaemia in insulin resistance.

    Funded by: British Heart Foundation: RG/05/014

    Atherosclerosis 2009;204;1;105-11

  • Low-density lipoprotein receptor-related protein 1 is an essential receptor for myelin phagocytosis.

    Gaultier A, Wu X, Le Moan N, Takimoto S, Mukandala G, Akassoglou K, Campana WM and Gonias SL

    Department of Pathology, University of California, San Diego, La Jolla, CA 92093, USA.

    Multiple sclerosis (MS) is an autoimmune disease in which myelin is progressively degraded. Because degraded myelin may both initiate and accelerate disease progression, clearing degraded myelin from extracellular spaces may be critical. In this study, we prepared myelin vesicles (MV) from rat brains as a model of degraded myelin. Murine embryonic fibroblasts (MEFs) rapidly internalized MVs, which accumulated in lysosomes only when these cells expressed low-density lipoprotein receptor-related protein (LRP1). Receptor-associated protein (RAP), which binds LRP1 and inhibits interaction with other ligands, blocked MV uptake by LRP1-expressing MEFs. As a complementary approach, we prepared primary cultures of rat astrocytes, microglia and oligodendrocytes. All three cell types expressed LRP1 and mediated MV uptake, which was inhibited by RAP. LRP1 gene-silencing in oligodendrocytes also blocked MV uptake. Myelin basic protein (MBP), which was expressed as a recombinant protein, bound directly to LRP1. MBP-specific antibody inhibited MV uptake by oligodendrocytes. In experimental autoimmune encephalomyelitis in mice, LRP1 protein expression was substantially increased in the cerebellum and spinal cord. LRP1 colocalized with multiple CNS cell types. These studies establish LRP1 as a major receptor for phagocytosis of degraded myelin, which may function alone or in concert with co-receptors previously implicated in myelin phagocytosis.

    Funded by: NHLBI NIH HHS: HL-60551; NINDS NIH HHS: NS-052189, NS-057456, R01 NS-054671

    Journal of cell science 2009;122;Pt 8;1155-62

  • Low-density lipoprotein receptor-related protein 1 promotes cancer cell migration and invasion by inducing the expression of matrix metalloproteinases 2 and 9.

    Song H, Li Y, Lee J, Schwartz AL and Bu G

    Edward Mallinckrodt Department of Pediatrics, Washington University School of Medicine, St Louis, Missouri 63110, USA.

    The low-density lipoprotein receptor-related protein 1 (LRP1) is a multifunctional endocytic receptor involved in the metabolism of various extracellular ligands, including proteinases, that play critical roles in tumor invasion. Although several studies have shown an increased expression of LRP1 in cancer cells, its function in tumor development and progression remains largely unclear. Here, we reveal a novel mechanism by which LRP1 induces the expression of matrix metalloproteinase 2 (MMP2) and MMP9 and thereby promotes the migration and invasion of human glioblastoma U87 cells. Knockdown of LRP1 expression greatly decreased U87 cell migration and invasion, which was rescued by the forced expression of a functional LRP1 minireceptor. Inhibition of ligand binding to LRP1 by a specific antagonist, receptor-associated protein, also led to reduced cancer cell migration and invasion. Because MMPs play critical roles in cancer cell migration and invasion, we examined the expression of several MMPs and found that the expression of functional MMP2 and MMP9 was selectively decreased in LRP1 knockdown cells. More importantly, decreased cell migration and invasion of LRP1 knockdown cells were completely rescued by exogenous expression of MMP2 or MMP9, suggesting that these MMPs are likely downstream targets of LRP1-mediated signaling. We further show that the level of phosphorylated extracellular signal-regulated kinase (ERK) was significantly decreased in LRP1-silenced cells, suggesting that ERK is a potential mediator of LRP1-regulated MMP2 and MMP9 expression in U87 cells. Together, our data strongly suggest that LRP1 promotes glioblastoma cell migration and invasion by regulating the expression and function of MMP2 and MMP9 perhaps via an ERK-dependent signaling pathway.

    Funded by: NCI NIH HHS: CA100520, R01 CA100520, R01 CA100520-01A2, R01 CA100520-02, R01 CA100520-03, R01 CA100520-04, R01 CA100520-05

    Cancer research 2009;69;3;879-86

  • LRP1 controls intracellular cholesterol storage and fatty acid synthesis through modulation of Wnt signaling.

    Terrand J, Bruban V, Zhou L, Gong W, El Asmar Z, May P, Zurhove K, Haffner P, Philippe C, Woldt E, Matz RL, Gracia C, Metzger D, Auwerx J, Herz J and Boucher P

    CNRS, UMR7175, Université de Strasbourg, Illkirch, F-67401 France.

    The low-density lipoprotein receptor-related protein LRP1 is a cell surface receptor with functions in diverse physiological pathways, including lipid metabolism. Here we show that LRP1-deficient fibroblasts accumulate high levels of intracellular cholesterol and cholesteryl-ester when stimulated for adipocyte differentiation. We demonstrate that LRP1 stimulates a canonical Wnt5a signaling pathway that prevents cholesterol accumulation. Moreover, we show that LRP1 is required for lipolysis and stimulates fatty acid synthesis independently of the noradrenergic pathway, through inhibition of GSK3beta and its previously unknown target acetyl-CoA carboxylase (ACC). As a result of ACC inhibition, mature LRP1-deficient adipocytes of adult mice are hypotrophic, and lower uptake of fatty acids into adipose tissue leads to their redistribution to the liver. These results establish LRP1 as a novel integrator of adipogenic differentiation and fat storage signals.

    Funded by: NHLBI NIH HHS: HL20948, HL63762, R37 HL063762; NIDDK NIH HHS: DK067320; NINDS NIH HHS: NS43408

    The Journal of biological chemistry 2009;284;1;381-8

  • Genetic interaction between tau and the apolipoprotein E receptor LRP1 Increases Alzheimer's disease risk.

    Vázquez-Higuera JL, Mateo I, Sánchez-Juan P, Rodríguez-Rodríguez E, Pozueta A, Infante J, Berciano J and Combarros O

    Neurology Service and Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas (CIBERNED), University Hospital Marqués de Valdecilla (University of Cantabria), Santander, Spain.

    Abnormal tau hyperphosphorylation is one of the central events in the development of neurofibrillary tangles (NFTs) in Alzheimer's disease (AD), and phosphorylation of tau is accelerated by the increase in the level of neuronal cholesterol. Apolipoprotein E (APOE) promotes the neuronal uptake of cholesterol via APOE receptors such as the low-density lipoprotein receptor-related protein 1 (LRP1), and the APOE epsilon4 allele is associated with an increase in NFT burden in AD brain. In a case-control study in 246 AD patients and 237 healthy controls, we examined whether the combined gene effects between tau (intron 9, rs2471738) polymorphism and LRP1 (exon 3, rs1799986) polymorphism might be responsible for susceptibility to AD, independently or in concert with the APOE epsilon4 allele. Subjects carrying both the tau (intron 9, rs2471738) T allele (CT and TT genotypes) and the LRP1 (exon 3, rs1799986) T allele (CT and TT genotypes) had a 6 times higher risk of developing AD than subjects without these risk genotypes (odds ration = 6.20, 95% confidence interval = 1.74-22.05, p = 0.005), and this genetic interaction was observed in either the presence or the absence of the APOE epsilon4 allele. These data suggest that the synergistic effects (epistasis) between tau and LRP1 might modify the risk of AD in an APOE epsilon4 allele-independent fashion.

    Dementia and geriatric cognitive disorders 2009;28;2;116-20

  • The low density lipoprotein receptor-related protein 1 mediates uptake of amyloid beta peptides in an in vitro model of the blood-brain barrier cells.

    Yamada K, Hashimoto T, Yabuki C, Nagae Y, Tachikawa M, Strickland DK, Liu Q, Bu G, Basak JM, Holtzman DM, Ohtsuki S, Terasaki T and Iwatsubo T

    Department of Neuropathology and Neuroscience, Graduate School of Pharmaceutical Sciences, University of Tokyo, Tokyo 113-0033, Japan.

    The metabolism of amyloid beta peptide (A beta) in the brain is crucial to the pathogenesis of Alzheimer disease. A body of evidence suggests that A beta is actively transported from brain parenchyma to blood across the blood-brain barrier (BBB), although the precise mechanism remains unclear. To unravel the cellular and molecular mechanism of A beta transport across the BBB, we established a new in vitro model of the initial internalization step of A beta transport using TR-BBB cells, a conditionally immortalized endothelial cell line from rat brain. We show that TR-BBB cells rapidly internalize A beta through a receptor-mediated mechanism. We also provide evidence that A beta internalization is mediated by LRP1 (low density lipoprotein receptor-related protein 1), since administration of LRP1 antagonist, receptor-associated protein, neutralizing antibody, or small interference RNAs all reduced A beta uptake. Despite the requirement of LRP1-dependent internalization, A beta does not directly bind to LRP1 in an in vitro binding assay. Unlike TR-BBB cells, mouse embryonic fibroblasts endogenously expressing functional LRP1 and exhibiting the authentic LRP1-mediated endocytosis (e.g. of tissue plasminogen activator) did not show rapid A beta uptake. Based on these data, we propose that the rapid LRP1-dependent internalization of A beta occurs under the BBB-specific cellular context and that TR-BBB is a useful tool for analyzing the molecular mechanism of the rapid transport of A beta across BBB.

    Funded by: NIA NIH HHS: AG13956, R01 AG027924

    The Journal of biological chemistry 2008;283;50;34554-62

  • C-terminal 37 residues of LRP promote the amyloidogenic processing of APP independent of FE65.

    Lakshmana MK, Chen E, Yoon IS and Kang DE

    Department of Neurosciences, University of California, San Diego, Gilman Drive, La Jolla, CA 92093, USA.

    The major defining pathological hallmark of Alzheimer's disease (AD) is the accumulation of amyloid beta protein (Abeta), a small peptide derived from beta- and gamma-secretase cleavages of the amyloid precursor protein (APP). Recent studies have shown that the Low-density lipoprotein receptor-related protein (LRP) plays a pivotal role in the trafficking of APP and generation of Abeta. In particular, we recently showed that the soluble cytoplasmic tail of LRP (LRP-ST) without a membrane tether was sufficient to promote Abeta generation. In this study, we demonstrate that the last 37 residues of LRP cytoplasmic tail (LRP-C37) lacking the NPxY motifs and FE65 binding mediate the core pro-amyloidogenic activity of LRP-ST. Moreover, we show that the conserved dileucine motif within the LRP-C37 region is a key determinant of its Abeta promoting activity. Finally, results from a yeast two-hybrid screen using LRP-C37 region as bait reveal four new LRP-binding proteins implicated in intracellular signalling and membrane protein trafficking. Our findings indicate that the LRP-C37 sequence represents a new protein-binding domain that may be useful as a therapeutic target and tool to lower Abeta generation in AD.

    Funded by: NIA NIH HHS: AG 005131-24S1, P50 AG005131, P50 AG005131-26

    Journal of cellular and molecular medicine 2008;12;6B;2665-74

  • Gamma-secretase limits the inflammatory response through the processing of LRP1.

    Zurhove K, Nakajima C, Herz J, Bock HH and May P

    Department of Medicine II, University Hospital, University of Freiburg, 79106 Freiburg, Germany.

    Inflammation is a potentially self-destructive process that needs tight control. We have identified a nuclear signaling mechanism through which the low-density lipoprotein receptor-related protein 1 (LRP1) limits transcription of lipopolysaccharide (LPS)-inducible genes. LPS increases the proteolytic processing of the ectodomain of LRP1, which results in the gamma-secretase-dependent release of the LRP1 intracellular domain (ICD) from the plasma membrane and its translocation to the nucleus, where it binds to and represses the interferon-gamma promoter. Basal transcription of LPS target genes and LPS-induced secretion of proinflammatory cytokines are increased in the absence of LRP1. The interaction between LRP1-ICD and interferon regulatory factor 3 (IRF-3) promotes the nuclear export and proteasomal degradation of IRF-3. Feedback inhibition of the inflammatory response through intramembranous processing of LRP1 thus defines a physiological role for gamma-secretase.

    Funded by: NHLBI NIH HHS: P01 HL020948, P01 HL020948-210015, P01 HL020948-220015, P01 HL020948-230015, P01 HL020948-240015, P01 HL020948-250015, P01 HL020948-310005, P01 HL020948-320005, R01 HL063762, R01 HL063762-01, R01 HL063762-02, R01 HL063762-03, R01 HL063762-04, R01 HL063762-05, R01 HL063762-06, R01 HL063762-07, R01 HL063762-08, R01 HL063762-09, R01 HL063762-10, R37 HL063762; NINDS NIH HHS: R01 NS043408, R01 NS043408-01, R01 NS043408-02, R01 NS043408-03, R01 NS043408-04, R01 NS043408-05

    Science signaling 2008;1;47;ra15

  • Human plasmacytoid dendritic cells interact with gp96 via CD91 and regulate inflammatory responses.

    De Filippo A, Binder RJ, Camisaschi C, Beretta V, Arienti F, Villa A, Della Mina P, Parmiani G, Rivoltini L and Castelli C

    Unit of Immunotherapy of Human Tumours, Fondazione Istituto di Ricovero e Cura a Carattere Scientifico, Istituto Nazionale dei Tumori, Milano, Italy.

    Glucose-regulated stress protein gp96 is known to be involved in the host response to pathogens and to cancer. Our study explored the relationships between gp96 and human blood plasmacytoid dendritic cells (pDC) and proved that gp96 directly targets pDC by a receptor-dependent interaction. Competition studies identified CD91 as a gp96 receptor on pDC, and laser confocal imaging indicated that CD91 triggering was followed by gp96 endocytosis and trafficking into early endosomes and later into the endoplasmic reticulum compartment. Using two alternative Abs, we showed that human blood pDC reproducibly expressed CD91, although different levels of expression were detectable among the analyzed donors. Moreover, CpG-matured pDC displayed CD91 receptor up-regulation that correlated with an increased gp96 binding. Functionally, gp96-pDC interaction activated the NF-kappaB pathway, leading to the nuclear translocation of the NF-kappaB complex. gp96-treated pDC maintained an immature phenotype, while they down-modulated the release of IL-8, suggesting an anti-inflammatory role of this pathway, and they strongly up-regulated the cell surface expression of the gp96 receptor CD91. CpG-matured or gp96-treated pDC, expressing high levels of the gp96 receptor CD91, antagonized the gp96-induced activation of monocyte-derived dendritic cells in terms of cell surface phenotype and cytokine production. Altogether, these results suggest that gp96-pDC interaction might represent an active mechanism controlling the strength of the immune response to free, extracellular available gp96; this mechanism could be particularly relevant in wounds and chronic inflammation.

    Journal of immunology (Baltimore, Md. : 1950) 2008;181;9;6525-35

  • Thrombospondin 1 binding to calreticulin-LRP1 signals resistance to anoikis.

    Pallero MA, Elzie CA, Chen J, Mosher DF and Murphy-Ullrich JE

    Department of Pathology, VH 668 1530 3rd Ave., South, Birmingham, AL 35294-0019, USA.

    Anoikis, apoptotic cell death due to loss of cell adhesion, is critical for regulation of tissue homeostasis in tissue remodeling. Fibrogenesis is associated with reduced fibroblast apoptosis. The matricellular protein thrombospondin 1 (TSP1) regulates cell adhesion and motility during tissue remodeling and in fibrogenesis. The N-terminal domain of TSP1 binds to the calreticulin-LRP1 receptor co-complex to signal down-regulation of cell adhesion and increased cell motility through focal adhesion disassembly. TSP1 signaling through calreticulin-LRP1 activates cell survival signals such as PI3-kinase. Therefore, we tested the hypothesis that TSP1 supports cell survival under adhesion-independent conditions to facilitate tissue remodeling. Here, we show that platelet TSP1, its N-terminal domain (NoC1) as a recombinant protein, or a peptide comprising the calreticulin-LRP1 binding site [amino acids 17-35 (hep I)] in the N-terminal domain promotes fibroblast survival under anchorage-independent conditions. TSP1 activates Akt and decreases apoptotic signaling through caspase 3 and PARP1 in suspended fibroblasts. Inhibition of PI3K/Akt activity blocks TSP1-mediated anchorage-independent survival. Fibroblasts lacking LRP1 or expressing calreticulin lacking the TSP1 binding site do not respond to TSP1 with anchorage-independent survival. These data define a novel role for TSP1 signaling through the calreticulin/LRP1 co-complex in tissue remodeling and fibrotic responses through stimulation of anoikis resistance.-Pallero, M. A., Elzie, C. A., Chen, J., Mosher, D. F., Murphy-Ullrich, J. E. Thrombospondin 1 binding to calreticulin-LRP1 signals resistance to anoikis.

    Funded by: NCRR NIH HHS: C06 RR 15490, C06 RR015490; NHLBI NIH HHS: HL079644, R01 HL079644; NIAMS NIH HHS: T32 AR047512, T32AR47512-01A1; NIDCR NIH HHS: T32 DE014300, T32DE014300

    FASEB journal : official publication of the Federation of American Societies for Experimental Biology 2008;22;11;3968-79

  • Genetic association between low-density lipoprotein receptor-related protein gene polymorphisms and Alzheimer's disease in Chinese Han population.

    Zhou YT, Zhang ZX, Chan P, He XM, Tang MN, Wu CB and Hong Z

    Department of Neurology and Neurobiology, Key Laboratory of Neurodegenerative Diseases for Ministry of Education, Beijing Institute of Geriatrics and Xuanwu Hospital of Capital University of Medical Sciences, 100053 Beijing, China.

    Alzheimer's disease (AD) is the most common neurodegenerative disorders in the elderly. Low-density lipoprotein receptor-related protein (LRP), as a receptor of apolipoprotein E (APOE), APP, and alpha2 macroglobulin (alpha2-M), keeps the balance between degeneration and production of beta-amyloid protein (Abeta) clearance. Its gene had been defined as a candidate gene for AD, but the results were not universal. Total 496 AD patients and 478 controls were recruited in Chinese Han population and real-time PCR was used to detect the polymorphism of LRP C766T. Multiple logistic regression, Chi-square test and survival analysis were performed to explore the association. The distribution of LRP genotypes and alleles was significantly different between cases and controls, and T allele could reduce the risk for developing AD (OR of CT genotype: 0.57; 95% CI: 0.38-0.85, rho=0.003; OR of T allele: 0.57; 95% CI: 0.39-0.83, rho=0.003). TT genotype carriers had 5 years later for developing AD compared with CC genotype carriers, but survival analysis did not conform this (LRP TT vs. CT and CC log rank chi(2)=2.71, rho=0.26). The distribution of LRP C766T genotypes and alleles was different among different severity stratified by MMSE yet (rho=0.26). Our data suggested that the polymorphism of LRP C766T was strongly associated with AD and T allele might be a protective factor for AD in Chinese Han population.

    Neuroscience letters 2008;444;1;109-11

  • Effects of six APOA5 variants, identified in patients with severe hypertriglyceridemia, on in vitro lipoprotein lipase activity and receptor binding.

    Dorfmeister B, Zeng WW, Dichlberger A, Nilsson SK, Schaap FG, Hubacek JA, Merkel M, Cooper JA, Lookene A, Putt W, Whittall R, Lee PJ, Lins L, Delsaux N, Nierman M, Kuivenhoven JA, Kastelein JJ, Vrablik M, Olivecrona G, Schneider WJ, Heeren J, Humphries SE and Talmud PJ

    Department of Medicine, Division of Cardiovascular Genetics, UCL, London, UK.

    Objective: The purpose of this study was to identify rare APOA5 variants in 130 severe hypertriglyceridemic patients by sequencing, and to test their functionality, since no patient recall was possible.

    We studied the impact in vitro on LPL activity and receptor binding of 3 novel heterozygous variants, apoAV-E255G, -G271C, and -H321L, together with the previously reported -G185C, -Q139X, -Q148X, and a novel construct -Delta139 to 147. Using VLDL as a TG-source, compared to wild type, apoAV-G255, -L321 and -C185 showed reduced LPL activation (-25% [P=0.005], -36% [P<0.0001], and -23% [P=0.02]), respectively). ApoAV-C271, -X139, -X148, and Delta139 to 147 had little affect on LPL activity, but apoAV-X139, -X148, and -C271 showed no binding to LDL-family receptors, LR8 or LRP1. Although the G271C proband carried no LPL and APOC2 mutations, the H321L carrier was heterozygous for LPL P207L. The E255G carrier was homozygous for LPL W86G, yet only experienced severe hypertriglyceridemia when pregnant.

    Conclusions: The in vitro determined function of these apoAV variants only partly explains the high TG levels seen in carriers. Their occurrence in the homozygous state, coinheritance of LPL variants or common APOA5 TG-raising variant in trans, appears to be essential for their phenotypic expression.

    Funded by: British Heart Foundation: PG 04/110/17827, RG/08/008/25291, RG2005/014

    Arteriosclerosis, thrombosis, and vascular biology 2008;28;10;1866-71

  • Interaction of coagulation factor VIII with members of the low-density lipoprotein receptor family follows common mechanism and involves consensus residues within the A2 binding site 484-509.

    Ananyeva NM, Makogonenko YM, Sarafanov AG, Pechik IV, Gorlatova N, Radtke KP, Shima M and Saenko EL

    Center for Vascular and Inflammatory Diseases, University of Maryland School of Medicine, 800 West Baltimore Street, Baltimore, MD 21201, USA. NAnanyeva@som.umaryland.edu

    Coagulation factor VIII interacts with several members of the low-density lipoprotein receptor family including low-density lipoprotein receptor-related protein, low-density lipoprotein receptor, and very low-density lipoprotein receptor. The present study was aimed to compare the mechanisms of factor VIII interaction with low-density lipoprotein receptor-related protein, megalin, low-density lipoprotein receptor, and very low-density lipoprotein receptor in order to reveal a general mode of these interactions. Binding of plasma-derived factor VIII and its fragments to recombinant soluble ligand-binding domain of low-density lipoprotein receptor (sLDLR1-7) and purified megalin was studied in solid phase and surface plasmon resonance assays. Full-length factor VIII and its light chain bound to the receptors with similar affinities (KD = 260 +/- 9 and 156 +/- 4 nmol/l, respectively, for megalin and KD = 210 +/- 3 and 174 +/- 13 nmol/l, respectively, for sLDLR1-7). Von Willebrand factor inhibited factor VIII binding to both receptors. In contrast to the light chain, exposure of the high-affinity receptor-binding site within the heavy chain (KD = 22 +/- 4 nmol/l for megalin and 17 +/- 3 nmol/l for sLDLR1-7) required proteolytic cleavage by thrombin. This site was mapped to the A2 domain residues 484-509, based on the inhibitory effects of anti-A2 monoclonal antibody 413, and is shared by all four receptors. Using a panel of A2 mutants, we identified key amino acid residues- positively charged K466, R471, R489 and R490, and hydrophilic residues Y487 and S488- which form the frame of this 'consensus' binding site. We conclude that interaction of factor VIII with the members of the low-density lipoprotein receptor family follows the general mode, requires dissociation of factor VIII from von Willebrand factor, and is activation sensitive.

    Funded by: NHLBI NIH HHS: R01 HL72929

    Blood coagulation & fibrinolysis : an international journal in haemostasis and thrombosis 2008;19;6;543-55

  • [Association of the low density lipoprotein receptor-related protein gene 766C/T polymorphism with Alzheimer's disease in Xinjiang Uygurs and Hans].

    Zhou XH, Yue YH, Miao HJ, Hong Y and Ka-Bi N

    The First Cadres Ward, the First Affiliated Hospital, Xinjiang Medical University. Urumqi, Xinjiang, 830054 People's Republic of China. zhouxiaohui858@sinac.com

    Objective: To investigate the relationship between the low density lipoprotein receptor-related protein gene (LRP) 766C/T polymorphisms and Alzheimer's disease (AD) in Xinjiang Uygurs and Hans populations.

    Methods: Those included in the study were > or = 50 years of age and of either Xinjiang Uygur or Han descents. Two hundred and nine individuals had AD and 220 were healthy controls. They were recruited according to ADRDA-NINCDS criteriaîThe polymorphisms of the LRP gene were determined by the PCR-restriction fragment length polymorphism technique. The case-control analysis was adopted to analyze the frequencies of genotypes and alleles.

    Results: (1) The distribution of genotypes or alleles of LRP gene had significant differences between the AD group and the control group in both the Xinjiang Uygurs and Hans populations (P < 0.05). (2) The frequencies of genotypes and alleles were significantly different between the Han AD and Han control group (P < 0.05). (3) The frequencies of genotypes and alleles in those > or = 65 years were significantly different from that in others (P < 0.05). There was a significant increase of AD in the C allele carriers (OR=1.98, P < 0.05). (4) The frequencies of the CC genotype and C allele in female AD patients were higher than that in female controls (P < 0.05), and the C allele carriers had significant increase of AD (OR=2.927, P < 0.05).

    Conclusion: The LRP 766C/T polymorphisms were significantly different between the Chinese Xinjiang Uygur and Han populations. The LRP 766C/T polymorphisms might be associated with AD in the Han population, in females and those of > or = 65 years old.

    Zhonghua yi xue yi chuan xue za zhi = Zhonghua yixue yichuanxue zazhi = Chinese journal of medical genetics 2008;25;4;455-8

  • Involvement of the low-density lipoprotein receptor-related protein in the transcytosis of the brain delivery vector angiopep-2.

    Demeule M, Currie JC, Bertrand Y, Ché C, Nguyen T, Régina A, Gabathuler R, Castaigne JP and Béliveau R

    Angiochem, Montreal, Quebec, Canada.

    The blood-brain barrier (BBB) restricts the entry of proteins as well as potential drugs to cerebral tissues. We previously reported that a family of Kunitz domain-derived peptides called Angiopeps can be used as a drug delivery system for the brain. Here, we further characterize the transcytosis ability of these peptides using an in vitro model of the BBB and in situ brain perfusion. These peptides, and in particular Angiopep-2, exhibited higher transcytosis capacity and parenchymal accumulation than do transferrin, lactoferrin, and avidin. Angiopep-2 transport and accumulation in brain endothelial cells were unaffected by the P-glycoprotein inhibitor, cyclosporin A, indicating that this peptide is not a substrate for the efflux pump P-glycoprotein. However, competition studies show that activated alpha(2)-macroglobulin, a specific ligand for the low-density lipoprotein receptor-related protein-1 (LRP1) and Angiopep-2 can share the same receptor. In addition, LRP1 was detected in glioblastomas and brain metastases from lung and skin cancers. Fluorescent microscopy also revealed that Alexa488-Angiopep-2 co-localized with LRP1 in brain endothelial cell monolayers. Overall, these results suggest that Angiopep-2 transport across the BBB is, in part, mediated by LRP1.

    Journal of neurochemistry 2008;106;4;1534-44

  • Expression of heat shock protein receptors on fibroblast-like synovial cells derived from rheumatoid arthritis-affected joints.

    Hromadnikova I, Nguyen TT, Zlacka D, Sedlackova L, Popelka S, Veigl D, Pech J, Vavrincova P and Sosna A

    Department of Molecular Biology and Cell Pathology, 3rd Medical Faculty, Charles University, Ruska 87, Prague 10, 10000, Czech Republic. ilona.hromadnikova@lf3.cuni.cz

    We examined the membrane expression of inducible Hsp70 and HSP receptors like TLR2, TLR4, CD14, CD36, CD40 and CD91 on fibroblast-like synovial cells (SC) derived from synovial tissue in 23 patients with rheumatoid arthritis (RA), who underwent synovectomy by using flow cytometric analysis. For comparison, autologous skin fibroblasts (SF) derived from the operation wound were tested. Significantly higher Hsp70 expression was found on synovial cells than on skin fibroblasts (median SC 21.4% x SF 5.0%, P < 0.001). Both synovial cells and skin fibroblasts expressed high levels of cell surface CD91 (median SC 80.2% x SF 79.2%), however, no or low levels of CD14, CD40, TLR2, TLR4 and CD36. Further, we observed high co-expression of CD91 and Hsp70 on RA synovial cells (median 18.6%), while skin fibroblasts showed only background Hsp70 expression (median 3.9%, P < 0.001). Since we demonstrated the high prevalence of inducible Hsp70 in RA synovial fluids, we speculate that Hsp70 might be captured onto the membrane of synovial cells from the extracellular space via the CD91 receptor. The significance of the Hsp70 interaction with synovial cells via CD91 remains undefined, but may mediate other non-immune purposes.

    Rheumatology international 2008;28;9;837-44

  • Structural and functional consequences of tyrosine phosphorylation in the LRP1 cytoplasmic domain.

    Betts GN, van der Geer P and Komives EA

    Department of Chemistry and Biochemistry, University of California, San Diego, La Jolla, California 92093-0378, USA.

    The cytoplasmic domain of LRP1 contains two NPXY motifs that have been shown to interact with signaling proteins. In previous work, we showed that Tyr(4507) in the distal NPXY motif is phosphorylated by v-Src, whereas denaturation of the protein was required for phosphorylation of Tyr(4473) in the membraneproximal NPXY motif. Amide H/D exchange studies reveal that the distal NPXY motif is fully solvent-exposed, whereas the proximal one is not. Phosphopeptide mapping combined with in vitro and in vivo kinase experiments show that Tyr(4473) can be phosphorylated, but only if Tyr(4507) is phosphorylated or substituted with glutamic acid. Amide H/D exchange experiments indicate that solvent accessibility increases across the entire LRP1 cytoplasmic region upon phosphorylation at Tyr(4507); in particular the NPXY(4473) motif becomes much more exposed. This differential phosphorylation is functionally relevant: binding of Snx17, which is known to bind at the proximal NPXY motif, is inhibited by phosphorylation at Tyr(4473). Conversely, Shp2 binds most strongly when both of the NPXY motifs in LRP1 are phosphorylated.

    Funded by: NCI NIH HHS: CA78629; NIA NIH HHS: AG025343; NIDDK NIH HHS: T32-DK07233

    The Journal of biological chemistry 2008;283;23;15656-64

  • Angiotensin II upregulates LDL receptor-related protein (LRP1) expression in the vascular wall: a new pro-atherogenic mechanism of hypertension.

    Sendra J, Llorente-Cortés V, Costales P, Huesca-Gómez C and Badimon L

    Barcelona Cardiovascular Research Center, CSIC-ICCC, Hospital de la Santa Creu i Sant Pau, Av. S. Antoni M. Claret 167, 08025 Barcelona, Spain.

    Aims: Hypertension is a risk factor for atherothrombotic vascular events. Angiotensin II (Ang II), one of the main vasoactive hormones of the renin-angiotensin system, has been associated with the development and progression of atherosclerosis. However, it is not fully known how Ang II contributes to lipid-enriched atherosclerotic lesion formation. In human vascular smooth muscle cells (VSMC), low density lipoprotein (LDL) receptor-related protein (LRP1) internalizes cholesteryl esters (CE) from extracellular matrix-bound aggregated LDL (agLDL). The aim of this study was to investigate the effect of Ang II on LRP1 expression and function in VSMC.

    Here, we report for the first time that Ang II induces the upregulation of LRP1 expression in VSMC. Ang II (1 microM) induced maximal LRP1 mRNA expression at 12 h and maximal protein overexpression (by 4.10-fold) at 24 h in cultured human VSMC. Ang II effects were functionally translated into an increased CE accumulation from agLDL uptake (by two-fold at 50 microg/mL) that was prevented by the LRP1 ligand lactoferrin and by siRNA-LRP1 treatment. Ang II-LRP1 upregulation and excess CE accumulation from agLDL were prevented by losartan (an AT1 blocker) but not by PD123319 (a specific AT2 blocker). Additionally, in a normolipidaemic rat model, Ang II infusion produced a significant increase in aortic LRP1 expression and lipid infiltration in the arterial intima.

    Conclusion: The in vitro and in vivo data reported here indicate that Ang II upregulates LRP1 receptor expression and LRP1-mediated aggregated LDL uptake in vascular cells.

    Cardiovascular research 2008;78;3;581-9

  • The functional role of the second NPXY motif of the LRP1 beta-chain in tissue-type plasminogen activator-mediated activation of N-methyl-D-aspartate receptors.

    Martin AM, Kuhlmann C, Trossbach S, Jaeger S, Waldron E, Roebroek A, Luhmann HJ, Laatsch A, Weggen S, Lessmann V and Pietrzik CU

    Institute of Physiological Chemistry and Pathobiochemistry, Molecular Neurodegeneration and Institute of Physiology, Johannes-Gutenberg-University Mainz, D-55099 Mainz, Germany.

    The low density lipoprotein receptor-related protein 1 (LRP1) emerges to play fundamental roles in cellular signaling pathways in the brain. One of its prominent ligands is the serine proteinase tissue-type plasminogen activator (tPA), which has been shown to act as a key activator of neuronal mitogen-activated protein kinase pathways via the N-methyl-D-aspartate (NMDA) receptor. However, here we set out to examine whether LRP1 and the NMDA receptor might eventually act in a combined fashion to mediate tPA downstream signaling. By blocking tPA from binding to LRP1 using the receptor-associated protein, we were able to completely inhibit NMDA receptor activation. Additionally, inhibition of NMDA receptor calcium influx with MK-801 resulted in dramatic reduction of tPA-mediated downstream signaling. This indicates a functional interaction between the two receptors, since both experimental approaches resulted in strongly reduced calcium influx and Erk1/2 phosphorylation. Additionally, we were able to inhibit Erk1/2 activation by competing for the LRP1 C-terminal binding motif with a truncated PSD95 construct resembling its PDZ III domain. Furthermore, we identified the distal NPXY amino acid motif in the C terminus of LRP1 as the crucial element for LRP1-NMDA receptor interaction via the adaptor protein PSD95. These results provide new insights into the mechanism of a tPA-induced, LRP1-mediated gating mechanism for NMDA receptors.

    The Journal of biological chemistry 2008;283;18;12004-13

  • A unique function for LRP-1: a component of a two-receptor system mediating specific endocytosis of plasma-derived factor V by megakaryocytes.

    Bouchard BA, Meisler NT, Nesheim ME, Liu CX, Strickland DK and Tracy PB

    Department of Biochemistry, University of Vermont College of Medicine, Burlington, VT, USA. beth.bouchard@uvm.edu

    Background: Factor V is endocytosed by megakaryocytes from plasma via a specific, receptor-mediated, clathrin-dependent mechanism to form the unique platelet-derived FV pool.

    Objective: The role of low-density lipoprotein (LDL) receptor-related protein-1 (LRP-1), or a related family member, in FV endocytosis by megakaryocytes was examined because of its known interactions with other proteins involved in hemostasis.

    Methods: LRP-1 expression by megakaryocytes and its functional role in FV endocytosis was confirmed using reverse transcription polymerase chain reaction (RT-PCR) and specific antibodies. FV binding to megakaryocytes was performed under Ca(2+)-free conditions to quantify binding in the absence of endocytosis.

    Cell surface expression of LRP-1 by CD34+ ex vivo-derived megakaryocytes and the megakaryocyte-like cell line CMK was confirmed using anti-LRP-1 antibodies and was consistent with the detection of LRP-1 message in these cells. All cells capable of endocytosing FV expressed LRP-1. Anti-LRP-1 antibodies and receptor-associated protein (RAP), a known antagonist of LDL receptor family members, displaced only 50% of the [(125)I]FV bound to megakaryocytes. FV binding to megakaryocytes showed positive cooperativity (Hill coefficient = 1.92 +/- 0.18) that was substantially reduced in the presence of RAP (1.47 +/- 0.26). As FV endocytosis is specific to this cofactor, a model is hypothesized where FV binding to a specific receptor facilitates binding and endocytosis of a second FV molecule by LRP-1, or a related family member. These combined observations describe a unique role for LRP-1 in endocytosis of a coagulation protein trafficked to alpha-granules and not destined for lysosomal degradation.

    Funded by: NHLBI NIH HHS: HL46703, HL50784, HL54710, HL70826

    Journal of thrombosis and haemostasis : JTH 2008;6;4;638-44

  • The binding sites for the very low density lipoprotein receptor and low-density lipoprotein receptor-related protein are shared within coagulation factor VIII.

    Ananyeva NM, Makogonenko YM, Kouiavskaia DV, Ruiz J, Limburg V, Meijer AB, Khrenov AV, Shima M, Strickland DK and Saenko EL

    Center for Vascular and Inflammatory Diseases, Department of Biochemistry and Molecular Biology, University of Maryland School of Medicine, Baltimore, Maryland, USA. NAnanyeva@som.umaryland.edu

    Coagulation factor VIII (FVIII) is a ligand for two members of the low-density lipoprotein receptor family, low-density lipoprotein receptor-related protein (LRP) and low-density lipoprotein receptor, which cooperate in regulating clearance of FVIII from the circulation. This study was aimed to explore the mechanism of interaction of FVIII with very low density lipoprotein receptor (VLDLR), another member of the family, and map receptor-binding sites. Binding of plasma-derived FVIII and its fragments to recombinant soluble ectodomain of VLDLR (sVLDLR) was studied in solid-phase and surface plasmon resonance assays. Full-length FVIII and its light chain bound to sVLDLR with similar affinities (KD = 114 +/- 14 and 95 +/- 11 nmol/l, respectively); in contrast, exposure of high-affinity VLDLR-binding site within the heavy chain (KD = 30 +/- 2 nmol/l) required proteolytic cleavage by thrombin. The VLDLR-binding sites within heavy and light chains were mapped to the A2 domain residues 484-509 and the A3-C1 fragment, based on the inhibitory effects of anti-A2 monoclonal antibody 413 and anti-A3-C1 antibody fragment scFv KM33, respectively, previously shown to inhibit FVIII/LRP interaction. Soluble ligand-binding fragment of VLDLR inhibited activation of factor X by the intrinsic Xase in purified system. In cell culture, a higher Xase activity was associated with wild-type human embryonic kidney cells compared with transfected cells that express VLDLR on the cell surface. We conclude that the binding sites for VLDLR and LRP within FVIII overlap and the A2 site becomes exposed upon physiological activation of FVIII. A functional role of FVIII/VLDLR interaction may be related to regulation of intrinsic Xase activity.

    Funded by: NHLBI NIH HHS: HL054710, HL50784, HL66101, HL72929

    Blood coagulation & fibrinolysis : an international journal in haemostasis and thrombosis 2008;19;2;166-77

  • Perindoprilat modulates the activity of lipoprotein receptor-related protein in human mesangial cells.

    Pawluczyk IZ, Patel SR and Harris KP

    John Walls Renal Unit, Leicester General Hospital, and Department of Infection, Immunity, and Inflammation, University of Leicester, UK. izap1@le.ac.uk

    Low density lipoprotein receptor-related protein (LRP) is a multifunctional endocytic receptor implicated in the modulation of a number of cellular processes, including the turnover of proteases and the degradation of extracellular matrix proteins. As such, it can play a key role in the control of fibrosis. The aim of this investigation was to ascertain whether the anti-fibrotic effects exerted by the angiotensin-converting enzyme inhibitor (ACE-I) perindoprilat on macrophage-conditioned medium (MPCM)-injured human mesangial cells can be modulated by this receptor. Addition of receptor-associated protein to MPCM-injured mesangial cells with and without ACE-I increased the amount of tissue plasminogen activator protein detected in mesangial cell culture supernatants without affecting the protein levels of plasminogen activator inhibitor-1. The ability of ACE-I to reduce fibronectin was diminished in the presence of receptor-associated protein. ACE-I induced an increase in mesangial cell MMP9 mRNA, but reduced the MMP9 enzyme activity detected in mesangial cell supernatants. Mesangial cell lysates from ACE-I-treated cells were able to bind immobilized fibronectin at higher dilutions than cell lysates from untreated cells. Flow cytometry showed that MPCM induced an increase in LRP surface expression in mesangial cells over that in control cells and that this expression was further increased by ACE-I treatment. The increase in LRP expression in response to ACE-I was also observed by Western blotting. Northern blot analysis of RNA extracted from cells following a 24-h exposure to MPCM with and without ACE-I demonstrated that there was no change in LRP mRNA expression upon ACE-I treatment. In conclusion, we show that ACE-I treatment is able to modulate mesangial cell-surface expression of LRP, providing an additional mechanism whereby ACE-Is can mediate anti-fibrotic actions independent of their hemodynamic actions.

    The Journal of biological chemistry 2008;283;8;4588-94

  • Functional role of lipoprotein receptors in Alzheimer's disease.

    Jaeger S and Pietrzik CU

    Institute of Physiological Chemistry and Pathobiochemistry, Molecular Neurodegeneration, Johannes-Gutenberg-University Mainz, Mainz, Germany.

    The LDL receptor gene family constitutes a class of structurally closely related cell surface receptors fulfilling diverse functions in different organs, tissues, and cell types. The LDL receptor is the prototype of this family, which also includes the VLDLR, ApoER2/LRP8, LRP1 and LRP1B, as well as Megalin/GP330, SorLA/LR11, LRP5, LRP6 and MEGF7. Recently several lines of evidence have positioned the LDL receptor gene family as one of the key players in Alzheimer's disease (AD) research. Initially this receptor family was of high interest due to its key function in cholesterol/apolipoprotein E (ApoE) uptake, with the epsilon4 allele of ApoE as the strongest genetic risk factor for late-onset AD. It has been established that the cholesterol metabolism of the cell has a strong impact on the production of Abeta, the major component of the plaques found in the brain of AD-patients. The original report that soluble amyloid precursor protein (APP) containing the kunitz proteinase inhibitor (KPI) domain might act as a ligand for LRP1 led to a complex investigation of the interaction of both proteins and their potential function in AD development. Meanwhile, it has been demonstrated that LRP1 might bind to APP independent of the KPI domain in APP. This APP - LRP1 interaction is facilitated through a trimeric complex of APP-FE65-LRP1, which has a functional role in APP processing. Along with LRP1, APP is transported from the early secretory compartments to the cell surface and subsequently internalised into the endosomal / lysosomal compartments. Recent investigations indicate that ApoER2 and SorLA fulfil a similar role in shifting APP localisation in the cell, which affects APP processing and the production of the APP derived amyloid beta-peptide (Abeta). In addition to the effect of lipoprotein receptors on APP processing and Abeta production, LRP1 has been shown to bind Abeta directly or indirectly through Abeta-lactoferrin, Abeta-alpha2M and Abeta-ApoE complexes in vitro and in vivo. Based on these observations two LRP1 mediated clearance mechanisms of Abeta are proposed to play a crucial role in the prevention of AD: either Abeta-uptake into a cell with its subsequent degradation or its transport out of the brain over the blood brain barrier into the periphery. Following this export Abeta is degraded in the liver, where LRP1 potentially conducts the removal of Abeta from the blood stream. Although the involvement of LDLR family members in AD is not yet fully understood it becomes clear that they can directly affect APP production, Abeta-clearance and Abeta-transport over the blood brain barrier.

    Current Alzheimer research 2008;5;1;15-25

  • Mutational analysis of the FXNPXY motif within LDL receptor-related protein 1 (LRP1) reveals the functional importance of the tyrosine residues in cell growth regulation and signal transduction.

    Zhang H, Lee JM, Wang Y, Dong L, Ko KW, Pelletier L and Yao Z

    Department of Biochemistry, Microbiology and Immunology, University of Ottawa, Ottawa, ON, Canada K1H 8M5.

    LRP1 [LDL (low-density lipoprotein) receptor-related protein 1]-null CHO cells (Chinese-hamster ovary cells) (13-5-1 cells) exhibited accelerated cell growth and severe tumour progression after they were xenografted into nude mice. Reconstitution of LRP1 expression in these cells, either with the full-length protein or with a minireceptor, reduced growth rate as well as suppressed tumour development. We tested the role of the tyrosine residue in the FXNPXY63 motif within the LRP1 cytoplasmic domain in signal transduction and cell growth inhibition by site-specific mutagenesis. The LRP1 minireceptors harbouring Tyr63 to alanine or Tyr63 to phenylalanine substitution had diametrically opposite effects on cell growth, cell morphology and tumour development in mice. The Y63F-expressing cells showed suppressed cell growth and tumour development, which were associated with decreased beta-catenin and cadherin concentrations in the cells. On the other hand, the Y63A-expressing cells lacked inhibition on cell growth and tumour development, which were associated with hyperactivation of ERKs (extracellular-signal-regulated kinases), FAK (focal adhesion kinase) and cyclin D1 in the cells. The mutant Y63A minireceptor also exhibited reduced capacity in binding to the Dab2 (disabled 2) adaptor protein. In addition, the Y63A mutant showed increased caveolar localization, and cells expressing Y63A had altered caveolae architecture. However, tyrosine to alanine substitution at the other NPXY29 motif had no effect on cell growth or tumorigenesis. These results suggest that the FXNPXY63 motif of LRP1 not only governs cellular localization of the receptor but also exerts multiple functional effects on signalling pathways involved in cell growth regulation.

    The Biochemical journal 2008;409;1;53-64

  • Factor VIII bypasses CD91/LRP for endocytosis by dendritic cells leading to T-cell activation.

    Dasgupta S, Navarrete AM, André S, Wootla B, Delignat S, Repessé Y, Bayry J, Nicoletti A, Saenko EL, d'Oiron R, Jacquemin M, Saint-Remy JM, Kaveri SV and Lacroix-Desmazes S

    Unité 872, Institut National de la Santé et de la Recherche Médicale, Paris, France.

    Background: The development of factor VIII (FVIII) inhibitors remains the major hurdle in the clinical management of patients with hemophilia A. FVIII uptake by professional antigen-presenting cells (APC) is the first step involved in initiation of immune responses to FVIII. Studies on FVIII catabolism have highlighted the role played by CD91/LRP as a potential target for increasing FVIII half-life in patients and prolonging treatment efficiency. We investigated the involvement of CD91 in FVIII endocytosis by human dendritic cells (DC), a model of professional APC.

    Immature DC were generated from circulating monocytes from healthy donors. Surface expression of CD91 was assessed by flow cytometry. Uptake of fluorescein isothiocyanate-conjugated ligands by immature DC was studied in the presence of various blocking agents.

    Results: CD91 was expressed on approximately 20% of DC and mediated the internalization of its model ligand, alpha2-macroglobulin. DC internalized FVIII and activated a human FVIII-specific T-cell clone in a dose-dependent manner. FVIII uptake by DC and subsequent T-cell activation were not inhibited by receptor-associated protein.

    Conclusions: Our results indicate that CD91 and other members of the LDL receptor family are not strongly implicated in FVIII internalization by monocyte-derived DC, and suggest the involvement of alternative divalent ion-dependent endocytic receptors.

    Haematologica 2008;93;1;83-9

  • Polymorphisms of APOE and LRP genes in Brazilian individuals with Alzheimer disease.

    Bahia VS, Kok F, Marie SN, Shinjo SO, Caramelli P and Nitrini R

    Behavioral and Cognitive Neurology Unit, Department of Neurology, Hospital das Clínicas, University of São Paulo School of Medicine, São Paulo, Brazil. vs.bahia@uol.com.br

    Alzheimer disease (AD) is the most frequent cause of dementia in Western countries. Putative genetic risk factors for AD are polymorphisms in the apolipoprotein E (APOE) gene and in the low-density lipoprotein receptor-related protein (LRP) gene. Our objective was to investigate the role of the APOE coding region polymorphisms epsilon 2, epsilon 3, and epsilon 4 and APOE promoter variants A/T at position -491 and G/T at -219, as well as LRP polymorphism C/T, as risk factors for AD in Brazilian individuals. One hundred and twenty patients with probable AD, along with 120 controls were analyzed. A significant difference between patients and controls for epsilon 4 alleles was observed: frequency of this allele in AD was 0.31, and 0.10 in controls. Individuals with 2 epsilon 4 alleles had a higher risk for AD than subjects with only 1 such allele; presence of 1 epsilon 2 allele proved protective. The presence of the T allele of the -219 polymorphism was also associated with an increased risk of AD, but this polymorphism is in linkage disequilibrium with APOE epsilon polymorphisms. No significant differences between patients and controls were observed for -491 APOE or LRP polymorphisms. In this Brazilian population, both the epsilon 4 allele and T -219 polymorphism were associated with an increased risk for AD.

    Alzheimer disease and associated disorders 2008;22;1;61-5

  • Binding of alpha2ML1 to the low density lipoprotein receptor-related protein 1 (LRP1) reveals a new role for LRP1 in the human epidermis.

    Galliano MF, Toulza E, Jonca N, Gonias SL, Serre G and Guerrin M

    UMR5165 UDEAR-CNRS/UPS, CHU PURPAN, Toulouse, France.

    Background: The multifunctional receptor LRP1 has been shown to bind and internalize a large number of protein ligands with biological importance such as the pan-protease inhibitor alpha2-macroglobulin (alpha2M). We recently identified Alpha2ML1, a new member of the alpha2M gene family, expressed in epidermis. alpha2ML1 might contribute to the regulation of desquamation through its inhibitory activity towards proteases of the chymotrypsin family, notably KLK7. The expression of LRP1 in epidermis as well as its ability to internalize alpha2ML1 was investigated.

    In human epidermis, LRP1 is mainly expressed within the granular layer of the epidermis, which gathers the most differentiated keratinocytes, as shown by immunohistochemistry and immunofluorescence using two different antibodies. By using various experimental approaches, we show that the receptor binding domain of alpha2ML1 (RBDl) is specifically internalized into the macrophage-like cell line RAW and colocalizes with LRP1 upon internalization. Coimmunoprecipitation assays demonstrate that RBDl binds LRP1 at the cell surface. Addition of RAP, a universal inhibitor of ligand binding to LRP1, prevents RBDl binding at the cell surface as well as internalization into RAW cells. Silencing Lrp1 expression with specific siRNA strongly reduces RBDl internalization.

    Keratinocytes of the upper differentiated layers of epidermis express LRP1 as well as alpha2ML1. Our study also reveals that alpha2ML1 is a new ligand for LRP1. Our findings are consistent with endocytosis by LRP1 of complexes formed between alpha2ML1 and proteases. LRP1 may thus control desquamation by regulating the biodisponibility of extracellular proteases.

    PloS one 2008;3;7;e2729

  • Clathrin and LRP-1-independent constitutive endocytosis and recycling of uPAR.

    Cortese K, Sahores M, Madsen CD, Tacchetti C and Blasi F

    Centro di Ricerca MicroSCoBio/IFOM, FIRC Institute of Molecular Oncology, Dipartimento di Medicina Sperimentale, Sezione di Anatomia Umana, Università di Genova, Genova, Italy.

    Background: The urokinase receptor (uPAR/CD87) is highly expressed in malignant tumours. uPAR, as a GPI anchored protein, is preferentially located at the cell surface, where it interacts with its ligands urokinase (uPA) and the extracellular matrix protein vitronectin, thus promoting plasmin generation, cell-matrix interactions and intracellular signalling events. Interaction with a complex formed by uPA and its inhibitor PAI-1 induces cell surface down regulation and recycling of the receptor via the clathrin-coated pathway, a process dependent on the association to LRP-1.

    In this study, we have found that along with the ligand-induced down-regulation, uPAR also internalizes and recycles constitutively through a second pathway that is independent of LRP-1 and clathrin but shares some properties with macropinocytosis. The ligand-independent route is amiloride-sensitive, does not require uPAR partitioning into lipid rafts, is independent of the activity of small GTPases RhoA, Rac1 and Cdc42, and does not require PI3K activity. Constitutively endocytosed uPAR is found in EEA1 positive early/recycling endosomes but does not reach lysosomes in the absence of ligands. Electron microscopy analysis reveals the presence of uPAR in ruffling domains at the cell surface, in macropinosome-like vesicles and in endosomal compartments.

    These results indicate that, in addition to the ligand-induced endocytosis of uPAR, efficient surface expression and membrane trafficking might also be driven by an uncommon macropinocytic mechanism coupled with rapid recycling to the cell surface.

    PloS one 2008;3;11;e3730

  • Low density lipoprotein receptor-related protein polymorphisms are not risk factors for venous thromboembolism.

    Mello TB, Siqueira LH, Montavão SA, Ozello MC and Annichino-Bizzacchi JM

    Hematology and Hemotherapy Center, Faculty of Medical Sciences, State University of Campinas, Campinas, SP, Brazil. g.mello@uol.com.br

    Low-density lipoprotein receptor-related protein is a receptor involved in factor VIII catabolism. In spite of elevated factor VIII coagulant activity levels being a well-established independent risk factor for venous thrombosis, the origin of this abnormality is unknown. In this study, we investigated the role of polymorphisms C220T (exon 22), A775P (exon 14) and D2080N (exon 39) in the gene coding for this receptor in 249 patients (100 males/149 female, mean age 36.5 SD:11 years) with venous thromboembolism and 366 controls (155 male/211 females, mean age 38 SD:11 years ) matched by age and gender. The polymorphisms C220T (CT OR: 0.9, 95%CI: 0.6-1.2; TT OR: 1.0, 95%CI: 0.6-1.5) and D2080N (OR: 0.8, 95%CI: 0.3-2.4) were not associated to thrombosis susceptibility while the polymorphism A775P was identified in neither patients or controls. D2080N polymorphism was associated with factor VIII and von Willebrand factor levels, in the control group. Heterozygous individuals (DN), compared with homozygotes individuals (DD), presented lower levels of factor VIII (mean difference: 42.3 IU/dL, 95%CI: 5.7-78.9) and von Willebrand factor (mean difference: 34.8 IU/dL, 95%CI: 4.9-64.6). In conclusion, we demonstrated an association between D2080N polymorphism with factor VIII and von Willebrand factor levels in Brazilian normal individuals. However, none of the three polymorphisms in low-density lipoprotein receptor related protein gene were related to venous thrombosis risk in these patients.

    Thrombosis research 2008;121;5;625-9

  • Multidrug resistance proteins in renal cell carcinoma.

    Hodorová I, Rybárová S, Solár P, Vecanová J, Mihalik J, Bohus P, Mellová Y and Kluchová D

    Department of Anatomy, Faculty of Medicine, P. J. Safárik University in Kosice, Kosice, Slovak Republic. ingrid.hodorova@post.sk

    A large number of renal cancer patients show poor or partial response to chemotherapy and the precise mechanism has not been understood yet. MDR is the principal mechanism by which many cancers develop resistance to chemotherapeutic drugs and is associated with the elevated expression of MDR proteins. These are divided into two groups: ABC transporters and non-ABC transporters. The aim of our study was to determine the expression of MDR1/Pgp, MRP1 and LRP in 47 samples of renal cell carcinomas using immunohistochemical assay. Our results were analysed in relation to nuclear grade and other clinical and pathological parameters to see the possible correlation between the expression of MDR proteins and factors mentioned above. The majority of renal carcinoma specimens showed positivity for MDR proteins. In this regard, 21 % of samples revealed positive results for MDR1, 62 % for MRP1 and 76.6 % for LRP protein. Furthermore, our study displayed significant differences between MDR1, LRP and nuclear grade. On the other hand, no association was found between MRP1 and nuclear grade, as well as between the expression of three MDR proteins and other clinically relevant parameters.

    Folia biologica 2008;54;6;187-92

  • Tissue-type plasminogen activator promotes murine myofibroblast activation through LDL receptor-related protein 1-mediated integrin signaling.

    Hu K, Wu C, Mars WM and Liu Y

    Department of Pathology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15261, USA.

    The activation of interstitial fibroblasts to become alpha-SMA-positive myofibroblasts is an essential step in the evolution of chronic kidney fibrosis, as myofibroblasts are responsible for the production and deposition of the ECM components that are a hallmark of the disease. Here we describe a signaling pathway that leads to this activation. Tissue-type plasminogen activator (tPA) promoted TGF-beta1-mediated alpha-SMA and type I collagen expression in rat kidney interstitial fibroblasts. This fibrogenic effect was independent of its protease activity but required its membrane receptor, the LDL receptor-related protein 1 (LRP-1). In rat kidney fibroblasts, tPA induced rapid LRP-1 tyrosine phosphorylation and enhanced beta1 integrin recruitment by facilitating the LRP-1/beta1 integrin complex formation. Blockade or knockdown of beta1 integrin abolished type I collagen and alpha-SMA expression. Furthermore, inhibition of the integrin-linked kinase (ILK), a downstream effector of beta1 integrin, or disruption of beta1 integrin/ILK engagement, abrogated the tPA action, whereas ectopic expression of ILK mimicked tPA in promoting myofibroblast activation. In murine renal interstitium after obstructive injury, tPA and alpha-SMA colocalized with LRP-1, and tPA deficiency reduced LRP-1/beta1 integrin interaction and myofibroblast activation. These findings show that tPA induces LRP-1 tyrosine phosphorylation, which in turn facilitates the LRP-1-mediated recruitment of beta1 integrin and downstream ILK signaling, thereby leading to myofibroblast activation. This study implicates tPA as a fibrogenic cytokine that promotes the progression of kidney fibrosis.

    Funded by: NIDDK NIH HHS: DK054639, DK061408, DK064005, DK071040

    The Journal of clinical investigation 2007;117;12;3821-32

  • Protein kinase C-alpha-mediated regulation of low-density lipoprotein receptor related protein and urokinase increases astrocytoma invasion.

    Amos S, Mut M, diPierro CG, Carpenter JE, Xiao A, Kohutek ZA, Redpath GT, Zhao Y, Wang J, Shaffrey ME and Hussaini IM

    Department of Pathology, University of Virginia Health System, Charlottesville, VA 22908, USA. sa7h@virginia.edu

    Aggressive and infiltrative invasion is one of the hallmarks of glioblastoma. Low-density lipoprotein receptor-related protein (LRP) is expressed by glioblastoma, but the role of this receptor in astrocytic tumor invasion remains poorly understood. We show that activation of protein kinase C-alpha (PKC-alpha) phosphorylated and down-regulated LRP expression. Pretreatment of tumor cells with PKC inhibitors, phosphoinositide 3-kinase (PI3K) inhibitor, PKC-alpha small interfering RNA (siRNA), and short hairpin RNA abrogated phorbol 12-myristate 13-acetate-induced down-regulation of LRP and inhibited astrocytic tumor invasion in vitro. In xenograft glioblastoma mouse model and in vitro transmembrane invasion assay, LRP-deficient cells, which secreted high levels of urokinase-type plasminogen activator (uPA), invaded extensively the surrounding normal brain tissue, whereas the LRP-overexpressing and uPA-deficient cells did not invade into the surrounding normal brain. siRNA, targeted against uPA in LRP-deficient clones, attenuated their invasive potential. Taken together, our results strongly suggest the involvement of PKC-alpha/PI3K signaling pathways in the regulation of LRP-mediated astrocytoma invasion. Thus, a strategy of combining small molecule inhibitors of PKC-alpha and PI3K could provide a new treatment paradigm for glioblastomas.

    Funded by: NCI NIH HHS: CA090851; NHLBI NIH HHS: P01HL48807; NINDS NIH HHS: NS035122, R01 NS035122-10

    Cancer research 2007;67;21;10241-51

  • The low-density lipoprotein receptor-related protein regulates cancer cell survival and metastasis development.

    Montel V, Gaultier A, Lester RD, Campana WM and Gonias SL

    Department of Pathology, University of California San Diego School of Medicine, La Jolla, California 92093-0612, USA.

    Low-density lipoprotein receptor-related protein-1 (LRP-1) is a multifunctional receptor involved in receptor-mediated endocytosis and cell signaling. In this study, we show that LRP-1 is abundantly expressed in severe combined immunodeficient (SCID) mouse xenografts by various human cancer cell lines that express very low or undetectable levels of LRP-1 when cultured in 21% O2 in vitro (standard cell culture conditions). To test whether LRP-1 expression in vivo may be explained by hypoxia in the xenografts, CL16 cells, which are derived from the MDA-MB-435 cell line, were cultured in 1.0% O2. A substantial increase in LRP-1 expression was observed. To test the activity of LRP-1 in cancer progression in vivo, LRP-1 expression was silenced in CL16 cells with short hairpin RNA. These cells formed tumors in SCID mice, in which LRP-1 expression remained silenced. Although LRP-1 gene silencing did not inhibit CL16 cell dissemination from the primary tumors to the lungs, the pulmonary metastases failed to enlarge, suggesting compromised survival or growth at the implantation site. In cell culture experiments, significantly increased cell death was observed when LRP-1-silenced CL16 cells were exposed to CoCl2, which models changes that occur in hypoxia. Furthermore, LRP-1-silenced cells expressed decreased levels of vascular endothelial growth factor in response to 1.0% O2. These results suggest mechanisms by which LRP-1 may facilitate the development and growth of cancer metastases in vivo.

    Funded by: NCI NIH HHS: CA-94900; NHLBI NIH HHS: HL-60551

    Cancer research 2007;67;20;9817-24

  • Adipocyte differentiation-related protein is induced by LRP1-mediated aggregated LDL internalization in human vascular smooth muscle cells and macrophages.

    Llorente-Cortés V, Royo T, Juan-Babot O and Badimon L

    Cardiovascular Research Center, Consejo Superior de Investigaciones Científicas-Instituto Catalán de Ciencias Cardiovasculares, Hospital de la Santa Creu i Sant Pau, Barcelona, Spain.

    Aggregated LDL (agLDL) is internalized by LDL receptor-related protein (LRP1) in vascular smooth muscle cells (VSMCs) and human monocyte-derived macrophages (HMDMs). AgLDL is, therefore, a potent inducer of massive intracellular cholesteryl ester accumulation in lipid droplets. The adipocyte differentiation-related protein (ADRP) has been found on the surface of lipid droplets. The objectives of this work were to analyze whether agLDL uptake modulates ADRP expression levels and whether the effect of agLDL internalization on ADRP expression depends on LRP1 in human VSMCs and HMDMs. AgLDL strongly upregulates ADRP mRNA (real-time PCR) and protein expression (Western blot) in human VSMCs (mRNA: by 3.06-fold; protein: 8.58-fold) and HMDMs (mRNA: by 3.5-fold; protein: by 3.71-fold). Treatment of VSMCs and HMDMs with small anti-LRP1-interfering RNA (siRNA-LRP1) leads to specific inhibition of LRP1 expression. siRNA-LRP1 treatment significantly reduced agLDL-induced ADRP overexpression in HMDMs (by 69%) and in VSMCs (by 53%). Immunohystochemical studies evidence a colocolocalization between ADRP/macrophages and ADRP/VSMCs in advanced lipid-enriched atherosclerotic plaques. These results demonstrate that agLDL-LRP1 engagement induces ADRP overexpression in both HMDMs and human VSMCs and that ADRP is highly expressed in advanced lipid-enriched human atherosclerotic plaques. Therefore, LRP1-mediated agLDL uptake might play a pivotal role in vascular foam cell formation.

    Journal of lipid research 2007;48;10;2133-40

  • Cell-surface transglutaminase undergoes internalization and lysosomal degradation: an essential role for LRP1.

    Zemskov EA, Mikhailenko I, Strickland DK and Belkin AM

    Department of Biochemistry and Molecular Biology, University of Maryland School of Medicine, Baltimore, MD 21201, USA.

    Tissue transglutaminase functions as a protein crosslinking enzyme and an integrin-binding adhesion co-receptor for fibronectin on the cell surface. These activities of transglutaminase and the involvement of this protein in cell-matrix adhesion, integrin-mediated signaling, cell migration and matrix organization suggest a precise and efficient control of its cell-surface expression. We report a novel mechanism of regulation of surface transglutaminase through internalization and subsequent lysosomal degradation. Constitutive endocytosis of cell-surface transglutaminase depends on plasma membrane cholesterol and the activity of dynamin-2, and involves both clathrin-coated pits and lipid rafts or caveolae. Furthermore, the key matrix ligands of transglutaminase, fibronectin and platelet-derived growth factor, promote its endocytosis from the cell surface. Our results also indicate that transglutaminase interacts in vitro and on the cell surface with the major endocytic receptor, low-density lipoprotein receptor-related protein 1, and demonstrate the requirement for this receptor in the endocytosis of transglutaminase. Finally, a deficiency of this endocytic receptor or blockade of endo-lysosomal function upregulate transglutaminase expression on the cell surface, leading to increased cell adhesion and matrix crosslinking. These findings characterize a previously unknown pathway of transglutaminase internalization and degradation that might be crucial for regulation of its adhesive and signaling functions on the cell surface and reveal a novel functional link between cell-matrix adhesion and endocytosis.

    Funded by: NHLBI NIH HHS: HL50784; NIGMS NIH HHS: GM62895

    Journal of cell science 2007;120;Pt 18;3188-99

  • Clearance of amyloid-beta by circulating lipoprotein receptors.

    Sagare A, Deane R, Bell RD, Johnson B, Hamm K, Pendu R, Marky A, Lenting PJ, Wu Z, Zarcone T, Goate A, Mayo K, Perlmutter D, Coma M, Zhong Z and Zlokovic BV

    Frank P. Smith Laboratory for Neuroscience and Neurosurgical Research, Department of Neurosurgery, University of Rochester Medical School, Rochester, New York 14642, USA.

    Low-density lipoprotein receptor-related protein-1 (LRP) on brain capillaries clears amyloid beta-peptide (Abeta) from brain. Here, we show that soluble circulating LRP (sLRP) provides key endogenous peripheral 'sink' activity for Abeta in humans. Recombinant LRP cluster IV (LRP-IV) bound Abeta in plasma in mice and Alzheimer's disease-affected humans with compromised sLRP-mediated Abeta binding, and reduced Abeta-related pathology and dysfunction in a mouse model of Alzheimer disease, suggesting that LRP-IV can effectively replace native sLRP and clear Abeta.

    Funded by: NIA NIH HHS: P50 AG005681, P50 AG05681, R37 AG023084, R37 AG023084-02; NINDS NIH HHS: R01 NS034467, R01 NS034467-07, R37 NS034467, R37 NS34467

    Nature medicine 2007;13;9;1029-31

  • LDL-receptor mRNA expression in men is downregulated within an hour of an acute fat load and is influenced by genetic polymorphism.

    Pocathikorn A, Taylor RR, James I and Mamotte CD

    Department of Clinical Immunology, Royal Perth Hospital, Perth, Australia 6000.

    Little is known about the immediate effects of dietary fat on the expression of genes involved in cholesterol metabolism in humans. We investigated the effects of a high-fat meal on circulating mononuclear cell messenger RNA (mRNA) for the LDL receptor (LDLR), LDLR-related protein (LRP), and 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR) over 10 h. Selection of 12 C and 7 T homozygotes for the LRP exon 22 C200T polymorphism for the study also enabled us to examine the influence of this polymorphism on postprandial mRNA expression and lipoproteins, of relevance because of LRP's role in postprandial lipoprotein metabolism and association of the polymorphism with coronary artery disease. We found a postprandial decrease in LDLR mRNA abundance relative to the reference beta-actin (BA) mRNA. The decreased LDLR/BA mRNA value was apparent at 1 h (P < 0.005) and decreased to 25% of baseline at 6 h (P < 0.005). The LRP/BA mRNA value was also lower at 6 h (16% decrease, P < 0.05). HMGCR mRNA expression was unchanged. C homozygotes for the C200T polymorphism had higher LDLR/BA values than T homozygotes (P = 0.01) and although plasma LDL cholesterol (LDL-C) concentrations decreased in the postprandial period (P < 0.002), the decrease was less in C than in T homozygotes (P < 0.05). This study constitutes the first observation, to our knowledge, of postprandial changes in LDLR and LRP mRNA expression. It documents immediate effects of a fatty meal on these mRNA as well as an LRP genotype effect on LDLR mRNA and LDL-C.

    The Journal of nutrition 2007;137;9;2062-7

  • Plasminogen-dependent internalization of soluble melanotransferrin involves the low-density lipoprotein receptor-related protein and annexin II.

    Michaud-Levesque J, Demeule M and Béliveau R

    Laboratoire de Médecine Moléculaire, Service d'Hémato-Oncologie, Hôpital Ste-Justine, Université du Québec à Montréal, Montréal, Québec, Canada.

    We investigated the effect of plasminogen (Plg) on the internalization of recombinant soluble melanotransferrin (sMTf) using U87 human glioblastoma cells and murine embryonic fibroblasts (MEF) deficient in the low-density lipoprotein receptor-related protein (LRP). Using biospecific interaction analysis, both Glu- and Lys-Plg were shown to interact with immobilized sMTf. The binding of sMTf at the cell surface increased in the presence of both forms of Plg in control and in LRP-deficient MEF cells, whereas the uptake was strongly stimulated only by Lys-Plg in control MEF and U87 cells. In addition, in the presence of Lys-Plg, the internalization of sMTf was a saturable process, sensitive to temperature and dependent on the integrity of lysine residues. The addition of the receptor-associated protein, lactoferrin and aprotinin, as well as a monoclonal antibody (mAb) directed against LRP, inhibited the Lys-Plg-dependent uptake of sMTf. These results suggest an important role for LRP in this process. In addition, using binding and uptake assays in the presence of anti-annexin II mAb, we showed that annexin II might be responsible for the initial binding of sMTf in the presence of Plg. Our results suggest a Plg-mediated internalization mechanism for the clearance of sMTf via annexin II and LRP.

    Biological chemistry 2007;388;7;747-54

  • Apolipoprotein A-V interaction with members of the low density lipoprotein receptor gene family.

    Nilsson SK, Lookene A, Beckstead JA, Gliemann J, Ryan RO and Olivecrona G

    Department of Medical Biosciences/Physiological Chemistry, Umeå University, SE-901 87 Umeå, Sweden.

    Apolipoprotein A-V is a potent modulator of plasma triacylglycerol levels. To investigate the molecular basis for this phenomenon we explored the ability of apolipoprotein A-V, in most experiments complexed to disks of dimyristoylphosphatidylcholine, to interact with two members of the low density lipoprotein receptor family, the low density lipoprotein receptor-related protein and the mosaic type-1 receptor, SorLA. Experiments using surface plasmon resonance showed specific binding of both free and lipid-bound apolipoprotein A-V to both receptors. The binding was calcium dependent and was inhibited by the receptor associated protein, a known ligand for members of the low density lipoprotein receptor family. Preincubation with heparin decreased the receptor binding of apolipoprotein A-V, indicating that overlap exists between the recognition sites for these receptors and for heparin. A double mutant, apolipoprotein A-V (Arg210Glu/Lys211Gln), showed decreased binding to heparin and decreased ability to bind the low density lipoprotein receptor-related protein. Association of apolipoprotein A-V with the low density lipoprotein receptor-related protein or SorLA resulted in enhanced binding of human chylomicrons to receptor-covered sensor chips. Our results indicate that apolipoprotein A-V may influence plasma lipid homeostasis by enhancing receptor-mediated endocytosis of triacylglycerol-rich lipoproteins.

    Funded by: NHLBI NIH HHS: HL 073061, R01 HL073061

    Biochemistry 2007;46;12;3896-904

  • Low-density lipoprotein receptor-related protein 1 polymorphism 663 C > T affects clotting factor VIII activity and increases the risk of venous thromboembolism.

    Vormittag R, Bencur P, Ay C, Tengler T, Vukovich T, Quehenberger P, Mannhalter C and Pabinger I

    Division of Haematology and Haemostaseology, Department of Internal Medicine I, Medical University of Vienna, Vienna, Austria.

    Background: Clotting factor (F) VIII is an independent risk factor for primary and recurrent venous thromboembolism (VTE). The causes for high plasma FVIII levels are not fully understood, but an involvement of genetic factors has been demonstrated. A multifunctional endocytic receptor, low-density lipoprotein receptor-related protein 1 (LRP1), mediates cellular uptake and subsequent degradation of FVIII and may contribute to variations in FVIII levels.

    Objective: We assessed the association of a genetic variation of LRP1 (663C > T) with basal FVIII levels and the risk of venous thrombosis in a group of high-risk patients and in healthy controls.

    One hundred and fifty-two patients with a history of recurrent VTE (median age 56 years, 47% women) were compared with 198 age- and sex-matched controls (median age 53 years, 50% women). The LRP1 663C > T genotype was analyzed by mutagenic separated polymerase chain reaction assay and heterozygosity was confirmed by sequence analysis.

    Results: LRP1 663C > T genotype distribution differed significantly between patients (663CC n = 138, 663CT n = 14) and controls (663CC n = 190, 663CT n = 8; P = 0.048). In multivariable linear regression analysis including LRP1 663C > T, ABO blood group, von Willebrand factor antigen, C-reactive protein and age, LRP1 663CT was independently associated with FVIII activity (P = 0.02). LRP1 663CT was also associated with increased odds for VTE following adjustment for blood group O, FV Leiden and the prothrombin variation 20210G > A in multivariate analysis (odds ratio 3.3, 95% CI 1.3-8.5).

    Conclusions: According to our data the LRP1 663C > T polymorphism influences plasma FVIII levels independently of blood group, C-reactive protein and von Willebrand factor and is significantly associated with the risk of VTE.

    Journal of thrombosis and haemostasis : JTH 2007;5;3;497-502

  • The expression of low density lipoprotein receptor-related protein in colorectal carcinoma.

    Obermeyer K, Krueger S, Peters B, Falkenberg B, Roessner A and Röcken C

    Department of Pathology, Otto-von-Guericke-University, Magdeburg, and Charité-University Hospital, Berlin, Germany.

    Low density lipoprotein receptor-related protein (LRP) is a multifunctional cell surface receptor binding many different ligands including proteinases and their inhibitors, some of which are known to be involved in tumor biology. We studied the expression of LRP and its putative role in colorectal carcinoma. Tissue samples were obtained from 50 patients with colorectal carcinoma and fixed in formalin and embedded in paraffin. Immunohistochemical staining was performed using antibodies directed against LRP, cathepsin B and urokinase-type plasminogen activator (u-PA). The expression of LRP was further studied by polymerase chain reaction. The TNM stage was determined according to UICC guide lines and was based upon histological analysis. LRP was primarily expressed in stroma cells [36 patients (72%)] and less frequently in tumor cells [6 patients (12%)]. In 22% of all cases LRP was prominent at the invasion front. Cathepsin B was found both in the tumor stroma [50 (100%)] and in the tumor cells [46 (92%)]. u-PA was present in the tumor stroma [44 (88%)] and in the tumor cells [44 (88%)]. In stromal cells the expression of LRP correlated significantly with the expression of u-PA (p=0.043). Furthermore, the expression of LRP and of u-PA in tumor cells correlated with the tumor stage according to UICC (p=0.038 and 0.018, respectively). We provide evidence that LRP is expressed in colorectal cancer. As LRP forms complexes with u-PA and its inhibitor, we suspect that LRP can influence the known effects of u-PA on tumor biology.

    Oncology reports 2007;17;2;361-7

  • Identification of the ligands of protein interaction domains through a functional approach.

    Caratù G, Allegra D, Bimonte M, Schiattarella GG, D'Ambrosio C, Scaloni A, Napolitano M, Russo T and Zambrano N

    CEINGE Biotecnologie Avanzate, Dipartimento di Biochimica e Biotecnologie Mediche, Università di Napoli Federico II, 80131 Napoli, Italy.

    The identification of protein-protein interaction networks has often given important information about the functions of specific proteins and on the cross-talk among metabolic and regulatory pathways. The availability of entire genome sequences has rendered feasible the systematic screening of collections of proteins, often of unknown function, aimed to find the cognate ligands. Once identified by genetic and/or biochemical approaches, the interaction between two proteins should be validated in the physiologic environment. Herein we describe an experimental strategy to screen collections of protein-protein interaction domains to find and validate candidate interactors. The approach is based on the assumption that the overexpression in cultured cells of protein-protein interaction domains, isolated from the context of the whole protein, could titrate the endogenous ligand and, in turn, exert a dominant negative effect. The identification of the ligand could provide us with a tool to check the relevance of the interaction because the contemporary overexpression of the isolated domain and of its ligand could rescue the dominant negative phenotype. We explored this approach by analyzing the possible dominant negative effects on the cell cycle progression of a collection of phosphotyrosine binding (PTB) domains of human proteins. Of 47 PTB domains, we found that the overexpression of 10 of them significantly interfered with the cell cycle progression of NIH3T3 cells. Four of them were used as baits to identify the cognate interactors. Among these proteins, CARM1, interacting with the PTB domain of RabGAP1, and EF1alpha, interacting with RGS12, were able to rescue the block of the cell cycle induced by the isolated PTB domain of the partner protein, thus confirming in vivo the relevance of the interaction. These results suggest that the described approach can be used for the systematic screening of the ligands of various protein-protein interaction domains also by using different biological assays.

    Molecular & cellular proteomics : MCP 2007;6;2;333-45

  • Apolipoprotein E and low density lipoprotein receptor-related protein facilitate intraneuronal Abeta42 accumulation in amyloid model mice.

    Zerbinatti CV, Wahrle SE, Kim H, Cam JA, Bales K, Paul SM, Holtzman DM and Bu G

    Department of Pediatrics, Washington University School of Medicine, St. Louis, Missouri 63110, USA.

    The low density lipoprotein receptor-related protein (LRP) is highly expressed in the brain and has been shown to alter the metabolism of amyloid precursor protein and amyloid-beta peptide (Abeta) in vitro. Previously we developed mice that overexpress a functional LRP minireceptor (mLRP2) in their brains and crossed them to the PDAPP mouse model of Alzheimer disease. Overexpression of mLRP2 in 22-month-old PDAPP mice with amyloid plaques increased a pool of carbonate-soluble Abeta in the brain and worsened memory-related behavior. In the current study, we examined the effects of mLRP2 overexpression on 3-month-old PDAPP mice that had not yet developed amyloid plaques. We found significantly higher levels of membrane-associated Abeta42 in the hippocampus of mice that overexpressed mLRP2. Using immunohistochemical methods, we observed significant intraneuronal Abeta42 in the hippocampus and frontal cortex of PDAPP mice, which frequently co-localized with the lysosomal marker LAMP-1. Interestingly, PDAPP mice lacking apolipoprotein E (apoE) had much less intraneuronal Abeta42. We also found that PC12 cells overexpressing mLRP2 cleared Abeta42 and Abeta40 more rapidly from media than PC12 cells transfected with the vector only. Preincubation of apoE3 or apoE4 with Abeta42 increased the rate of Abeta clearance, and this effect was partially blocked by receptor-associated protein. Our results support the hypothesis that LRP binds and endocytoses Abeta42 both directly and via apoE but that endocytosed Abeta42 is not completely degraded and accumulates in intraneuronal lysosomes.

    Funded by: NIA NIH HHS: R01-AG027924; NINDS NIH HHS: F32-NS41872

    The Journal of biological chemistry 2006;281;47;36180-6

  • IAP family protein expression correlates with poor outcome of multiple myeloma patients in association with chemotherapy-induced overexpression of multidrug resistance genes.

    Nakagawa Y, Abe S, Kurata M, Hasegawa M, Yamamoto K, Inoue M, Takemura T, Suzuki K and Kitagawa M

    Department of Comprehensive Pathology, Aging and Developmental Sciences, Graduate School, Tokyo Medical and Dental University, Tokyo, Japan.

    Multidrug-resistant (MDR) multiple myeloma (MM) patients who fail chemotherapy frequently express MDR1 protein, which serves as an efflux pump that protects neoplastic cells. The expression of lung resistance protein (LRP), which mediates intercellular and nucleocytoplasmic transport, is also correlated with chemotherapy resistance and shorter survival of MM patients. Here, we investigated the chemotherapy-induced change of MDR expression in MM patients using quantitative RT-PCR. Overall expression levels of MDR1 and LRP in MM patients were significantly higher than those in control subjects and increased after chemotherapy. More than half of the patients exhibited increased expression of MDR1 (14/26) or LRP (17/26) after chemotherapy. Also, the expression of inhibitor of apoptosis proteins (IAP) was determined in association with the prognosis of the patients. Among patients with increased MDR1-expression after chemotherapy, those with a poor outcome exhibited significant increases in survivin, cIAP1, cIAP2, and XIAP expression by chemotherapy compared with those with a good prognosis. Similarly, in the LRP expression-increased group, patients with a poor outcome showed significant increases of cIAP1 and cIAP2 expression compared with those with longer survival. In patients with reduced-MDR1 or LRP expression after chemotherapy, changes in the expression of IAPs induced by chemotherapy did not correlate with their prognosis. These findings indicate that IAP family proteins might play a role in worsening the prognosis of MM patients in association with chemotherapy-induced overexpression of MDR1 or LRP.

    American journal of hematology 2006;81;11;824-31

  • Association study of polymorphisms in LRP1, tau and 5-HTT genes and Alzheimer's disease in a sample of Colombian patients.

    Forero DA, Arboleda G, Yunis JJ, Pardo R and Arboleda H

    Grupo de Neurociencias, Facultad de Medicina e Instituto de Genética, Universidad Nacional de Colombia, Bogotá, Colombia.

    Analysis of genetic susceptibility factors for Alzheimer's disease (AD) in populations with different genetic and environmental background may be useful to understand AD etiology. There are few genetic association studies of AD in Latin America. In the present work, we analyzed polymorphisms in 3 candidate genes; the LDL receptor related protein-1, the microtubule-associated protein Tau and the serotonin transporter genes in a sample of 106 Colombian AD patients and 97 control subjects. We did not find a significant allelic or genotypic association with any of the three polymorphisms analyzed using different statistical analysis, including a neural network model or different sample stratifications. To date, APOE polymorphisms are the only genetic risk factors identified for AD in the Colombian population. It may be factible that future combination of high-throughput genotyping platforms and multivariate analysis models may lead to the identification of other genetic susceptibility factors for AD in the Colombian population.

    Journal of neural transmission (Vienna, Austria : 1996) 2006;113;9;1253-62

  • Contribution of low density lipoprotein receptor-related protein genotypes to coagulation factor VIII levels in thrombotic women.

    Marchetti G, Lunghi B, Legnani C, Cini M, Pinotti M, Mascoli F and Bernard F

    Department of Biochemistry and Molecular Biology, University of Ferrara, via Fossato di Mortara n 74, I-44100 Ferrara, Italy.

    The contribution of low density lipoprotein (LDL) receptor-related protein (LRP) to variance of factor VIII (FVIII) levels in plasma was investigated in thrombotic women through analysis of frequent LRP genotypes. The G allele of the LRP -25C/G polymorphism, predicting increased LRP expression, was associated with 15% and 18% mean reductions of FVIII activity and protein levels, respectively. The combination of -25C/G LRP polymorphism with FVIII D1241E and ABO polymorphisms produced a gradient of FVIII levels, thus supporting the notion that several factors, acting in FVIII biosynthesis, post-translational modification and removal from circulation, have additive effects on the variance of FVIII levels in plasma.

    Haematologica 2006;91;9;1261-3

  • Proteolytic cleavage of factor VIII heavy chain is required to expose the binding-site for low-density lipoprotein receptor-related protein within the A2 domain.

    Bovenschen N, van Stempvoort G, Voorberg J, Mertens K and Meijer AB

    Department of Plasma Proteins, Sanquin Research, Plesmanlaan 125, 1066 CX Amsterdam, The Netherlands.

    Background: Low-density lipoprotein receptor-related protein (LRP) is an endocytic receptor that contributes to the clearance of coagulation factor (F) VIII from the circulation. Previously, we have demonstrated that region Glu(1811)-Lys(1818) within FVIII light chain constitutes an important binding region for this receptor. We have further found that FVIII light chain and intact FVIII are indistinguishable in their LRP-binding affinities. In apparent contrast to these observations, a second LRP-binding region has been identified within A2 domain region Arg(484)-Phe(509) of FVIII heavy chain.

    Objective: In this study, we addressed the relative contribution of FVIII heavy chain in binding LRP.

    Surface plasmon resonance analysis unexpectedly showed that FVIII heavy chain poorly associated to the receptor. The binding to LRP was, however, markedly enhanced upon cleavage of the heavy chain by thrombin. The A2 domain, purified from thrombin-activated FVIII, also showed efficient binding to LRP. Competition studies employing a recombinant antibody fragment demonstrated that region Arg(484)-Phe(509) mediates the enhanced LRP binding after thrombin cleavage.

    Conclusions: We propose that LRP binding of non-activated FVIII is mediated via the FVIII light chain while in activated FVIII both the heavy and light chain contribute to LRP binding.

    Journal of thrombosis and haemostasis : JTH 2006;4;7;1487-93

  • Sterol regulatory element-binding protein-2 negatively regulates low density lipoprotein receptor-related protein transcription.

    Llorente-Cortés V, Costales P, Bernués J, Camino-Lopez S and Badimon L

    Cardiovascular Research Center, CSIC-ICCC, Hospital de la Santa Creu i Sant Pau, Barcelona, 08025, Spain.

    Low density lipoprotein receptor-related protein (LRP1) binds aggregated LDL (agLDL) leading to a high intracellular cholesteryl ester (CE) accumulation. AgLDL up-regulates LRP1 expression concomitantly with an LDL receptor (LDLR) and sterol regulatory element binding protein (SREBP-2) down-regulation. The objectives were to investigate whether SREBP-2 regulates LRP1 transcription and determine the molecular mechanisms involved in the process. Down-regulation of active SREBP-2 by nLDL and agLDL led to LDLR down-regulation and LRP1 up-regulation. Enforced expression of an active form of SREBP-2 (SREBP-2-NT, amino acid residues 1-468) decreased LRP1 expression and LRP1 promoter (WT-LRP1) luciferase activity in a dose-dependent manner. LDL did not exert any significant effect on LRP1 promoter activity when a putative sterol regulatory element (SRE) (5-GTGGGGTGA-3'; +225 to +233) was mutated (SRE-MT-LRP1). SREBP-2 overexpression exerted stronger down-regulatory effects on WT-LRP1 than on SRE-MT-LRP1 promoter activity both in control, nLDL- and agLDL-exposed HeLa cells. Gel mobility shift assays showed that recombinant SREBP-2-NT protein (1-468) binds to a double-stranded LRP1 DNA fragment (215 to 245) containing a wild-type (wt) SRE sequence but not to a mutated SRE (mt) sequence (5-GAATTCGA-3'). Our results demonstrate that LDL stimulates LRP1 transcription and decreases SREBP-2 active form which negatively regulates LRP1 transcription. SRE sequence (+225 to +233) plays a pivotal role for the down-regulatory effect of SREBP-2 on LRP1 promoter activity.

    Journal of molecular biology 2006;359;4;950-60

  • Association between single nucleotide polymorphisms of drug resistance-associated genes and response to chemotherapy in advanced ovarian cancer.

    Obata H, Yahata T, Quan J, Sekine M and Tanaka K

    Division of Molecular Genetics, Department of Obstetrics and Gynecology, Niigata University Graduate School of Medical and Dental Science, Niigata, Japan.

    Background: Single nucleotide polymorphisms (SNPs) may show clinicopathological importance as prognostic markers. This study examined the association of SNPs and the expression of drug resistance-associated markers with response to chemotherapy in advanced ovarian cancer (stages III and IV) patients.

    SNPs were analyzed for MDR1, MRP1, MRP2 and LRP in 60 advanced ovarian cancer patients. The protein expression of each factor was analyzed by immunohistochemistry in all patients.

    Results: As a result of examining the relevance of SNP genotypes to the response to chemotherapy, a significant relevance (p=0.01) was observed regarding MRP1 exon-17 SNP (G2168A) involving amino acid substitution. No significant relationship was observed between protein expression and the response to chemotherapy or disease-free survival time.

    Conclusion: Analysis of drug resistance gene polymorphism appears to be an indicator of the response to chemotherapy in advanced ovarian cancer.

    Anticancer research 2006;26;3B;2227-32

  • The urokinase/PAI-2 complex: a new high affinity ligand for the endocytosis receptor low density lipoprotein receptor-related protein.

    Croucher D, Saunders DN and Ranson M

    School of Biological Sciences, University of Wollongong, New South Wales 2522.

    The efficient inactivation of urokinase plasminogen activator (uPA) by plasminogen activator inhibitor type 2 (PAI-2) at the surface of carcinoma cells is followed by rapid endocytosis of the uPA-PAI-2 complex. We now show that one pathway of this receptor-mediated endocytosis is mediated via the low density lipoprotein receptor-related protein (LRP) in prostate cancer cells. Detailed biochemical analyses using ligand binding assays and surface plasmon resonance revealed a novel and distinct interaction mechanism between native, human LRP and uPA-PAI-2. As reported previously for PAI-1, inhibition of uPA by PAI-2 significantly increased the affinity of the complex for LRP (K(D) of 36 nm for uPA-PAI-2 versus 200 nm for uPA). This interaction was maintained in the presence of uPAR, confirming the validity of this interaction at the cell surface. However, unlike PAI-1, no interaction was observed between LRP and PAI-2 in either the stressed or the relaxed conformation. This suggests that the uPA-PAI-2-LRP interaction is mediated by site(s) within the uPA molecule alone. Thus, as inhibition of uPA by PAI-2 resulted in accelerated clearance of uPA from the cell surface possibly via its increased affinity for LRP, this represents a mechanism through which PAI-2 can clear proteolytic activity from the cell surface. Furthermore, lack of a direct interaction between PAI-2 and LRP implies that downstream signaling events initiated by PAI-1 may not be activated by PAI-2.

    Funded by: Wellcome Trust: 052458

    The Journal of biological chemistry 2006;281;15;10206-13

  • HIV tat and neurotoxicity.

    King JE, Eugenin EA, Buckner CM and Berman JW

    Department of Pathology, F727, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461, USA.

    HIV tat is the transactivator of HIV-1, supporting efficient viral replication by stabilizing the transcription of viral genes. Tat can be released from HIV-infected cells and alter several functions in uninfected cells. In the brain, tat induces neuronal dysfunction/toxicity, even though neurons cannot be directly infected with HIV, resulting in CNS pathology, such as the dementia and encephalitis associated with NeuroAIDS. This review discusses the most recent data addressing tat-induced neurotoxicity and integrates these new findings in the context of NeuroAIDS.

    Funded by: NIAID NIH HHS: AI-051519; NIGMS NIH HHS: 5 T32 GM007288; NIMH NIH HHS: K01 MH076679, MH0702297, MH52974; NINDS NIH HHS: NS07098, NS11920

    Microbes and infection 2006;8;5;1347-57

  • [Meta-analysis of the association of the LRP C766T polymorphism with the risk of Alzheimer's disease].

    Deng Y, Sun Y, Shi JJ and Zhang SZ

    West China Hospital, Sichuan University, Chengdu 610041, China. dengying03@126.com

    A C-to-T polymorphism in exon 3 of the low density lipoprotein receptor-related protein 1 (LPR-1) gene has been implicated as a risk factor for Alzheimer's disease (AD). The authors performed a meta-analysis to investigate the association between the C766T polymorphism in the LPR-1 gene and the risk for AD. Nineteen references were retrieved through Medline, Cochran Library and CBM search from 1997 to 2004. Similar search strategies were applied to each of these databases. Studies which were eligible for the meta-analysis should meet the following inclusion criteria: presentation of original data and a cross-sectional design, AD as the outcome of interest, an odds ratio (or enough information to calculate it) reported to quantify the association between the frequencies of genotypes and/or alleles of LPR-1 gene C766T polymorphism and the risk for AD. All analyses were performed with Review Manager 4.2. A total of 3,560 AD patients and 3,476 control subjects were analyzed according to the random effect model because some between-study heterogeneity was found (P<0.01). The combined data statistics revealed that there was no statistical difference (test for overall effect: Z=1.74, P=0.08, OR=1.17, 95% CI: 0.98-1.39; Z=1.31, P=0.19, OR=1.11, 95% CI: 0.95-1.31) in the frequencies of allele and genotype between the AD patients and the controls. The meta-analysis showed that the LPR-1 polymorphism was not a major risk factor for AD, although a small effect of the polymorphism for AD risk could not be excluded.

    Yi chuan = Hereditas 2006;28;4;393-8

  • Regulation of the composition of the extracellular matrix by low density lipoprotein receptor-related protein-1: activities based on regulation of mRNA expression.

    Gaultier A, Salicioni AM, Arandjelovic S and Gonias SL

    Department of Pathology, School of Medicine, University of California-San Diego, 9500 Gilman Drive, La Jolla, CA 92093, USA.

    Low density lipoprotein receptor-related protein-1 (LRP-1) is a catabolic receptor for extracellular matrix (ECM) structural proteins and for proteins that bind to ECM. LRP-1 also is implicated in integrin maturation. In this study, we applied a proteomics strategy to identify novel proteins involved in ECM modeling that are regulated by LRP-1. We show that LRP-1 deficiency in murine embryonic fibroblasts (MEFs) is associated with increased levels of type III collagen and pigment epithelium-derived factor, which accumulate in the substratum surrounding cells. The collagen receptor, uPAR-AP/Endo-180, is also increased in LRP-1-deficient MEFs. Human LRP-1 reversed the changes in protein expression associated with LRP-1 deficiency; however, the endocytic activity of LRP-1 was not involved. Instead, regulation occurred at the mRNA level. Inhibition of c-Jun amino-terminal kinase (JNK) blocked type III collagen expression in LRP-1-deficient MEFs, suggesting regulation of JNK activity as a mechanism by which LRP-1 controls mRNA expression. The ability of LRP-1 to regulate expression of the factors identified here suggests a role for LRP-1 in determining blood vessel structure and in angiogenesis.

    Funded by: NHLBI NIH HHS: R01 HL 60551

    The Journal of biological chemistry 2006;281;11;7332-40

  • Longitudinal memory performance during normal aging: twin association models of APOE and other Alzheimer candidate genes.

    Reynolds CA, Prince JA, Feuk L, Brookes AJ, Gatz M and Pedersen NL

    Department of Psychology, University of California--Riverside, 92521, USA. chandra.reynolds@ucr.edu

    The APOE gene (apolipoprotein E) is a major risk factor for Alzheimer's Disease (AD) but has been inconsistently associated with memory in nondemented adults. Two other genes with mixed support as genetic risk factors for AD, A2M (alpha-2-macroglobulin) and LRP (low-density lipoprotein receptor-related protein), have not been studied in relation to memory among nondemented adults. The present study examined these three genes and latent growth parameters estimated from memory performance spanning 13 years in 478 twins from the Swedish Adoption/Twin Study of Aging (SATSA). APOE was associated with working and recall memory ability levels and working memory rate of change, with e4 homozygotes exhibiting the worst performance at all ages. Homozygotes for the rare A2M insertion/deletion variant exhibited accelerating decline on delayed figural recognition. There were no significant findings for LRP. Dominance, often untested in previous studies, was important in the current study's findings.

    Funded by: NIA NIH HHS: AG10175, R01-AG04563, R01-AG17561

    Behavior genetics 2006;36;2;185-94

  • Loss of core fucosylation of low-density lipoprotein receptor-related protein-1 impairs its function, leading to the upregulation of serum levels of insulin-like growth factor-binding protein 3 in Fut8-/- mice.

    Lee SH, Takahashi M, Honke K, Miyoshi E, Osumi D, Sakiyama H, Ekuni A, Wang X, Inoue S, Gu J, Kadomatsu K and Taniguchi N

    Department of Biochemistry, Osaka University Graduate School of Medicine, B1, 2-2 Yamadaoka, Suita, Osaka 565-0871.

    alpha1,6-Fucosyltransferase (Fut8) catalyzes the transfer of a fucose residue from GDP-fucose to the innermost N-acetylglucosamine residue of N-glycans. Here we report that the loss of core fucosylation impairs the function of low-density lipoprotein (LDL) receptor-related protein-1 (LRP-1), a multifunctional scavenger and signaling receptor, resulting in a reduction in the endocytosis of insulin like growth factor (IGF)-binding protein-3 (IGFBP-3) in the cells derived from Fut8-null (Fut8-/-) mice. The reduced endocytosis was restored by the re-introduction of Fut8. Serum levels of IGFBP-3 were markedly upregulated in Fut8-/- mice. These data clearly indicate that core fucosylation is crucial for the scavenging activity of LRP-1 in vivo.

    Journal of biochemistry 2006;139;3;391-8

  • A 3'-UTR polymorphism in the oxidized LDL receptor 1 gene increases Abeta40 load as cerebral amyloid angiopathy in Alzheimer's disease.

    Shi J, Tian J, Pritchard A, Lendon C, Lambert JC, Iwatsubo T and Mann DM

    Clinical Neuroscience Research Group, University of Manchester, Hope Hospital, Salford, M6 8HD, UK.

    It is presently unclear whether polymorphic variations in the oxidized low-density lipoprotein receptor 1 (OLR1), or low-density lipoprotein receptor-related protein 1 (LRP1), genes act as risk factors for Alzheimer's disease (AD). In the present study, we have investigated the extent of amyloid beta protein (Abeta) deposition as cerebral amyloid angiopathy (CAA) or senile plaques (SP) in relationship to OLR1 +1071 and +1073 polymorphisms and LRP1 C766T polymorphism in patients with AD There was an increased Abeta40 load as CAA, but not as SP, in frontal cortex of AD patients carrying OLR1+1073 CC genotype, compared to those with CT, TT or CT+TT genotypes, but only in those individuals without apolipoprotein (APOE) epsilon4 allele. No differences in total Abeta or Abeta42 load as CAA or SP between OLR1+1073 genotypes was seen, nor were there any differences between OLR1+1071 and LRP1 genotypes for any measure of Abeta. Present data suggests that homozygosity for the C allele for OLR1+1073 polymorphism, selectively in individuals without APOE epsilon4 allele, may impair clearance of Abeta, and particularly Abeta40, from the brain across the blood-brain barrier, leading to its 'diversion' into perivascular drainage channels, thereby increasing the severity of CAA in such persons.

    Acta neuropathologica 2006;111;1;15-20

  • Human thyroid carcinoma cell invasion is controlled by the low density lipoprotein receptor-related protein-mediated clearance of urokinase plasminogen activator.

    Sid B, Dedieu S, Delorme N, Sartelet H, Rath GM, Bellon G and Martiny L

    Laboratoire de Biochimie, UMR CNRS 6198, Faculté des Sciences, 51687 Reims, France.

    The low density lipoprotein receptor-related protein (LRP), a large scavenger receptor reported to mediate the uptake and degradation of various ligands, emerges as a promising receptor for targeting the invasive behaviour of human cancer cells. However, the accurate function of LRP during tumor invasion seems to be highly dependent on cellular context and remains controversial. The expression patterns of both this receptor and the main proteolytic systems involved in cell invasion were examined in two follicular thyroid carcinoma cell lines exhibiting different invasive phenotypes. We established that a low expression of LRP at the cell surface was associated to elevated extracellular MMP2 and urokinase plasminogen activator (uPA) activities as well as to high invasiveness properties. Surprisingly, neither exogenously added receptor-associated protein, an antagonist of LRP, nor LRP blocking antibodies significantly modified the amount of extracellular MMP2. Furthermore, the invasive phenotype of thyroid carcinoma cells was not related to their matrix metalloproteinases amount since different specific inhibitors of these proteases failed to affect the invasive properties of both cell lines. Additionally, blocking LRP-mediated clearance led to a further increase of the uPA amount and activities and to increased invasiveness in both cell lines. Finally thyroid carcinoma cells aggressiveness was widely increased by exogenous uPA; and anti-uPA antibodies treatments abolished both basal and receptor-associated protein-induced thyroid cell invasion. Overall our results identified the LRP-mediated clearance of uPA as one of the mechanisms involved during the control of human thyroid carcinoma cell invasion.

    The international journal of biochemistry & cell biology 2006;38;10;1729-40

  • Association study of the A2M and LRP1 Genes with Alzheimer disease in the Han Chinese.

    Bian L, Yang JD, Guo TW, Duan Y, Qin W, Sun Y, Feng GY and He L

    Institute for Nutritional Sciences, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China; Bio-X Center, Shanghai Jiao Tong University, Shanghai, China.

    Background: Low-density lipoprotein receptor-related protein 1 (LRP1) and alpha-2-macroglobulin (A2M) are two plausible candidate genes for Alzheimer disease (AD) based on their important biological function and positional information. To date, numerous studies have investigated their possible association with AD but the results are controversial.

    Methods: To investigate the potential genetic contribution of the two genes in the Han Chinese population, we performed a case-control association study using 10 polymorphisms (4 in LRP1 and 6 in A2M) that span approximately the whole corresponding gene.

    Results: Comparison of allele, genotype, and haplotype frequencies for polymorphisms in A2M revealed no significant differences between patients and control subjects. For the LRP1 gene, however, we found an overrepresentation of the CTCG haplotype in the control group (p = .002). The difference was still of statistical significance in the apolipoprotein E (APOE) epsilon 4 negative subjects (p(CTCG) = .003). Multiple logistic regression analysis did not show any evidence of synergism between A2M, LRP1, and APOE.

    Conclusions: Our results indicate that the CTCG haplotype of LRP1 may reduce the risk of late-onset AD, but A2M is not associated with this disease in the Han Chinese population.

    Biological psychiatry 2005;58;9;731-7

  • Human plasma N-glycoproteome analysis by immunoaffinity subtraction, hydrazide chemistry, and mass spectrometry.

    Liu T, Qian WJ, Gritsenko MA, Camp DG, Monroe ME, Moore RJ and Smith RD

    Biological Sciences Division and Environmental Molecular Sciences Laboratory, Pacific Northwest National Laboratory, Richland, Washington 99354, USA.

    The enormous complexity, wide dynamic range of relative protein abundances of interest (over 10 orders of magnitude), and tremendous heterogeneity (due to post-translational modifications, such as glycosylation) of the human blood plasma proteome severely challenge the capabilities of existing analytical methodologies. Here, we describe an approach for broad analysis of human plasma N-glycoproteins using a combination of immunoaffinity subtraction and glycoprotein capture to reduce both the protein concentration range and the overall sample complexity. Six high-abundance plasma proteins were simultaneously removed using a pre-packed, immobilized antibody column. N-linked glycoproteins were then captured from the depleted plasma using hydrazide resin and enzymatically digested, and the bound N-linked glycopeptides were released using peptide-N-glycosidase F (PNGase F). Following strong cation exchange (SCX) fractionation, the deglycosylated peptides were analyzed by reversed-phase capillary liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). Using stringent criteria, a total of 2053 different N-glycopeptides were confidently identified, covering 303 nonredundant N-glycoproteins. This enrichment strategy significantly improved detection and enabled identification of a number of low-abundance proteins, exemplified by interleukin-1 receptor antagonist protein (approximately 200 pg/mL), cathepsin L (approximately 1 ng/mL), and transforming growth factor beta 1 (approximately 2 ng/mL). A total of 639 N-glycosylation sites were identified, and the overall high accuracy of these glycosylation site assignments as assessed by accurate mass measurement using high-resolution liquid chromatography coupled to Fourier transform ion cyclotron resonance mass spectrometry (LC-FTICR) is initially demonstrated.

    Funded by: NCRR NIH HHS: P41 RR018522, RR18522; NIGMS NIH HHS: U54 GM-62119-02, U54 GM062119

    Journal of proteome research 2005;4;6;2070-80

  • TGF-beta control of cell proliferation.

    Huang SS and Huang JS

    Department of Biochemistry and Molecular Biology, Saint Louis University School of Medicine, St. Louis, Missouri 63104, USA. huangss@slu.edu

    This article focuses on recent findings that the type V TGF-beta receptor (TbetaR-V), which co-expresses with other TGF-beta receptors (TbetaR-I, TbetaR-II, and TbetaR-III) in all normal cell types studied, is involved in growth inhibition by IGFBP-3 and TGF-beta and that TGF-beta activity is regulated by two distinct endocytic pathways (clathrin- and caveolar/lipid-raft-mediated). TGF-beta is a potent growth inhibitor for most cell types, including epithelial and endothelial cells. The signaling by which TGF-beta controls cell proliferation is not well understood. Many lines of evidence indicate that other signaling pathways, in addition to the prominent TbetaR-I/TbetaR-II/Smad2/3/4 signaling cascade, are required for mediating TGF-beta-induced growth inhibition. Recent studies revealed that TbetaR-V, which is identical to LRP-1, mediates IGF-independent growth inhibition by IGFBP-3 and mediates TGF-beta-induced growth inhibition in concert with TbetaR-I and TbetaR-II. In addition, IRS proteins and a Ser/Thr-specific protein phosphatase(s) are involved in the TbetaR-V-mediated growth inhibitory signaling cascade. The TbetaR-V signaling cascade appears to cross-talk with the TbetaR-I/TbetaR-II, insulin receptor (IR), IGF-I receptor (IGF-IR), integrin and c-Met signaling cascades. Attenuation or loss of the TbetaR-V signaling cascade may enable carcinoma cells to escape from TGF-beta growth control and may contribute to the aggressiveness and invasiveness of these cells via promoting epithelial-to-mesenchymal transdifferentiation (EMT). Finally, the ratio of TGF-beta binding to TbetaR-II and TbetaR-I is a signal controlling TGF-beta partitioning between two distinct endocytosis pathways and resultant TGF-beta responsiveness. These recent studies have provided new insights into the molecular mechanisms underlying TGF-beta-induced cellular growth inhibition, cross-talk between the TbetaR-V and other signaling cascades, the signal that controls TGF-beta responsiveness and the role of TbetaR-V in tumorigenesis.

    Funded by: NCI NIH HHS: CA 38808

    Journal of cellular biochemistry 2005;96;3;447-62

  • Low-density lipoprotein receptor-related protein polymorphisms in patients with elevated factor VIII coagulant activity and venous thrombosis.

    Cunningham N, Laffan MA, Manning RA and O'Donnell JS

    Haematology Department, Imperial College London, London, UK.

    Elevated factor VIII coagulant activity (FVIII:C) levels (>150 IU/dl) represent a prevalent independent risk factor for venous thromboembolism (VTE). Low-density lipoprotein receptor-related protein (LRP) is involved in factor VIII clearance in vivo, and elevated FVIII:C was a feature of the LRP knockout mouse model. Three coding polymorphisms of LRP1 (exon 3, C766T; exon 14, A217V; and exon 39, D2080N), together with an insertion/deletion polymorphism within the first intron of lipoprotein receptor-associated protein (LRPAP1), have been identified. In addition, LRP1 2080D was recently reported to be associated with increased plasma FVIII:C levels in normal individuals. In this study, we investigated the role of these four polymorphisms in patients with objectively confirmed VTE and elevated FVIII:C levels. In our control group, genotype distributions were consistent with previous reports. Neither the allele frequencies nor genotype distributions at LRP1 A217V, LRP1 D2080N and LRPAP1 intron 1 were significantly different between the elevated FVIII:C and control groups. In contrast to previous reports, we found no effect of LRP1 D2080N genotype on plasma FVIII:C levels in normal individuals. More importantly, prevalence of the LRP1 2080D allele was not increased in the group of patients with high FVIII:C and VTE. We conclude that LRP1 and LRPAP1 polymorphisms are not responsible for high FVIII:C levels in patients with VTE.

    Blood coagulation & fibrinolysis : an international journal in haemostasis and thrombosis 2005;16;7;465-8

  • The apoE isoform binding properties of the VLDL receptor reveal marked differences from LRP and the LDL receptor.

    Ruiz J, Kouiavskaia D, Migliorini M, Robinson S, Saenko EL, Gorlatova N, Li D, Lawrence D, Hyman BT, Weisgraber KH and Strickland DK

    Department of Surgery, University of Maryland School of Medicine, Rockville, MD 21201, USA.

    Apolipoprotein E (apoE) associates with lipoproteins and mediates their interaction with members of the LDL receptor family. ApoE exists as three common isoforms that have important distinct functional and biological properties. Two apoE isoforms, apoE3 and apoE4, are recognized by the LDL receptor, whereas apoE2 binds poorly to this receptor and is associated with type III hyperlipidemia. In addition, the apoE4 isoform is associated with the common late-onset familial and sporadic forms of Alzheimer's disease. Although the interaction of apoE with the LDL receptor is well characterized, the specificity of other members of this receptor family for apoE is poorly understood. In the current investigation, we have characterized the binding of apoE to the VLDL receptor and the LDL receptor-related protein (LRP). Our results indicate that like the LDL receptor, LRP prefers lipid-bound forms of apoE, but in contrast to the LDL receptor, both LRP and the VLDL receptor recognize all apoE isoforms. Interestingly, the VLDL receptor does not require the association of apoE with lipid for optimal recognition and avidly binds lipid-free apoE. It is likely that this receptor-dependent specificity for various apoE isoforms and for lipid-free versus lipid-bound forms of apoE is physiologically significant and is connected to distinct functions for these receptors.

    Funded by: NHLBI NIH HHS: 2P01 HL-41633, HL-50784, HL-54710, HL-72929; NIA NIH HHS: AG-12406

    Journal of lipid research 2005;46;8;1721-31

  • Beyond endocytosis: LRP function in cell migration, proliferation and vascular permeability.

    Lillis AP, Mikhailenko I and Strickland DK

    Department of Surgery, University of Maryland School of Medicine, Rockville, MD 20855, USA.

    The low-density lipoprotein (LDL) receptor related protein (LRP1 or LRP) is a large endocytic receptor widely expressed in several tissues and known to play roles in areas as diverse as lipoprotein metabolism, degradation of proteases, activation of lysosomal enzymes and cellular entry of bacterial toxins and viruses. This member of the LDL receptor superfamily is constitutively endocytosed from the membrane and recycled back to the cell surface. Its many functions were long thought to involve its ability to bind over 30 different ligands and deliver them to lysosomes for degradation. However, LRP has since been shown to interact with scaffolding and signaling proteins via its intracellular domain in a phosphorylation-dependent manner and to function as a co-receptor partnering with other cell surface or integral membrane proteins. This multi-talented receptor has been implicated in regulation of platelet derived growth factor receptor activity, integrin maturation and recycling, and focal adhesion disassembly. These functions may account for recent studies identifying LRP's role in protection of the vasculature, regulation of cell migration, and modulation of the integrity of the blood-brain barrier.

    Funded by: NHLBI NIH HHS: HL50784, HL54710, HL65939; NIGMS NIH HHS: 5T32GM07171

    Journal of thrombosis and haemostasis : JTH 2005;3;8;1884-93

  • Platelet-derived growth factor receptor-beta (PDGFR-beta) activation promotes its association with the low density lipoprotein receptor-related protein (LRP). Evidence for co-receptor function.

    Newton CS, Loukinova E, Mikhailenko I, Ranganathan S, Gao Y, Haudenschild C and Strickland DK

    Department of Surgery and Physiology, University of Maryland School of Medicine, 15601 Crabbs Branch Way, Rockville, Maryland 20855, USA.

    Activation of the platelet-derived growth factor receptor-beta (PDGFR-beta) leads to tyrosine phosphorylation of the cytoplasmic domain of LRP and alters its association with adaptor and signaling proteins, such as Shc. The mechanism of the PDGF-induced LRP tyrosine phosphorylation is not well understood, especially since PDGF not only activates PDGF receptor but also binds directly to LRP. To gain insight into this mechanism, we used a chimeric receptor in which the ligand binding domain of the PDGFR-beta was replaced with that from the macrophage colony-stimulating factor (M-CSF) receptor, a highly related receptor tyrosine kinase of the same subfamily, but with different ligand specificity. Activation of the chimeric receptor upon the addition of M-CSF readily mediated the tyrosine phosphorylation of LRP. Since M-CSF is not recognized by LRP, these results indicated that growth factor binding to LRP is not necessary for this phosphorylation event. Using a panel of cytoplasmic domain mutants of the chimeric M-CSF/PDGFR-beta, we confirmed that the kinase domain of PDGFR-beta is absolutely required for LRP tyrosine phosphorylation but that PDGFR-beta-mediated activation of phosphatidylinositol 3-kinase, RasGAP, SHP-2, phospholipase C-gamma, and Src are not necessary for LRP tyrosine phosphorylation. To identify the cellular compartment where LRP and the PDGFR-beta may interact, we employed immunofluorescence and immunogold electron microscopy. In WI-38 fibroblasts, these two receptors co-localized in coated pits and endosomal compartments following PDGF stimulation. Further, phosphorylated forms of the PDGFR-beta co-immunoprecipitated with LRP following PDGF treatment. Together, these studies revealed close association between activated PDGFR-beta and LRP, suggesting that LRP functions as a co-receptor capable of modulating the signal transduction pathways initiated by the PDGF receptor from endosomes.

    Funded by: NHLBI NIH HHS: HL50784, HL54710

    The Journal of biological chemistry 2005;280;30;27872-8

  • Association study and meta-analysis of low-density lipoprotein receptor related protein in Alzheimer's disease.

    Pritchard A, Harris J, Pritchard CW, St Clair D, Lemmon H, Lambert JC, Chartier-Harlin MC, Hayes A, Thaker U, Iwatsubo T, Mann DM and Lendon C

    Molecular Psychiatry Department, Division of Neuroscience, Queen Elizabeth Psychiatric Hospital, University of Birmingham, Birmingham B15 2QZ, UK. axp890@bham.ac.uk

    Genome scans in sporadic Alzheimer's disease (AD) have revealed a possible susceptibility locus on chromosome 12. The low density lipoprotein receptor related protein (LRP1) gene lies within this area of linkage. Eighteen previous AD case-control studies have investigated the C766T polymorphism in LRP1 with conflicting results, including a protective effect on AD of the T allele, an increased susceptibility towards AD with both the C and T alleles, or no association at all. We have now performed a case-control study based on a large UK cohort of 477 AD patients and 466 matched controls, and have included these data, with those drawn from the 18 previous studies, into in a meta-analysis of 4668 AD patients and 4473 controls. We find no evidence for influence on the risk for AD in either our own present cohort or in the combined data set. Furthermore, we investigated whether the C766T polymorphism might modify the clinical and pathological phenotype in our cohort. We found no association with AD when the cohort was stratified into those with early (<65 years) or late (>65 years) onset, or when split into Apolipoprotein E (APOE) epsilon4 bearers and epsilon4 non-bearers. In addition, the C766T polymorphism was shown not to influence the age onset of AD. In a separate autopsy-confirmed cohort of 130 AD cases, no association with genotype or allele was observed for tissue levels of beta-amyloid 40, beta-amyloid 42, total beta-amyloid, pathological tau proteins, microglial cells or extent of astrocytic activity. Therefore, in this present study, we find no evidence for the involvement of this polymorphism either in increasing the susceptibility to AD, or by acting as a phenotypic modifier.

    Neuroscience letters 2005;382;3;221-6

  • Expression of the common heat-shock protein receptor CD91 is increased on monocytes of exposed yet HIV-1-seronegative subjects.

    Kebba A, Stebbing J, Rowland S, Ingram R, Agaba J, Patterson S, Kaleebu P, Imami N and Gotch F

    Department of Immunology, Imperial College, Chelsea & Westminster Hospital, London, UK. a.kebba@ic.ac.uk

    The significantly higher surface expression of the surface heat-shock protein receptor CD91 on monocytes of human immunodeficiency virus type-1 (HIV-1)-infected, long-term nonprogressors suggests that HIV-1 antigen uptake and cross-presentation mediated by CD91 may contribute to host anti-HIV-1 defenses and play a role in protection against HIV-1 infection. To investigate this further, we performed phenotypic analysis to compare CD91 surface expression on CD14(+) monocytes derived from a cohort of HIV-1-exposed seronegative (ESN) subjects, their seropositive (SP) partners, and healthy HIV-1-unexposed seronegative (USN) subjects. The median fluorescent intensity (MFI) of CD91 on CD14(+) monocytes was significantly higher in ESN compared with SP (P = 0.028) or USN (P = 0.007), as well as in SP compared with USN subjects (P = 0.018). CD91 MFI was not normalized in SP subjects on highly active antiretroviral therapy (HAART) despite sustainable, undetectable plasma viraemia. Data in three SP subjects experiencing viral rebounds following interruption of HAART showed low CD91 MFI comparable with levels in USN subjects. There was a significant positive correlation between CD91 MFI and CD8(+) T cell counts in HAART-naïve SP subjects (r = 0.7, P = 0.015). Increased surface expression of CD91 on CD14(+) monocytes is associated with the apparent HIV-1 resistance that is observed in ESN subjects.

    Funded by: Wellcome Trust

    Journal of leukocyte biology 2005;78;1;37-42

  • Sequences from the low density lipoprotein receptor-related protein (LRP) cytoplasmic domain enhance amyloid beta protein production via the beta-secretase pathway without altering amyloid precursor protein/LRP nuclear signaling.

    Yoon IS, Pietrzik CU, Kang DE and Koo EH

    Department of Neurosciences, University of California, San Diego, La Jolla, 92093, USA.

    Increasing evidence suggests that the low density lipoprotein receptor-related protein (LRP) affects the processing of amyloid precursor protein (APP) and amyloid beta (Abeta) protein production as well as mediates the clearance of Abeta from the brain. Recent studies indicate that the cytoplasmic domain of LRP is critical for this modulation of APP processing requiring perhaps a complex between APP, the adaptor protein FE65, and LRP. In this study, we expressed a small LRP domain consisting of the C-terminal 97 amino acids of the cytoplasmic domain, or LRP-soluble tail (LRP-ST), in CHO cells to test the hypothesis that the APP.LRP complex can be disrupted. We anticipated that LRP-ST would inhibit the normal interaction between LRP and APP and therefore perturb APP processing to resemble a LRP-deficient state. Surprisingly, CHO cells expressing LRP-ST demonstrated an increase in both sAPP secretion and Abeta production compared with control CHO cells in a manner reminiscent of the cellular effects of the APP "Swedish mutation." The increase in sAPP secretion consisted mainly of sAPPbeta, consistent with the increase in Abeta release. Further, this effect is LRP-independent, as the same alterations remained when LRP-ST was expressed in LRP-deficient cells but not when the construct was membrane-anchored. Finally, deletion experiments suggested that the last 50 amino acid residues of LRP-ST contain the important domain for altering APP processing and Abeta production. These observations indicate that there are cellular pathways that may suppress Abeta generation but that can be altered to facilitate Abeta production.

    Funded by: NIA NIH HHS: AG12376

    The Journal of biological chemistry 2005;280;20;20140-7

  • The low density lipoprotein receptor-related protein (LRP) is a novel beta-secretase (BACE1) substrate.

    von Arnim CA, Kinoshita A, Peltan ID, Tangredi MM, Herl L, Lee BM, Spoelgen R, Hshieh TT, Ranganathan S, Battey FD, Liu CX, Bacskai BJ, Sever S, Irizarry MC, Strickland DK and Hyman BT

    Alzheimer Disease Research Laboratory, Massachusetts General Hospital, Harvard Medical School, Charlestown, Massachusetts 02129, USA.

    BACE is a transmembrane protease with beta-secretase activity that cleaves the amyloid precursor protein (APP). After BACE cleavage, APP becomes a substrate for gamma-secretase, leading to release of amyloid-beta peptide (Abeta), which accumulates in senile plaques in Alzheimer disease. APP and BACE are co-internalized from the cell surface to early endosomes. APP is also known to interact at the cell surface and be internalized by the low density lipoprotein receptor-related protein (LRP), a multifunctional endocytic and signaling receptor. Using a new fluorescence resonance energy transfer (FRET)-based assay of protein proximity, fluorescence lifetime imaging (FLIM), and co-immunoprecipitation we demonstrate that the light chain of LRP interacts with BACE on the cell surface in association with lipid rafts. Surprisingly, the BACE-LRP interaction leads to an increase in LRP C-terminal fragment, release of secreted LRP in the media and subsequent release of the LRP intracellular domain from the membrane. Taken together, these data suggest that there is a close interaction between BACE and LRP on the cell surface, and that LRP is a novel BACE substrate.

    Funded by: NHLBI NIH HHS: HL50784, HL54710; NIA NIH HHS: AG12406, AG15379, P50AG05134; NIBIB NIH HHS: EB00768

    The Journal of biological chemistry 2005;280;18;17777-85

  • Endothelial expression of E-selectin is induced by the platelet-specific chemokine platelet factor 4 through LRP in an NF-kappaB-dependent manner.

    Yu G, Rux AH, Ma P, Bdeir K and Sachais BS

    University of Pennsylvania, 207 John Morgan, Philadelphia, PA 19104, USA.

    The involvement of platelets in the pathogenesis of atherosclerosis has recently gained much attention. Platelet factor 4 (PF4), a platelet-specific chemokine released on platelet activation, has been localized to atherosclerotic lesions, including macrophages and endothelium. In this report, we demonstrate that E-selectin, an adhesion molecule involved in atherogenesis, is up-regulated in human umbilical vein endothelial cells exposed to PF4. Induction of E-selectin RNA is time and dose dependent. Surface expression of E-selectin, as measured by flow cytometry, is also increased by PF4. PF4 induces E-selectin expression by activation of transcriptional activity. Activation of nuclear factor-kappaB is critical for PF4-induced E-selectin expression, as demonstrated by promoter activation studies and electrophoretic mobility shift assays. Further, we have identified the low-density lipoprotein receptor-related protein as the cell surface receptor mediating this effect. These results demonstrate that PF4 is able to increase expression of E-selectin by endothelial cells and represents another potential mechanism by which platelets may participate in atherosclerotic lesion progression.

    Funded by: NHLBI NIH HHS: HL068631, K08 HL04245

    Blood 2005;105;9;3545-51

  • Rapid endocytosis of the low density lipoprotein receptor-related protein modulates cell surface distribution and processing of the beta-amyloid precursor protein.

    Cam JA, Zerbinatti CV, Li Y and Bu G

    Department of Pediatrics, Washington University School of Medicine, St. Louis, Missouri 63110, USA.

    The low density lipoprotein receptor-related protein (LRP) is a approximately 600-kDa multifunctional endocytic receptor that is highly expressed in the brain. LRP and its ligands apolipoprotein E, alpha2-macroglobulin, and beta-amyloid precursor protein (APP), are genetically linked to Alzheimer disease and are found in characteristic plaque deposits in brains of patients with Alzheimer disease. To identify which extracellular domains of LRP interact with APP, we used minireceptors of each of the individual LRP ligand binding domains and assessed their ability to bind and degrade a soluble APP fragment. LRP minireceptors containing ligand binding domains II and IV, but not I or III, interacted with APP. To test whether APP trafficking is directly related to the rapid endocytosis of LRP, we generated stable Chinese hamster ovary cell lines expressing either a wild-type LRP minireceptor or its endocytosis mutants. Chinese hamster ovary cells stably expressing wild-type LRP minireceptor had less cell surface APP than pcDNA3 vector-transfected cells, whereas those stably expressing endocytosis-defective LRP minireceptors accumulated APP at the cell surface. We also found that the steady-state levels of the amyloid beta-peptides (Abeta) is dictated by the relative expression levels of APP and LRP, probably reflecting the dual roles of LRP in both Abeta production and clearance. Together, these data establish a relationship between LRP rapid endocytosis and APP trafficking and proteolytic processing to generate Abeta.

    Funded by: NIA NIH HHS: P50-AG05681; NINDS NIH HHS: NS41872

    The Journal of biological chemistry 2005;280;15;15464-70

  • Functions of sorting nexin 17 domains and recognition motif for P-selectin trafficking.

    Knauth P, Schlüter T, Czubayko M, Kirsch C, Florian V, Schreckenberger S, Hahn H and Bohnensack R

    Centro de Investigación y Asistencia en Tecnología y Diseño del Estado de Jalisco (CIATEJ), Av. Normalistas 800, 44270 Guadalajara, México. pknauth@ciatej.net.mx

    SNX17 is a member of the sorting nexin family (SNX), a group of hydrophilic proteins whose common characteristic property is a phox homology (PX) domain. The PX domain directs SNXs to phosphatidylinositides containing membranes of the endosomal compartment, where the SNXs are involved in the sorting of transmembrane proteins. SNX17 is known to interact with P-selectin and the LDL receptor family. Here, we report that the PX domain of SNX17 specifically binds to phosphatidylinositol 3-phosphate-containing membranes. The functional part of SNX17 that binds P-selectin or Patched (PTCH) consists of a truncated FERM domain and a unique C terminus together (FC-unit). In a yeast two-hybrid analysis a putative recognition motif for the FC-unit was revealed within P-selectin as FxNaa(F/Y). When HepG2 cells overexpress P-selectin together with SNX17, SNX17 changes its distribution from early endosomes to lysobisphosphatidic acid-containing late endosomes. Furthermore, overexpressed SNX17 restrains P-selectin in the outer membrane of the late endosomal compartment, thus preventing the normal lysosomal accumulation of P-selectin. These results suggest that the PX domain is necessary for the intracellular localisation, while the FC-unit is required for cargo recognition. We hypothesise that the expression level of SNX17 may regulate the lysosomal degradation, at least for P-selectin, by suppressing its entry into the inner vesicles of the multi-vesicular bodies (MVBs).

    Journal of molecular biology 2005;347;4;813-25

  • LDL receptor-related protein and the vascular wall: implications for atherothrombosis.

    Llorente-Cortés V and Badimon L

    Cardiovascular Research Center, CSIC-ICCC, Hospital de la Santa Creu i Sant Pau, Barcelona, Spain.

    LDL receptor-related protein 1 (LRP1) is highly expressed in the vascular wall and is mainly associated with macrophages and vascular smooth muscle cells (VSMCs). Overexpression of LRP1 in atherosclerotic lesions has been demonstrated in several animal models and human lesions. Clinical studies have suggested a relation between alterations in LRP1 expression and coronary heart disease. Indeed, it has been demonstrated that LRP1 gene expression is increased in blood mononuclear cells from patients with coronary obstruction and that the LRP1 mRNA-protein expression ratio is altered in coronary patients. Taken together, these results seem to suggest that LRP1 may be a pivotal receptor in the etiology of atherosclerosis. Our group has contributed to the elucidation of the physiopathologic role of LRP1 in the vascular wall by demonstrating that LRP1-mediated, matrix-retained LDL internalization could be crucial for VSMC-foam cell formation, that LRP1 is upregulated by lipid during human atherosclerotic lesion progression, and that LRP1-mediated aggregated LDL uptake causes the prothrombotic transformation of the vascular wall. Therefore, LRP1 seems to play a pathologic function during atherosclerotic lesion progression; however, LRP1 also seems to be essential for embryonic development and for the maintenance of vascular integrity. The protective effect of LRP1 in the vessel wall seems to be mainly due to its role in controlling certain signaling pathways. In this review, we will focus on the description of the main physiopathologic functions of LRP1 in the vascular wall.

    Arteriosclerosis, thrombosis, and vascular biology 2005;25;3;497-504

  • Expression of LRP1 by human osteoblasts: a mechanism for the delivery of lipoproteins and vitamin K1 to bone.

    Niemeier A, Kassem M, Toedter K, Wendt D, Ruether W, Beisiegel U and Heeren J

    Department of Orthopaedics, University Hospital Hamburg-Eppendorf, Hamburg, Germany. niemeier@uke.uni-hamburg.de

    Unlabelled: Accumulating clinical and experimental data show the importance of dietary lipids and lipophilic vitamins, such as vitamin K1, for bone formation. The molecular mechanism of how they enter the osteoblast is unknown. Here we describe the expression of the multifunctional LRP1 by human osteoblasts in vitro and in vivo. We provide evidence that LRP1 plays an important role in the uptake of postprandial lipoproteins and vitamin K1 by human osteoblasts.

    Introduction: Chylomicrons (CM) and their remnants (CR) represent the postprandial plasma carriers of dietary lipids. Dietary vitamin K1 is known to be transported in the circulation as part of CM/CR and is required by osteoblasts as an essential co-factor for the gamma-carboxylation of bone matrix proteins. The molecular mechanisms underlying the delivery of lipophilic substances to bone are not understood. In this study, the expression and function of CM/CR receptors was examined in human osteoblasts.

    Four human osteoblast-like cell lines were analyzed: two osteosarcoma lines (MG63, SaOS-2) and two telomerase-immortalized human bone marrow stromal cell lines (hMSC-TERT [4] and [20]) after 1,25(OH)2 vitamin D3 induction of osteoblastic differentiation (hMSC-TERT-OB). Receptor expression was examined by Western blotting and immunohistochemistry of normal human bone sections. Endocytotic receptor function was analyzed by cellular uptake assays using fluorescent and radiolabeled human CR. Vitamin K1-enriched CR (CR-K1) were generated in vivo after oral vitamin administration and vitamin K1 uptake by osteoblasts was measured by HPLC. The effect of CR-K1 uptake on osteocalcin carboxylation was measured by ELISA.

    Results: Osteoblasts exhibit high levels of protein expression of the CR receptors LRP1 and LDLR. VLDLR is expressed to a lower degree. Immunohistochemistry of normal human bone sections showed strong LRP1 expression by osteoblasts and marrow stromal cells. Uptake of fluorescent CR by osteoblasts resulted in the typical pattern of receptor-mediated endocytosis. CR uptake was stimulated by the exogenous addition of the lipoprotein receptor ligands apolipoprotein E and lipoprotein lipase. Uptake was reduced by the known LRP1 inhibitors RAP, lactoferrin, and suramin, but not by LDL, which exclusively binds to the LDLR. Vitamin K1 uptake by hMSC-TERT-OB after incubation with CR-K1 was also shown to be sensitive to LPL stimulation and the LRP1 specific inhibitor lactoferrin. CR-K1 uptake into osteoblasts stimulated the gamma-carboxylation of osteocalcin.

    Conclusion: Human osteoblasts express receptors of the LDLR family with a capacity for vitamin K1 uptake through CR endocytosis, a novel mechanism for the delivery of dietary lipids and lipophilic vitamins to human bone. The current data suggest that, among the expressed receptors, LRP1 plays a predominant role.

    Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research 2005;20;2;283-93

  • Genetic-morphologic association study: association between the low density lipoprotein-receptor related protein (LRP) and cerebral amyloid angiopathy.

    Christoforidis M, Schober R and Krohn K

    Department of Neuropathology, University of Leipzip, Germany. mchristoforidis@web.de

    Accumulating evidence suggests that genetic factors such as apolipoprotein E (APOE), can act in different ways in the pathogenesis of cerebral amyloid angiopathy (CAA) and Alzheimer's disease (AD). The role of the low-density lipoprotein-receptor related protein (LRP), the major cerebral APOE receptor, in AD has been discussed controversially depending on data from different populations and methodological approaches. We examined the influence of LRP polymorphisms on CAA in 125 post-mortem cases genotyped for APOE and classified according to the neurofibrillary Braak and Braak staging of AD (indicating neurodegeneration grade). CAA was assessed separately for leptomeningeal (CAAlep.), noncapillary cortical (CAAcort.) and capillary cortical (CAAcap.) vessels in beta-amyloid stained sections. Our results suggest: (i) the 87 bp allele of LRP5' polymorphism (LRP5') is an independent predictive factor for CAAcort. and CAAlep.; (ii) the C/C genotype (C allele) of the LRP exon 3 polymorphism is positively associated with the severity of CAAlep. and CAAcort., implicating a younger age of CAA onset and/or faster CAA progression; (iii) as CAAcort. and CAAlep. showed different genetic associations in contrast to CAAcap., we can underscore the hypothesis that different molecular mechanisms are involved in CAA pathogenesis of noncapillary and capillary cerebral vessels. Our results lead us to postulate that the LRP5'87 bp and the LRP exon 3 C alleles of the LRP gene (or another locus that might be in linkage disequilibrium with these LRP polymorphic sites) could modify cerebrovascular LRP function or expression in noncapillary cerebral vessels, leading to an increased cerebrovascular amyloid deposition.

    Neuropathology and applied neurobiology 2005;31;1;11-9

  • LDL-receptor-related protein regulates beta2-integrin-mediated leukocyte adhesion.

    Spijkers PP, da Costa Martins P, Westein E, Gahmberg CG, Zwaginga JJ and Lenting PJ

    Laboratory for Thrombosis and Haemostasis, Department of Haematology, University Medical Center Utrecht, The Netherlands.

    Beta2-integrin clustering on activation is a key event in leukocyte adhesion to the endothelium during the inflammatory response. In the search for molecular mechanisms leading to this clustering, we have identified low-density lipoprotein (LDL) receptor-related protein (LRP) as a new partner for beta2-integrins at the leukocyte surface. Immobilized recombinant LRP fragments served as an adhesive surface for blood-derived leukocytes and the U937 cell line. This adhesion was decreased up to 95% in the presence of antibodies against beta2-integrins, pointing to these integrins as potential partners for LRP. Using purified proteins, LRP indeed associated with the alphaMbeta2 complex and the alphaM and alphaL I-domains (K(d, app) approximately 0.5 microM). Immunoprecipitation experiments and confocal microscopy revealed that endogenously expressed LRP and alphaLbeta2 colocalized in monocytes and U937 cells. Furthermore, activation of U937 cells resulted in clustering of alphaLbeta2 and LRP to similar regions at the cell surface, indicating potential cooperation between both proteins. This was confirmed by the lack of alphaLbeta2 clustering in U937 cells treated by antisense oligonucleotides to down-regulate LRP. In addition, the absence of LRP resulted in complete abrogation of beta2-integrin-dependent adhesion to endothelial cells in a perfusion system, demonstrating the presence of a previously unrecognized link between LRP and leukocyte function.

    Blood 2005;105;1;170-7

  • Immune function of C1q and its modulators CD91 and CD93.

    Tarr J and Eggleton P

    Institute of Biomedical and Clinical Sciences, Peninsula Medical School, Universities of Exeter and Plymouth, Exeter, Devon EX1 2LU, UK.

    C1q is a subcomponent of the first component of complement C1, which is a multimolecular complex comprising one molecule of C1q and two molecules each of the autoreactive proteases, C1r and C1s. This multimolecular complex triggers the classical pathway of complement. Advances in the past several years have provided a partial crystal structure of the C1q subunit. This, together with gene deletion of C1q, has allowed further insight into the multifunctional immune aspects of this molecule. Two C1q-mediated functions that have received intense scrutiny recently are C1q-mediated apoptotic clearance of cell debris and phagocytosis. This has led to a heightened search for specific receptors for the collagen-like region (CLR) as well as the globular heads. Two transmembrane proteins, CD91 and CD93, have been proposed to interact indirectly with the CLR of C1q, promoting apoptotic clearance and phagocytosis, respectively. The aim of this article is to provide an overview of the structural and functional information that implicates CD91 and CD93 in C1q-mediated functional effects.

    Critical reviews in immunology 2005;25;4;305-30

  • Serine and threonine phosphorylation of the low density lipoprotein receptor-related protein by protein kinase Calpha regulates endocytosis and association with adaptor molecules.

    Ranganathan S, Liu CX, Migliorini MM, Von Arnim CA, Peltan ID, Mikhailenko I, Hyman BT and Strickland DK

    Department of Surgery, University of Maryland School of Medicine, Baltimore, Maryland 21201, USA.

    The low density lipoprotein receptor-related protein (LRP) is a large receptor that participates in endocytosis, signaling pathways, and phagocytosis of necrotic cells. Mechanisms that direct LRP to function in these distinct pathways likely involve its association with distinct cytoplasmic adaptor proteins. We tested the hypothesis that the association of various adaptor proteins with the LRP cytoplasmic domain is modulated by its phosphorylation state. Phosphoamino acid analysis of metabolically labeled LRP revealed that this receptor is phosphorylated at serine, threonine, and tyrosine residues within its cytoplasmic domain, whereas inhibitor studies identified protein kinase Calpha (PKCalpha) as a kinase capable of phosphorylating LRP. Mutational analysis identified critical threonine and serine residues within the LRP cytoplasmic domain that are necessary for phosphorylation mediated by PKCalpha. Mutating these threonine and serine residues to alanines generated a receptor that was not phosphorylated and that was internalized more rapidly than wild-type LRP, revealing that phosphorylation reduces the association of LRP with adaptor molecules of the endocytic machinery. In contrast, serine and threonine phosphorylation was necessary for the interaction of LRP with Shc, an adaptor protein that participates in signaling events. Furthermore, serine and threonine phosphorylation increased the interaction of LRP with other adaptor proteins such as Dab-1 and CED-6/GULP. These results indicate that phosphorylation of LRP by PKCalpha modulates the endocytic and signaling function of LRP by modifying its association with adaptor proteins.

    Funded by: NHLBI NIH HHS: HL50784, HL54710; NIA NIH HHS: AG12406

    The Journal of biological chemistry 2004;279;39;40536-44

  • Identification and characterization of the acidic pH binding sites for growth regulatory ligands of low density lipoprotein receptor-related protein-1.

    Ling TY, Chen CL, Huang YH, Liu IH, Huang SS and Huang JS

    Institute of Biomedical Sciences, Academia Sinica, Taipei 115, Taiwan.

    The type V TGF-beta receptor (TbetaR-V) plays an important role in growth inhibition by IGFBP-3 and TGF-beta in responsive cells. Unexpectedly, TbetaR-V was recently found to be identical to the LRP-1/alpha(2)M receptor; this has disclosed previously unreported growth regulatory functions of LRP-1. Here we demonstrate that, in addition to expressing LRP-1, all cells examined exhibit low affinity but high density acidic pH binding sites for LRP-1 growth regulatory ligands (TGF-beta(1), IGFBP-3, and alpha(2)M(*)). These sites, like LRP-1, are sensitive to receptor-associated protein and calcium depletion but, unlike LRP-1, are also sensitive to chondroitin sulfate and heparin and capable of directly binding ligands, which do not bind to LRP-1. Annexin VI has been identified as a major membrane-associated protein capable of directly binding alpha(2)M(*) at acidic pH. This is evidenced by: 1) structural and Western blot analyses of the protein purified from bovine liver plasma membranes by alpha(2)M(*) affinity column chromatography at acidic pH, and 2) dot blot analysis of the interaction of annexin VI and (125)I-alpha(2)M(*). Cell surface annexin VI is involved in (125)I-TGF-beta(1) and (125)I-alpha(2)M(*) binding to the acidic pH binding sites and (125)I-alpha(2)M(*) binding to LRP-1 at neutral pH as demonstrated by the sensitivity of cells to pretreatment with anti-annexin VI IgG. Cell surface annexin VI is also capable of mediating internalization and degradation of cell surface-bound (125)I-TGF-beta(1) and (125)I-alpha(2)M(*) at pH 6 and of forming ternary complexes with (125)I-alpha(2)M(*) and LRP-1 at neutral pH as demonstrated by co-immunoprecipitation. Trifluoperazine and fluphenazine, which inhibit ligand binding to the acidic pH binding sites, block degradation after internalization of cell surface-bound (125)I-TGF-beta(1) or (125)I-alpha(2)M(*). These results suggest that cell surface annexin VI may function as an acidic pH binding site or receptor and may also function as a co-receptor with LRP-1 at neutral pH.

    Funded by: NCI NIH HHS: CA38808

    The Journal of biological chemistry 2004;279;37;38736-48

  • Genetic and functional characteristics of the human in vivo LRP1/A2MR receptor suggested as a risk marker for Alzheimer's disease and other complex (degenerative) diseases.

    Gläser C, Schulz S, Handschug K, Huse K and Birkenmeier G

    Institute of Human Genetics and Medical Biology, University of Halle, Magdeburger Str. 2, D-06097 Halle, Germany. christiane.glaeser@medizin.uni-halle.de

    LDL receptor-related protein/alpha2-macroglobulin receptor (LRP1/A2MR) a multiligand receptor is considered as not only being a possible risk factor of neurodegenerative diseases like Alzheimer's disease but also as determining the progression of other complex diseases like atherosclerosis and cancer. Although a large number of in vitro studies have highlighted its functional importance, as yet not enough is known about the clinical importance of the genetic background of LRP1 in human diseases. The aim of this ex vivo/in vivo study of 448 subjects was to present data on genetic LRP1 variants of healthy European Caucasians from Central Germany. Genotype-dependent LRP1 expression was analyzed in a representative subgroup (gene expression: n = 127, protein expression: n = 44). These data were evaluated in comparison to other published clinical LRP1 studies. For 15 functionally interesting genetic variants the genotype and allele distributions of the German Caucasians were presented in relation to their in vivo LRP1 gene and protein expression. A direct influence of the LRP1 promoter polymorphism c.1-25C>G on the human in vivo LRP1 expression level was demonstrated. In an analysis of 48 further studies genomic and functional results were evaluated. The analysis especially on Alzheimers's disease partly highlighted contradictory results, but suggested that ethnic as well as genomic characteristics determine LRP1 expression and must be considered in clinical investigations on human LRP1.

    Neuroscience research 2004;50;1;85-101

  • Effects of apolipoprotein E on the human immunodeficiency virus protein Tat in neuronal cultures and synaptosomes.

    Pocernich CB, Sultana R, Hone E, Turchan J, Martins RN, Calabrese V, Nath A and Butterfield DA

    Department of Chemistry, University of Kentucky, Lexington, Kentucky 40506, USA.

    Human immunodeficiency virus type 1 (HIV-1)-associated dementia is observed in 20-30% of patients with acquired immunodeficiency syndrome (AIDS). The epsilon4 allele of the apolipoprotein E (APOE) gene currently is thought to play a role as a risk factor for the development of HIV dementia. The HIV protein Tat is neurotoxic and binds to the same receptor as apoE, the low-density lipoprotein receptor-related protein (LRP). In this study, we investigated the role apoE plays in Tat toxicity. Synaptosomes from wild-type mice treated with Tat had increased reactive oxygen species (ROS), increased lipid and protein oxidation, and decreased mitochondrial membrane potential. Synaptosomes from APOE-knockout mice also had increased ROS, increased protein oxidation, and decreased mitochondrial membrane potential, but to a significantly lesser degree. Treatment of synaptosomes with heparinase and Tat increased Tat-induced oxidative stress, consistent with the notion of Tat requiring interaction with neuronal membranes to induce oxidative damage. Human lipidated apoE3 greatly protected neurons from Tat-induced toxicity, whereas human lipidated apoE4 showed no protection. We demonstrated that human apoE3 has antioxidant properties against Tat-induced toxicity. Taken together, the data suggest that murine apoE and human apoE4 act similarly and do not protect the cell from Tat-induced toxicity. This would allow excess Tat to remain outside the cell and interact with synaptosomal membranes, leading to oxidative stress and neurotoxicity, which could contribute to dementia associated with HIV. We show that the antioxidant properties of apoE3 greatly outweigh the competition for clearance in deterring Tat-induced oxidative stress.

    Funded by: NCRR NIH HHS: P20 RR-15592; NIA NIH HHS: AG-05119, AG-10836; NIMH NIH HHS: MH-64409; NINDS NIH HHS: R01 NS-39253

    Journal of neuroscience research 2004;77;4;532-9

  • LRP/amyloid beta-peptide interaction mediates differential brain efflux of Abeta isoforms.

    Deane R, Wu Z, Sagare A, Davis J, Du Yan S, Hamm K, Xu F, Parisi M, LaRue B, Hu HW, Spijkers P, Guo H, Song X, Lenting PJ, Van Nostrand WE and Zlokovic BV

    Frank P. Smith Laboratories for Neuroscience and Neurosurgical Research, Department of Neurosurgery, Arthur Kornberg Medical Research Building, University of Rochester Medical Center, Rochester, NY 14642, USA.

    LRP (low-density lipoprotein receptor-related protein) is linked to Alzheimer's disease (AD). Here, we report amyloid beta-peptide Abeta40 binds to immobilized LRP clusters II and IV with high affinity (Kd = 0.6-1.2 nM) compared to Abeta42 and mutant Abeta, and LRP-mediated Abeta brain capillary binding, endocytosis, and transcytosis across the mouse blood-brain barrier are substantially reduced by the high beta sheet content in Abeta and deletion of the receptor-associated protein gene. Despite low Abeta production in the brain, transgenic mice expressing low LRP-clearance mutant Abeta develop robust Abeta cerebral accumulations much earlier than Tg-2576 Abeta-overproducing mice. While Abeta does not affect LRP internalization and synthesis, it promotes proteasome-dependent LRP degradation in endothelium at concentrations > 1 microM, consistent with reduced brain capillary LRP levels in Abeta-accumulating transgenic mice, AD, and patients with cerebrovascular beta-amyloidosis. Thus, low-affinity LRP/Abeta interaction and/or Abeta-induced LRP loss at the BBB mediate brain accumulation of neurotoxic Abeta.

    Funded by: NIA NIH HHS: AG16223, AG23084; NINDS NIH HHS: NS34467, NS36645

    Neuron 2004;43;3;333-44

  • Mutants of plasminogen activator inhibitor-1 designed to inhibit neutrophil elastase and cathepsin G are more effective in vivo than their endogenous inhibitors.

    Stefansson S, Yepes M, Gorlatova N, Day DE, Moore EG, Zabaleta A, McMahon GA and Lawrence DA

    Department of Vascular Biology, J. H. Holland Laboratory, Rockville, Maryland 20855, USA. StefansS@usa.redcross.org

    Neutrophil elastase and cathepsin G are abundant intracellular neutrophil proteinases that have an important role in destroying ingested particles. However, when neutrophils degranulate, these proteinases are released and can cause irreparable damage by degrading host connective tissue proteins. Despite abundant endogenous inhibitors, these proteinases are protected from inhibition because of their ability to bind to anionic surfaces. Plasminogen activator inhibitor type-1 (PAI-1), which is not an inhibitor of these proteinases, possesses properties that could make it an effective inhibitor of neutrophil proteinases if its specificity could be redirected. PAI-1 efficiently inhibits surface-sequestered proteinases, and it efficiently mediates rapid cellular clearance of PAI-1-proteinase complexes. Therefore, we examined whether PAI-1 could be engineered to inhibit and clear neutrophil elastase and cathepsin G. By introducing specific mutations in the reactive center loop of wild-type PAI-1, we generated PAI-1 mutants that are effective inhibitors of both proteinases. Kinetic analysis shows that the inhibition of neutrophil proteinases by these PAI-1 mutants is not affected by the sequestration of neutrophil elastase and cathepsin G onto surfaces. In addition, complexes of these proteinases and PAI-1 mutants are endocytosed and degraded by lung epithelial cells more efficiently than either the neutrophil proteinases alone or in complex with their physiological inhibitors, alpha1-proteinase inhibitor and alpha1-antichymotrypsin. Finally, the PAI-1 mutants were more effective in reducing the neutrophil elastase and cathepsin G activities in an in vivo model of lung inflammation than were their physiological inhibitors.

    Funded by: NHLBI NIH HHS: 1 R43 HL69624-01, HL54710, HL55374, HL55747

    The Journal of biological chemistry 2004;279;29;29981-7

  • Functional proteomics mapping of a human signaling pathway.

    Colland F, Jacq X, Trouplin V, Mougin C, Groizeleau C, Hamburger A, Meil A, Wojcik J, Legrain P and Gauthier JM

    Hybrigenics SA, 75014 Paris, France. fcolland@hybrigenics.fr

    Access to the human genome facilitates extensive functional proteomics studies. Here, we present an integrated approach combining large-scale protein interaction mapping, exploration of the interaction network, and cellular functional assays performed on newly identified proteins involved in a human signaling pathway. As a proof of principle, we studied the Smad signaling system, which is regulated by members of the transforming growth factor beta (TGFbeta) superfamily. We used two-hybrid screening to map Smad signaling protein-protein interactions and to establish a network of 755 interactions, involving 591 proteins, 179 of which were poorly or not annotated. The exploration of such complex interaction databases is improved by the use of PIMRider, a dedicated navigation tool accessible through the Web. The biological meaning of this network is illustrated by the presence of 18 known Smad-associated proteins. Functional assays performed in mammalian cells including siRNA knock-down experiments identified eight novel proteins involved in Smad signaling, thus validating this integrated functional proteomics approach.

    Genome research 2004;14;7;1324-32

  • The low density lipoprotein receptor-related protein is a motogenic receptor for plasminogen activator inhibitor-1.

    Degryse B, Neels JG, Czekay RP, Aertgeerts K, Kamikubo Y and Loskutoff DJ

    Department of Cell Biology, The Scripps Research Institute, VB-3, La Jolla, California 92037, USA.

    Although plasminogen activator inhibitor-1 (PAI-1) is known to stimulate cell migration, little is known about underlying mechanisms. We show that both active and inactive (e.g. cleaved) PAI-1 can activate the Jak/Stat signaling system and stimulate cell migration in chemotaxis, haptotaxis, chemokinesis, and wound healing assays. Moreover, antibodies to the LDL receptor-related protein (LRP) and an LRP antagonist (RAP) blocked these motogenic effects of PAI-1, while a PAI-1 mutant that did not bind to LRP failed to activate the Jak/Stat signaling pathway or to stimulate cell migration. PAI-1 had no chemotactic effect on LRP-deficient cells. These results indicate that LRP is a signaling molecule, that it mediates the migration-promoting activity of PAI-1, and that this activity does not require intact, biologically active PAI-1. Activation of this LRP-dependent signaling pathway by PAI-1 may begin to explain how the inhibitor stimulates cell migration in a variety of normal and pathological processes.

    Funded by: NHLBI NIH HHS: HL31950

    The Journal of biological chemistry 2004;279;21;22595-604

  • Low-density lipoprotein receptor-related protein interacts with MafB, a regulator of hindbrain development.

    Petersen HH, Hilpert J, Jacobsen C, Lauwers A, Roebroek AJ and Willnow TE

    Max-Delbrueck-Center for Molecular Medicine, Berlin, Germany. petersen@mdc-berlin.de

    The intracellular domain (ICD) of the low-density lipoprotein receptor-related protein (LRP) functionally interacts with adaptor proteins both as an integral part of the receptor polypeptide and after proteolytic release. Identification of such adaptors has been difficult because the ICD is self-activating in conventional transcription factor-based yeast two-hybrid screens. We adopted an alternative screen for the ICD that depends on the activation of the Ras-signaling pathway and uncovered the transcription factor MafB as novel ICD interacting protein. MafB is a regulator of hindbrain segmentation and interacts with the ICD through a leucine zipper domain. The ICD co-localizes with MafB to the nucleus and negatively regulates its transcriptional activity, suggesting a possible role for LRP in brain development.

    FEBS letters 2004;565;1-3;23-7

  • The low density lipoprotein receptor-1, LRP1, interacts with the human frizzled-1 (HFz1) and down-regulates the canonical Wnt signaling pathway.

    Zilberberg A, Yaniv A and Gazit A

    Department of Human Microbiology, Sackler School of Medicine, Tel Aviv University, Tel Aviv 69978, Israel.

    Members of the low density lipoprotein receptor family (LDLR), LRP5/6, were shown to interact with the Frizzled (Fz) receptors and to function as Wnt coreceptors. Here we show that mLRP4T100, a minireceptor of LRP1, another member of the LDLR family, interacts with the human Fz-1 (HFz1), previously shown to serve as a receptor transmitting the canonical Wnt-3a-induced signaling cascade. However, in contrast to LRP5/6, mLRP4T100, as well as the full-length LRP1, did not cooperate with HFz1 in transmitting the Wnt-3a signaling but rather repressed it. mLRP4T100 inhibitory effect was displayed also by endocytosis-defective mLRP4T100 mutants, suggesting that LRP1 repressive effect is not attributable to LRP1-mediated enhanced HFz1 internalization and subsequent degradation. Enforced expression of mLRP4T100 decreased the capacity of HFz1 cysteine-rich domain (CRD) to interact with LRP6, in contrast to HFz1-CRD/Wnt-3a interaction that was not disrupted by overexpressing mLRP4T100. These data suggest that LRP1, by sequestering HFz1, disrupts the receptor/coreceptor complex formation, leading to the repression of the canonical Wnt signaling. Thus, this study implies that the ability to interact with Fz receptors is shared by several members of the LDLR family. However, whereas some members of the LDLR family, such as LRP5/6, interact with Fz and serve as Wnt coreceptors, others negatively regulate Wnt signaling, presumably by sequestering Fz.

    The Journal of biological chemistry 2004;279;17;17535-42

  • Regional European differences in allele and genotype frequencies of low density lipoprotein receptor-related protein 1 polymorphism in Alzheimer's disease.

    Panza F, D'Introno A, Colacicco AM, Capurso C, Basile AM, Capurso S, Capurso A and Solfrizzi V

    Department of Geriatrics, Center for Aging Brain, Memory Unit, University of Bari, Policlinico, Piazza Giulio Cesare 11, 70124 Bari, Italy. geriat.dot@geriatria.uniba.it

    The low density lipoprotein receptor-related protein 1 (LRP1 gene) is a candidate gene for Alzheimer's disease (AD), because it is a ligand for proteins involved in AD pathogenesis, such as apolipoprotein E (APOE), alpha2-macroglobulin (A2M), amyloid precursor protein (APP), and is located on chromosome 12, within a region linked with AD. An association between a silent polymorphism (C/T) in exon 3 and late onset AD has been reported, with an increased frequency of the C allele, although with conflicting results. We examined this polymorphism in a cohort of 166 sporadic AD patients and 225 sex- and age-matched nondemented controls from Southern Italy. No statistically significant differences were found in LRP1 genotype and allele frequencies between the whole AD sample and controls, nor in early- and late-onset subsets of AD patients. No statistically significant differences in frequencies between LRP1 alleles and AD among APOE allele, age, or gender strata were found. Finally, comparing our results with the findings from other European populations, the LRP1 C allele frequency showed a statistically significant decreasing trend from Northern to Southern regions of Europe, with a concomitant increase in LRP1 T allele frequency, but in AD patients only. Finally, in the AD sample, a decreasing geographical trend from North to South of Europe was found for LRP1 CC genotype, and an inverse trend for LRP1 CT genotype frequency. We suggest that these regional variations in LRP1 genotype and allele frequencies in AD could be related to the different patterns of association between this polymorphism and the disease in various European studies.

    American journal of medical genetics. Part B, Neuropsychiatric genetics : the official publication of the International Society of Psychiatric Genetics 2004;126B;1;69-73

  • LRP-1/TbetaR-V mediates TGF-beta1-induced growth inhibition in CHO cells.

    Tseng WF, Huang SS and Huang JS

    Department of Biochemistry and Molecular Biology, Saint Louis University School of Medicine, 1402 South Grand Boulevard, St. Louis, MO 63104, USA.

    The type V transforming growth factor-beta (TGF-beta) receptor (TbetaR-V) is hypothesized to be involved in cellular growth inhibition by TGF-beta(1). Recently, TbetaR-V was found to be identical to low density lipoprotein receptor-related protein-1 (LRP-1). Here we demonstrate that TGF-beta(1) inhibits growth of wild-type CHO cells but not LRP-1-deficient mutant cells (CHO-LRP-1(-) cells). Stable transfection of CHO-LRP-1(-) cells with LRP-1 cDNA restores the wild-type morphology and the sensitivity to growth inhibition by TGF-beta(1). In addition, overexpression of LRP-1 minireceptors exerts a dominant negative effect and attenuates the growth inhibitory response to TGF-beta(1) in wild-type CHO cells. These results suggest that LRP-1/TbetaR-V is critical for TGF-beta(1)-mediated growth inhibition in CHO cells.

    Funded by: NCI NIH HHS: CA 38808

    FEBS letters 2004;562;1-3;71-8

  • Low density lipoprotein receptor-related protein-1 promotes beta1 integrin maturation and transport to the cell surface.

    Salicioni AM, Gaultier A, Brownlee C, Cheezum MK and Gonias SL

    Department of Pathology, University of Virginia School of Medicine, Charlottesville 22908, USA.

    Low density lipoprotein receptor-related protein-1 (LRP-1) mediates the endocytosis of multiple plasma membrane proteins and thereby models the composition of the cell surface. LRP-1 also functions as a catabolic receptor for fibronectin, limiting fibronectin accumulation in association with cells. The goal of the present study was to determine whether LRP-1 regulates cell surface levels of the beta(1) integrin subunit. We hypothesized that LRP-1 may down-regulate cell surface beta(1) by promoting its internalization; however, unexpectedly, LRP-1 expression was associated with a substantial increase in cell surface beta(1) integrin in two separate cell lines, murine embryonic fibroblasts (MEFs) and CHO cells. The total amount of beta(1) integrin was unchanged because LRP-1-deficient cells retained increased amounts of beta(1) in the endoplasmic reticulum (ER). Expression of human LRP-1 in LRP-1-deficient MEFs reversed the shift in subcellular beta(1) integrin distribution. Metabolic labeling experiments demonstrated that the precursor form of newly synthesized beta(1) integrin (p105) is converted into mature beta(1) (p125) more slowly in LRP-1-deficient cells. Although low levels of cell surface beta(1) integrin, in LRP-1-deficient MEFs, were associated with decreased adhesion to fibronectin, the subcellular distribution of beta(1) integrin was most profoundly dependent on LRP-1 only after the cell cultures became confluent. A mutagen-treated CHO cell line, in which LRP-1 is expressed but retained in the secretory pathway, also demonstrated nearly complete ER retention of beta(1) integrin. These studies support a model in which LRP-1 either directly or indirectly promotes maturation of beta(1) integrin precursor and thereby increases the level of beta(1) integrin at the cell surface.

    Funded by: NHLBI NIH HHS: R01 HL60551

    The Journal of biological chemistry 2004;279;11;10005-12

  • The low-density lipoprotein receptor-related protein associates with calnexin, calreticulin, and protein disulfide isomerase in receptor-associated-protein-deficient fibroblasts.

    Orlando RA

    Department of Biochemistry and Molecular Biology, School of Medicine, University of New Mexico, Health Sciences Center, Albuquerque, NM 87131-5221, USA. rorlando@salud.unm.edu

    The low-density lipoprotein receptor-related protein (LRP) is a large (>600 kDa) multi-ligand-binding cell surface receptor that is now known to participate in a diverse range of cellular events. To accomplish this diverse role, LRP is composed of repetitive amino acid motifs consisting of complement-type and EGF precursor-type repeats. Within these repeats are six conserved cysteine residues that form the core disulfide bond structure of each repeat. To accommodate the intricate folding that such a complex structure dictates, a specialized chaperone is present in the endoplasmic reticulum (ER) called the receptor-associated protein (RAP) that binds to LRP immediately following its biosynthesis and assists in its exocytic transport. Interestingly, RAP -/- mice show reduced LRP expression in certain cell types, but not a more global affect on LRP expression that was expected. Such a tissue-restricted effect by RAP prompted an investigation if other ER chaperones associate with LRP to assist in its complex folding requirements and compensate for the absence of RAP in RAP -/- cells. Fibroblasts obtained from RAP -/- mice demonstrate similar LRP expression levels and subcellular distribution as RAP +/+ fibroblasts. Moreover, RAP -/- cells show an identical exocytic trafficking rate for LRP as RAP +/+ cells and comparable cell surface internalization kinetics. In RAP -/- cells, three well-known ER chaperones, calnexin, calreticulin, and protein disulfide isomerase (PDI), associate with LRP and likely compensate for the absence of RAP.

    Funded by: NHLBI NIH HHS: HL63291

    Experimental cell research 2004;294;1;244-53

  • Internalization but not binding of thrombospondin-1 to low density lipoprotein receptor-related protein-1 requires heparan sulfate proteoglycans.

    Wang S, Herndon ME, Ranganathan S, Godyna S, Lawler J, Argraves WS and Liau G

    Department of Vascular Biology, Jerome H. Holland Laboratory, American Red Cross, Rockville, Maryland 20855, USA. swang@path.uab.edu

    The amino-terminal domain of the extracellular matrix (ECM) protein thrombospondin-1 (TSP-1) mediates binding to cell surface heparan sulfate proteoglycans (HSPG) as well as binding to the endocytic receptor, low density lipoprotein-related protein (LRP-1). We previously found that recombinant TSP-1 containing the amino-terminal residues 1-214, retained both of these interactions (Mikhailenko et al. [1997]: J Biol Chem 272:6784-6791). Here, we examined the activity of a recombinant protein containing amino-terminal residues 1-90 of TSP-1 and found that this domain did not retain high-affinity heparin-binding. The loss of heparin-binding correlated with decreased binding to the fibroblast cell surface. However, both ligand blotting and solid phase binding studies indicate that this truncated fragment of TSP-1 retained high-affinity binding to LRP-1. Consistent with this, it also retained the ability to block the uptake and degradation of (125)I-TSP-1. However, TSP-1(1-90) itself was poorly endocytosed and this truncated amino-terminal domain was considerably more effective than the full-length heparin-binding domain (HBD) of TSP-1 in blocking the catabolism of endogenously expressed TSP-1. These results indicate that TSP-1 binding to LRP-1 does not require prior or concomitant interaction with cell surface HSPG but suggest subsequent endocytosis requires high-affinity heparin-binding.

    Funded by: NHLBI NIH HHS: HL56063, HL68003

    Journal of cellular biochemistry 2004;91;4;766-76

  • The low density lipoprotein receptor-related protein LRP is regulated by membrane type-1 matrix metalloproteinase (MT1-MMP) proteolysis in malignant cells.

    Rozanov DV, Hahn-Dantona E, Strickland DK and Strongin AY

    Cancer Research Center, the Burnham Institute, La Jolla, California 92037, USA.

    We demonstrate that the presentation of LRP and the subsequent uptake of its ligands by malignant cells are both strongly regulated by MT1-MMP. Because LRP is essential for the clearance of multiple ligands, these findings have important implications for many pathophysiological processes including the pericellular proteolysis in neoplastic cells as well as the fate of the soluble matrix-degrading proteases such as MMP-2. MT1-MMP is a key protease in cell invasion and a physiological activator of MMP-2. Cellular LRP consists of a non-covalently associated 515-kDa extracellular alpha-chain (LRP-515) and an 85-kDa membrane-spanning beta-chain, and plays a dual role as a multifunctional endocytic receptor and a signaling molecule. Through the capture and uptake of several soluble proteases, LRP is involved in the regulation of matrix proteolysis. LRP-515 associates with the MT1-MMP catalytic domain and is highly susceptible to MT1-MMP proteolysis in vitro. Similar to MT1-MMP, the metalloproteinases MT2-MMP, MT3-MMP and MT4-MMP also degrade LRP. The N-terminal and C-terminal parts of the LRP-515 subunit are resistant and susceptible, respectively, to MT1-MMP proteolysis. In cells co-expressing LRP and MT1-MMP, the proteolytically competent protease decreases the levels of cellular LRP and releases its N-terminal portion in the extracellular milieu while the catalytically inert protease co-precipitates with LRP. These events implicate MT1-MMP, not only in the activation of MMP-2, but also in the mechanisms that control the subsequent fate of MMP-2 in cells and tissues.

    Funded by: NCI NIH HHS: CA77470, CA83017; NHLBI NIH HHS: HL50784, HL54710

    The Journal of biological chemistry 2004;279;6;4260-8

  • Localization of low density lipoprotein receptor-related protein 1 to caveolae in 3T3-L1 adipocytes in response to insulin treatment.

    Zhang H, Links PH, Ngsee JK, Tran K, Cui Z, Ko KW and Yao Z

    Lipoprotein and Atherosclerosis Group, University of Ottawa Heart Institute, Ottawa K1Y 4W7, Ontario, Canada..

    The insulin-induced translocation of low density lipoprotein receptor-related protein 1 (LRP1) from intracellular membranes to the cell surface in 3T3-L1 adipocytes was differentiation-dependent and did not occur in 3T3-L1 fibroblasts. Prompted by findings that the plasma membrane of 3T3-L1 adipocytes was rich in caveolae, we determined whether LRP1 became caveolae-associated upon insulin stimulation. The caveolae domain was isolated by the well characterized detergent solubilization and sucrose density ultracentrifugation methodology. Under basal conditions, only a trace amount of LRP1 was caveolae-associated despite the markedly elevated caveolin-1 and caveolae after adipocytic cell differentiation. Upon insulin treatment, the amount of LRP1 associated with caveolae was increased by 4-fold within 10 min, which was blocked completely by pretreatment with wortmannin prior to insulin. The caveolar localization of LRP1 in adipocytes was specific to insulin; treatment with platelet-derived growth factor-bb isoform did not promote but rather decreased caveolar localization of LRP1 below basal levels. The insulin-induced caveolar localization of LRP1 was also observed in 3T3-L1 fibroblasts where translocation of LRP1 from intracellular membranes to the cell surface was absent, suggesting that association of LRP1 with caveolae was achieved, at least in part, through lateral transmigration along the plane of plasma membranes. Immunocytochemistry studies revealed partial co-localization of LRP1 (either endogenous LRP1 or an epitope-tagged minireceptor) with caveolin-1 in cells treated with insulin, which was confirmed by co-immunoprecipitation of LRP1 with caveolin-1 in cells treated with insulin but not platelet-derived growth factor-bb. These results suggest that the localization of LRP1 to caveolae responds selectively to extracellular signals.

    The Journal of biological chemistry 2004;279;3;2221-30

  • Complete sequencing and characterization of 21,243 full-length human cDNAs.

    Ota T, Suzuki Y, Nishikawa T, Otsuki T, Sugiyama T, Irie R, Wakamatsu A, Hayashi K, Sato H, Nagai K, Kimura K, Makita H, Sekine M, Obayashi M, Nishi T, Shibahara T, Tanaka T, Ishii S, Yamamoto J, Saito K, Kawai Y, Isono Y, Nakamura Y, Nagahari K, Murakami K, Yasuda T, Iwayanagi T, Wagatsuma M, Shiratori A, Sudo H, Hosoiri T, Kaku Y, Kodaira H, Kondo H, Sugawara M, Takahashi M, Kanda K, Yokoi T, Furuya T, Kikkawa E, Omura Y, Abe K, Kamihara K, Katsuta N, Sato K, Tanikawa M, Yamazaki M, Ninomiya K, Ishibashi T, Yamashita H, Murakawa K, Fujimori K, Tanai H, Kimata M, Watanabe M, Hiraoka S, Chiba Y, Ishida S, Ono Y, Takiguchi S, Watanabe S, Yosida M, Hotuta T, Kusano J, Kanehori K, Takahashi-Fujii A, Hara H, Tanase TO, Nomura Y, Togiya S, Komai F, Hara R, Takeuchi K, Arita M, Imose N, Musashino K, Yuuki H, Oshima A, Sasaki N, Aotsuka S, Yoshikawa Y, Matsunawa H, Ichihara T, Shiohata N, Sano S, Moriya S, Momiyama H, Satoh N, Takami S, Terashima Y, Suzuki O, Nakagawa S, Senoh A, Mizoguchi H, Goto Y, Shimizu F, Wakebe H, Hishigaki H, Watanabe T, Sugiyama A, Takemoto M, Kawakami B, Yamazaki M, Watanabe K, Kumagai A, Itakura S, Fukuzumi Y, Fujimori Y, Komiyama M, Tashiro H, Tanigami A, Fujiwara T, Ono T, Yamada K, Fujii Y, Ozaki K, Hirao M, Ohmori Y, Kawabata A, Hikiji T, Kobatake N, Inagaki H, Ikema Y, Okamoto S, Okitani R, Kawakami T, Noguchi S, Itoh T, Shigeta K, Senba T, Matsumura K, Nakajima Y, Mizuno T, Morinaga M, Sasaki M, Togashi T, Oyama M, Hata H, Watanabe M, Komatsu T, Mizushima-Sugano J, Satoh T, Shirai Y, Takahashi Y, Nakagawa K, Okumura K, Nagase T, Nomura N, Kikuchi H, Masuho Y, Yamashita R, Nakai K, Yada T, Nakamura Y, Ohara O, Isogai T and Sugano S

    Helix Research Institute, 1532-3 Yana, Kisarazu, Chiba 292-0812, Japan.

    As a base for human transcriptome and functional genomics, we created the "full-length long Japan" (FLJ) collection of sequenced human cDNAs. We determined the entire sequence of 21,243 selected clones and found that 14,490 cDNAs (10,897 clusters) were unique to the FLJ collection. About half of them (5,416) seemed to be protein-coding. Of those, 1,999 clusters had not been predicted by computational methods. The distribution of GC content of nonpredicted cDNAs had a peak at approximately 58% compared with a peak at approximately 42%for predicted cDNAs. Thus, there seems to be a slight bias against GC-rich transcripts in current gene prediction procedures. The rest of the cDNAs unique to the FLJ collection (5,481) contained no obvious open reading frames (ORFs) and thus are candidate noncoding RNAs. About one-fourth of them (1,378) showed a clear pattern of splicing. The distribution of GC content of noncoding cDNAs was narrow and had a peak at approximately 42%, relatively low compared with that of protein-coding cDNAs.

    Nature genetics 2004;36;1;40-5

  • Cellular growth inhibition by IGFBP-3 and TGF-beta1 requires LRP-1.

    Huang SS, Ling TY, Tseng WF, Huang YH, Tang FM, Leal SM and Huang JS

    Department of Biochemistry, Saint Louis University School of Medicine, 1402 South Grand Blvd., St. Louis, Missouri 63104, USA. huangss@slu.edu

    The type V TGF-beta receptor (TbetaR-V)/IGFBP-3 receptor mediates the IGF-independent growth inhibition induced by IGFBP-3. It also mediates the growth inhibitory response to TGF-beta1 in concert with other TGF-beta receptor types, and its loss may contribute to the malignant phenotype of human carcinoma cells. Here we demonstrate that TbetaR-V is identical to LRP-1/alpha2M receptor as shown by MALDI-TOF analysis of tryptic peptides of TbetaR-V purified from bovine liver. In addition, 125I-IGFBP-3 affinity-labeled TbetaR-V in Mv1Lu cells is immunoprecipitated by antibodies to LRP-1 and TbetaR-V. RAP, an LRP-1 antagonist, inhibits binding of 125I-TGF-beta1 and 125I-IGFBP-3 to TbetaR-V and diminishes IGFBP-3-induced growth inhibition in Mv1Lu cells. Absent or low levels of LRP-1, as with TbetaR-V, have been linked to the malignant phenotype of carcinoma cells. Mutagenized Mv1Lu cells selected for reduced expression of LRP-1 have an attenuated growth inhibitory response to TGF-beta1 and IGFBP-3. LRP-1-deficient mouse embryonic fibroblasts lack a growth inhibitory response to TGF-beta1 and IGFBP-3. On the other hand, stable transfection of H1299 human lung carcinoma cells with LRP-1 cDNA restores the growth inhibitory response. These results suggest that the LRP-1/TbetaR-V/IGFBP-3 receptor is required for the growth inhibitory response to IGFBP-3 and TGF-beta1.

    Funded by: NCI NIH HHS: CA 38808

    FASEB journal : official publication of the Federation of American Societies for Experimental Biology 2003;17;14;2068-81

  • Low density lipoprotein receptor-related protein (LRP) is a heparin-dependent adhesion receptor for connective tissue growth factor (CTGF) in rat activated hepatic stellate cells.

    Gao R and Brigstock DR

    Department of Surgery, The Ohio State University, 700 Children's Drive, 43212, Columbus, OH, USA

    Connective tissue growth factor (CTGF) is a cysteine-rich, extracellular matrix-associated heparin-binding protein implicated in a variety of fibrotic disorders. CTGF is initially synthesized as a mosaic protein containing four discrete structural modules (CTGF(1-4)) but this is susceptible to proteolytic cleavage yielding isoforms comprising modules 3 and 4 (CTGF(3-4)) or module 4 alone (CTGF(4)). In this study, we show that cultured rat hepatic stellate cells (HSCs) produce CTGF(1-4) and CTGF(3-4) following treatment with transforming growth factor-beta and that CTGF is a cell adhesion factor for activated HSCs. Low density lipoprotein receptor-associated protein (LRP) is a receptor for CTGF(1-4) or CTGF(3-4), but not CTGF(4), whereas cell surface heparan sulfate proteoglycans (HSPGs) are binding sites for all CTGF isoforms. Prior occupancy of LRP with other LRP ligands, receptor associated protein, anti-LRP, or a thrombospondin type I peptide (TEWSACSKTCG) resulted in a 50% decrease in the adhesion of activated HSCs to CTGF(1-4) or CTGF(3-4) whereas there was no effect on CTGF(4)-mediated adhesion. Co-incubation of CTGF with heparin or perturbation of cell surface HSPGs with heparinase or sodium chlorate completely blocked adhesion of activated HSCs to all CTGF isoforms. Freshly isolated HSCs demonstrated only weak binding to CTGF but strong binding to fibronectin. Thus HSC adhesion is at least partially promoted by CTGF through its binding to LRP, a process that is heparin-dependent. CTGF-LRP interactions are likely mediated by module 3 and CTGF-heparin interactions occur principally in module 4, although additional motifs may account for the heparin-dependency of LRP binding. These data show that LRP and HSPGs are utilized by HSCs for binding to CTGF and suggest that these cell surface molecules may be involved in mediating CTGF activity or adhesive signaling during the activation process.

    Hepatology research : the official journal of the Japan Society of Hepatology 2003;27;3;214-220

  • The intracellular domain of the low density lipoprotein receptor-related protein modulates transactivation mediated by amyloid precursor protein and Fe65.

    Kinoshita A, Shah T, Tangredi MM, Strickland DK and Hyman BT

    Alzheimer Disease Research Laboratory, Massachusetts General Hospital, Harvard Medical School, Charlestown, Massachusetts 02129, USA.

    Low density lipoprotein-related protein (LRP) is a transmembrane receptor, localized mainly in hepatocytes, fibroblasts, and neurons. It is implicated in diverse biological processes both as an endocytic receptor and as a signaling molecule. Recent reports show that LRP undergoes sequential proteolytic cleavage in the ectodomain and transmembrane domain. The latter cleavage, mediated by the Alzheimer-related gamma-secretase activity that also cleaves amyloid precursor protein (APP) and Notch, results in the release of the LRP cytoplasmic domain (LRPICD) fragment. This relatively small cytoplasmic fragment has several motifs by which LRP interacts with various intracellular adaptor and scaffold proteins. However, the function of this fragment is largely unknown. Here we show that the LRPICD is translocated to the nucleus, where it colocalizes in the nucleus with a transcription modulator, Tip60, which is known to interact with Fe65 and with the APP-derived intracellular domain. LRPICD dramatically inhibits APP-derived intracellular domain/Fe65 transactivation mediated by Tip60. LRPICD has a close interaction with Tip60 in the nucleus, as shown by a fluorescence resonance energy transfer assay. These observations suggest that LRPICD has a novel signaling function, negatively impacting transcriptional activity of the APP, Fe65, and Tip60 complex in the nucleus, and shed new light on the function of LRP in transcriptional modulation.

    Funded by: NIA NIH HHS: AG12406, AG15379, P01AG05134

    The Journal of biological chemistry 2003;278;42;41182-8

  • Lipoprotein receptor-mediated induction of matrix metalloproteinase by tissue plasminogen activator.

    Wang X, Lee SR, Arai K, Lee SR, Tsuji K, Rebeck GW and Lo EH

    Neuroprotection Research Laboratory, Departments of Radiology and Neurology, Massachusetts General Hospital, Charlestown, and Program in Neuroscience, Harvard Medical School, Boston, Massachusetts, USA. wangxi@helix.mgh.harvard.edu

    Although thrombolysis with tissue plasminogen activator (tPA) is a stroke therapy approved by the US Food and Drug Administration, its efficacy may be limited by neurotoxic side effects. Recently, proteolytic damage involving matrix metalloproteinases (MMPs) have been implicated. In experimental embolic stroke models, MMP inhibitors decreased cerebral hemorrhage and injury after treatment with tPA. MMPs comprise a family of zinc endopeptidases that can modify several components of the extracellular matrix. In particular, the gelatinases MMP-2 and MMP-9 can degrade neurovascular matrix integrity. MMP-9 promotes neuronal death by disrupting cell-matrix interactions, and MMP-9 knockout mice have reduced blood-brain barrier leakage and infarction after cerebral ischemia. Hence it is possible that tPA upregulates MMPs in the brain, and that subsequent matrix degradation causes brain injury. Here we show that tPA upregulates MMP-9 in cell culture and in vivo. MMP-9 levels were lower in tPA knockouts compared with wild-type mice after focal cerebral ischemia. In human cerebral microvascular endothelial cells, MMP-9 was upregulated when recombinant tPA was added. RNA interference (RNAi) suggested that this response was mediated by the low-density lipoprotein receptor-related protein (LRP), which avidly binds tPA and possesses signaling properties. Targeting the tPA-LRP signaling pathway in brain may offer new approaches for decreasing neurotoxicity and improving stroke therapy.

    Funded by: NIA NIH HHS: R01-AG14473; NINDS NIH HHS: P50-NS10828, R01-NS37074, R01-NS38731, R01-NS40529

    Nature medicine 2003;9;10;1313-7

  • Apolipoprotein E binding to low density lipoprotein receptor-related protein-1 inhibits cell migration via activation of cAMP-dependent protein kinase A.

    Zhu Y and Hui DY

    Department of Pathology and Laboratory Medicine, University of Cincinnati College of Medicine, Cincinnati, Ohio 45267-0529, USA.

    Smooth muscle cell migration and proliferation contribute to neointimal hyperplasia and vascular stenosis after endothelial denudation. Previous studies revealed that apolipoprotein E (apoE) is an effective inhibitor of platelet-derived growth factor-directed smooth muscle cell migration and proliferation and that the anti-migratory function is mediated via apoE binding to low density lipoprotein receptor-related protein-1 (LRP-1). This study was undertaken to identify the intracellular pathway by which apoE binding to LRP-1 results in inhibition of smooth muscle cell migration. The results showed that apoE increased intracellular cAMP levels 3-fold after 5 min, and the increase was sustained for more than 1 h. As a consequence, apoE also increased protein kinase A (PKA) activity in smooth muscle cells. Importantly, suppression of PKA activity with a cell-permeable peptide inhibitor of PKA abolished the inhibitory effect of apoE on smooth muscle cell migration. These results indicated that apoE inhibition of smooth muscle cell migration is mediated via the activation of cAMP-dependent PKA. Additional experiments revealed that apoE also inhibited fibroblasts migration toward platelet-derived growth factor by a similar mechanism of cAMP-dependent PKA activation. It is noteworthy that apoE failed to increase cAMP levels or inhibit migration of LRP-1-negative mouse embryonic fibroblasts and LRP-1-deficient smooth muscle cells. Taken together, these findings established the mechanism by which apoE inhibits cell migration, i.e. via cAMP-dependent protein kinase A activation as a consequence of its binding to LRP-1.

    Funded by: NHLBI NIH HHS: HL61332

    The Journal of biological chemistry 2003;278;38;36257-63

  • Dimers of beta 2-glycoprotein I increase platelet deposition to collagen via interaction with phospholipids and the apolipoprotein E receptor 2'.

    Lutters BC, Derksen RH, Tekelenburg WL, Lenting PJ, Arnout J and de Groot PG

    Department of Haematology, University Medical Center, and Institute of Biomembranes, Utrecht University, 3508GA Utrecht, The Netherlands.

    Patients with prolonged clotting times caused by lupus anticoagulant (LAC) are at risk for thrombosis. This paradoxal association is not understood. LAC is frequently caused by anti-beta2-glycoprotein I (beta 2GPI) antibodies. Antibody-induced dimerization of beta 2GPI increases the affinity of beta 2GPI for phospholipids, explaining the observed prolonged clotting times. We constructed dimers of beta 2GPI that mimic effects of beta 2GPI-anti-beta 2GPI antibody complexes, and we studied their effects on platelet adhesion and thrombus formation in a flow system. Dimeric beta 2GPI increased platelet adhesion to collagen by 150% and increased the number of large aggregates. We also observed increased platelet adhesion to collagen when whole blood was spiked with patient-derived polyclonal anti-beta 2GPI or some, but not all, monoclonal anti-beta 2GPI antibodies with LAC activity. These effects could be abrogated by inhibition of thromboxane synthesis. A LAC-positive monoclonal anti-beta 2GPI antibody, which did not affect platelet adhesion, prevented the induced increase in platelet adhesion by beta 2GPI dimers. Furthermore, increased platelet adhesion disappeared after preincubation with receptor-associated protein, a universal inhibitor of interaction of ligands with members of the low density lipoprotein receptor family. Using co-immunoprecipitation, it was shown that dimeric beta 2GPI can interact with apolipoprotein E receptor 2 (apoER2'), a member of the low density lipoprotein receptor family present on platelets. These results demonstrate that dimeric beta 2GPI induces increased platelet adhesion and thrombus formation, which depends on activation via apoER2'.

    The Journal of biological chemistry 2003;278;36;33831-8

  • Disease-associated dendritic cells respond to disease-specific antigens through the common heat shock protein receptor.

    Stebbing J, Gazzard B, Portsmouth S, Gotch F, Kim L, Bower M, Mandalia S, Binder R, Srivastava P and Patterson S

    Department of Immunology, Chelsea and Westminster Hospital, 369 Fulham Rd, London SW10 9NH, United Kingdom. j.stebbing@imperial.ac.uk

    The most abundant intracellular proteins, heat shock proteins (HSPs), serve as molecular chaperones for regulatory and maturation pathways. Diverse families of HSPs have been shown to bind antigenic peptides and to play major roles in innate and adaptive immune responses through the common HSP receptor, CD91. HIV-1+ patients with Kaposi sarcoma (KS) were matched for CD4 count and HIV-1 RNA viral load to HIV-1+ patients without Kaposi sarcoma (and negative for Kaposisarcoma-associated herpesvirus). We then investigated the pathways used by tumor lysates, viral lysates, and viral particles in their activation. In particular, we observed immune responses after HSP depletion using antitumor antibiotics and blockade of the common HSP receptor, CD91. Despite the impaired functional capacity of dendritic cells (DCs) derived from patients with KS, DCs retain the ability to prime the adaptive arm of the immune system through the common HSP receptor, leading to phenotypic activation and stimulation of tetramer-positive CD8+ cytotoxic T cells. We also show that interferon-producing plasmacytoid DCs are selectively depleted in KS-positive compared with matched KS-negative HIV-1-infected patients. Functionally impaired DCs can effectively cross-present immune responses through the common HSP receptor. These results have important implications for the etiopathogenesis of KS and for the development and design of any compounds, including vaccines, derived from cellular lysates.

    Blood 2003;102;5;1806-14

  • Low density lipoprotein receptor-related protein is a calreticulin coreceptor that signals focal adhesion disassembly.

    Orr AW, Pedraza CE, Pallero MA, Elzie CA, Goicoechea S, Strickland DK and Murphy-Ullrich JE

    Department of Pathology, Division of Molecular and Cellular Pathology and The Cell Adhesion and Matrix Research Center, University of Alabama at Birmingham, VH 668 1530, 3rd Ave. South, Birmingham, AL 35294-0019, USA.

    Thrombospondin (TSP) signals focal adhesion disassembly (the intermediate adhesive state) through interactions with cell surface calreticulin (CRT). TSP or a peptide (hep I) of the active site induces focal adhesion disassembly through binding to CRT, which activates phosphoinositide 3-kinase (PI3K) and extracellular signal-related kinase (ERK) through Galphai2 proteins. Because CRT is not a transmembrane protein, it is likely that CRT signals as part of a coreceptor complex. We now show that low density lipoprotein receptor-related protein (LRP) mediates focal adhesion disassembly initiated by TSP binding to CRT. LRP antagonists (antibodies, receptor-associated protein) block hep I/TSP-induced focal adhesion disassembly. LRP is necessary for TSP/hep I signaling because TSP/hep I is unable to stimulate focal adhesion disassembly or ERK or PI3K signaling in fibroblasts deficient in LRP. LRP is important in TSP-CRT signaling, as shown by the ability of hep I to stimulate association of Galphai2 with LRP. The isolated proteins LRP and CRT interact, and LRP and CRT are associated with hep I in molecular complexes extracted from cells. These data establish a mechanism of cell surface CRT signaling through its coreceptor, LRP, and suggest a novel function for LRP in regulating cell adhesion.

    Funded by: NHLBI NIH HHS: HL44575, HL50784, HL54710, R01 HL044575, R01 HL079644, T 32 HL07918; NIAMS NIH HHS: T32 AR47512

    The Journal of cell biology 2003;161;6;1179-89

  • Low density lipoprotein receptor-related protein (LRP) is required for lactoferrin-enhanced collagen gel contractile activity of human fibroblasts.

    Takayama Y, Takahashi H, Mizumachi K and Takezawa T

    Functional Biomolecules Laboratory, National Institute of Livestock and Grassland Science, 2 Ikenodai, Tsukuba, Ibaraki 305-0901 Japan. takay@affrc.go.jp

    Fibroblasts plated on a type I collagen gel can reduce the size of the gel in a way that mimics the reorganization of the collagen matrix that accompanies the wound healing process. We demonstrated previously that lactoferrin (Lf) specifically binds to WI-38 human fibroblasts and enhances their collagen gel contractile activity. The effect of Lf correlated with the phosphorylation of myosin light chain (MLC), suggesting that Lf promotes fibroblast contractile activity by regulating MLC phosphorylation. We found here that the binding of Lf to WI-38 cells was inhibited by recombinant receptor-associated protein (RAP), a universal competitor for ligand binding to LRP (LDL receptor-related protein), and RAP can also promote the collagen gel contractile activity. These observations suggest that LRP is a receptor that mediates the Lf-induced enhancement of collagen gel contractile activity in WI-38 fibroblasts. To confirm the hypothesis, we utilized LRP antisense oligonucleotide, which was modified by morpholino linkage. Suppression of LRP expression abrogated the Lf-induced enhancement the contractile activity in fibroblasts. Treatment of fibroblasts with Lf enhanced the phosphorylation of ERK1/2 and the activation of MLC kinase (MLCK). These effects were attenuated by suppression of LRP expression. These findings suggest that LRP is involved in the Lf-enhanced collagen gel contractile activity of WI-38 fibroblasts by converting the Lf binding signal into the activation of ERK1/2 and MLCK.

    The Journal of biological chemistry 2003;278;24;22112-8

  • v-Src induces Shc binding to tyrosine 63 in the cytoplasmic domain of the LDL receptor-related protein 1.

    Barnes H, Ackermann EJ and van der Geer P

    Department of Chemistry and Biochemistry, University of California, San Diego, 9500 Gilman Dr., La Jolla, CA 92093-0601, USA.

    We recently observed that the LDL receptor-related protein 1 (LRP-1) is tyrosine phosphorylated in v-Src-transformed cells. Using a GST-fusion protein containing the cytoplasmic domain of LRP-1, we show that LRP-1 is a direct substrate for v-Src in vitro. To study LRP-1 phosphorylation in vivo, we constructed an LRP-1 minireceptor composed of the beta chain linked at the amino-terminus to a Myc epitope (Myc-LRPbeta). When expressed together with v-Src, Myc-LRPbeta becomes phosphorylated on tyrosine. Of the four tyrosine residues present in the cytoplasmic domain of LRP-1, only Tyr 63 is phosphorylated by v-Src in vivo or in vitro. Using fibroblasts deficient in Src, Yes and Fyn, we were able to show that there are multiple kinases present in the cell that can phosphorylate LRP-1. Tyrosine-phosphorylated LRP-1 associates with Shc, a PTB and SH2 domain containing signaling protein that is involved in the activation of Ras. Binding of the purified Shc PTB domain to Tyr 63 containing peptides shows that the interaction between LRP-1 and Shc is direct. We found that DAB, a PTB domain containing signaling protein that is involved in signaling by LDL receptor-related proteins in the nervous system, did not bind to full-length LRP-1. Our observations suggest that LRP-1 may be involved in normal and malignant signal transduction through a direct interaction with Shc adaptor proteins.

    Funded by: NCI NIH HHS: R29 CA78629; NIDDK NIH HHS: DK 07233

    Oncogene 2003;22;23;3589-97

  • Allosteric modulation of ligand binding to low density lipoprotein receptor-related protein by the receptor-associated protein requires critical lysine residues within its carboxyl-terminal domain.

    Migliorini MM, Behre EH, Brew S, Ingham KC and Strickland DK

    Department of Vascular Biology, Jerome H. Holland Laboratory for the Biomedical Sciences, American Red Cross, Rockville, Maryland 20855, USA.

    The low density lipoprotein receptor-related protein (LRP) is a large endocytic receptor that recognizes more than 30 different ligands and plays important roles in protease and lipoprotein catabolism. Ligand binding to newly synthesized LRP is modulated by the receptor-associated protein (RAP), an endoplasmic reticulum-resident protein that functions as a molecular chaperone and prevents ligands from associating with LRP via an allosteric-type mechanism. RAP is a multidomain protein that contains two independent LRP binding sites, one located at the amino-terminal portion of the molecule and the other at the carboxyl-terminal portion of the molecule. The objective of the present investigation was to gain insight into how these two regions of RAP interact with LRP and function to modulate its ligand binding properties. These objectives were accomplished by random mutagenesis of RAP, which identified two critical lysine residues, Lys-256 and Lys-270, within the carboxyl-terminal domain that are necessary for binding of this region of RAP to LRP and to heparin. RAP molecules in which either of these two lysine residues was mutated still bound LRP but with reduced affinity. Furthermore, the mutant RAPs were significantly impaired in their ability to inhibit alpha(2)M* binding to LRP via allosteric mechanisms. In contrast, the mutant RAP molecules were still effective at inhibiting uPA.PAI-1 binding to LRP. These results confirm that both LRP binding sites within RAP cooperate to inhibit ligand binding via an allosteric mechanism.

    Funded by: NHLBI NIH HHS: HL50784, HL54710

    The Journal of biological chemistry 2003;278;20;17986-92

  • The heat-shock protein receptor CD91 is up-regulated in monocytes of HIV-1-infected "true" long-term nonprogressors.

    Stebbing J, Gazzard B, Kim L, Portsmouth S, Wildfire A, Teo I, Nelson M, Bower M, Gotch F, Shaunak S, Srivastava P and Patterson S

    Department of Immunology, Division of Investigative Science, Faculty of Medicine, Imperial College of Science, Technology and Medicine, The Chelsea and Westminster Hospital, London, United Kingdom. j.stebbing@ic.ac.uk

    A small proportion of patients with human immunodeficiency virus type 1 (HIV-1) remains asymptomatic for a long period after infection. It is thought that a vigorous immune response may contribute to long-term nonprogression, though studies are confounded by heterogeneity among patients. We studied the levels of HIV-1 receptors, costimulatory T-cell molecules, and dendritic cell (DC) numbers in 18 patients with long-term infection, CD4 counts greater than 400 cells/mm(3), and HIV-1 viral loads lower than 50 copies/mL. These patients were further differentiated through the presence or absence of 2-LTR DNA circles, a possible marker for residual ongoing HIV-1 replication. A statistically significant increase in levels of CD91, the heat-shock protein (HSP) receptor, was observed in therapy-naive patients who had no evidence of ongoing viral replication (P =.01). This difference was most notable on their monocytes. High levels of CD91 may be a host factor that contributes to the maintenance of long-term nonprogression. The ability of CD91 to internalize alpha-defensins and to cross-present exogenous antigen to cytotoxic T lymphocytes through major histocompatibility complex (MHC) class 1 may maintain CD8(+) responses in these patients.

    Blood 2003;101;10;4000-4

  • Influence of exonic polymorphisms in the gene for LDL receptor-related protein (LRP) on risk of coronary artery disease.

    Pocathikorn A, Granath B, Thiry E, Van Leuven F, Taylor R and Mamotte C

    Department of Clinical Immunology and Biochemical Genetics, Royal Perth Hospital, GPO Box X2213, WA, Australia.

    The low density lipoprotein (LDL) receptor-related protein (LRP) is a multifunctional receptor involved in numerous biological processes relevant to vascular biology including lipoprotein metabolism. Several polymorphisms in the LRP gene have been described and in this study we examined their influence on coronary artery disease (CAD). We compared the frequencies of the exon 3 (C766T), exon 6 (C663T), exon 22 (C200T), and four rarer and more recently described polymorphisms in approximately 600 Caucasian subjects aged <50 years with angiographic CAD and approximately 700 similarly aged subjects without symptomatic CAD randomly selected from the community. We found the distribution of exon 22 C200T genotypes to differ significantly between the CAD (CC: 52%, CT: 39%, TT: 9%) and control subjects (CC: 43%, CT: 46%, TT: 11%, P=0.005), with the CC genotype conferring an odds ratio (OR) for CAD of 1.5 (95% CI: 1.2-1.8, P=0.001) despite a lack of significant influence on plasma cholesterol or triglyceride. The other LRP polymorphisms were less common. Two showed an association with CAD; for the exon 3 C766T polymorphism the TT genotype was significantly lower (1.0 vs. 2.7%; OR: 0.36; P=0.04) and, for the exon 6 C663T polymorphism, the heterozygote frequency was higher (6.2 vs. 3.4%; OR: 1.9; P=0.03) in CAD subjects. In conclusion, LRP gene polymorphisms, particularly the relatively common exon 22 C200T polymorphism, are a significant risk factor for premature CAD in Caucasians.

    Atherosclerosis 2003;168;1;115-21

  • LDL receptor-related proteins in neurodevelopment.

    May P and Herz J

    Department of Molecular Genetics, University of Texas Southwestern Medical Center, Dallas, TX 75390-9046, USA.

    Low-density lipoprotein receptor-related proteins (LRPs) are evolutionarily ancient cell-surface receptors with diverse biological functions. All are expressed in the central nervous system and, for most receptors, animal models have shown that they are indispensable for successful neurodevelopment. The mechanisms by which they regulate the formation of the nervous system are varied and include the transduction of extracellular signals and the modulation of intracellular signal propagation, as well as cargo transport, the function most commonly attributed to this gene family. Here, we will summarize recent advances in our understanding of the molecular basis on which these receptors function during development.

    Funded by: NHLBI NIH HHS: R37 HL063762

    Traffic (Copenhagen, Denmark) 2003;4;5;291-301

  • Low density lipoprotein receptor-related protein and factor IXa share structural requirements for binding to the A3 domain of coagulation factor VIII.

    Bovenschen N, Boertjes RC, van Stempvoort G, Voorberg J, Lenting PJ, Meijer AB and Mertens K

    Department of Plasma Proteins, Sanquin Research at CLB, 1066 CX Amsterdam, The Netherlands.

    Low-density lipoprotein receptor-related protein (LRP) is an endocytic receptor that binds multiple distinct ligands, including blood coagulation factor VIII (FVIII). FVIII is a heterodimeric multidomain protein that consists of a heavy chain (domains A1, a1, A2, a2, and B) and a light chain (domains a3, A3, C1, and C2). Both chains contribute to high-affinity interaction with LRP. One LRP-interactive region has previously been located in the C2 domain, but its affinity is low in comparison with that of the entire FVIII light chain. We now have compared a variety of FVIII light chain derivatives with the light chain of its homolog FVa for LRP binding. In surface plasmon resonance studies employing LRP cluster II, the FVa and FVIII light chains proved different in that only FVIII displayed high-affinity binding. Because the FVIII a3-A3-C1 fragment was effective in associating with LRP, this region was explored for structural elements that are exposed but not conserved in FV. Competition studies using synthetic peptides suggested that LRP binding involves the FVIII-specific region Lys(1804)-Ala(1834) in the A3 domain. In line with this observation, LRP binding was inhibited by a recombinant antibody fragment that specifically binds to the FVIII sequence Glu(1811)-Lys(1818). The role of this sequence in LRP binding was further tested using a FVIII/FV chimera in which sequence Glu(1811)-Lys(1818) was replaced with the corresponding sequence of FV. Although this chimera still displayed residual binding to LRP cluster II, its affinity was reduced. This suggests that multiple sites in FVIII contribute to high-affinity LRP binding, one of which is the FVIII A3 domain region Glu(1811)-Lys(1818). This suggests that LRP binding to the FVIII A3 domain involves the same structural elements that also contribute to the assembly of FVIII with FIXa.

    The Journal of biological chemistry 2003;278;11;9370-7

  • Residues Phe342-Asn346 of activated coagulation factor IX contribute to the interaction with low density lipoprotein receptor-related protein.

    Rohlena J, Kolkman JA, Boertjes RC, Mertens K and Lenting PJ

    Department of Plasma Proteins, Sanquin Research at CLB, 1066 CX Amsterdam, The Netherlands.

    When blood coagulation factor IX is converted to activated factor IX (factor IXa), it develops enzymatic activity and exposes the binding sites for both activated factor VIII and the endocytic receptor low density lipoprotein receptor-related protein (LRP). In the present study we investigated the interaction between factor IXa and LRP in more detail, using an affinity-purified soluble form of LRP (sLRP). Purified sLRP and full-length LRP displayed similar binding to factor IXa. An anti-factor IX monoclonal antibody CLB-FIX 13 inhibited factor IXa.sLRP complex formation. Both the antibody and a soluble recombinant fragment of LRP (i.e. cluster IV) interfered with factor IXa amidolytic activity, suggesting that the antibody and LRP share similar binding regions near the active site of factor IXa. Next, a panel of recombinant factor IXa variants with amino acid replacements in the surface loops bordering the active site was tested for binding to antibody CLB-FIX 13 and sLRP in a solid phase binding assay. Factor IXa variants with mutations in the region Phe(342)-Asn(346), located between the active site of factor IXa and factor VIII binding helix, showed reduced binding to both antibody CLB-FIX 13 and sLRP. Surface plasmon resonance analysis revealed that the variant with Asn(346) replaced by Asp displayed slower association to sLRP, whereas the variant with residues Phe(342)-Tyr(345) replaced by the corresponding residues of thrombin showed faster dissociation. Recombinant soluble LRP fragment cluster IV inhibited factor IXa-mediated activation of factor X with IC(50) values of 5 and 40 nm in the presence and absence of factor VIII, respectively. This inhibition thus seems to occur via two mechanisms: by interference with factor IXa.factor VIIIa complex assembly and by direct inhibition of factor IXa enzymatic activity. Accordingly, we propose that LRP may function as a regulator of blood coagulation.

    The Journal of biological chemistry 2003;278;11;9394-401

  • Association of the 3' UTR transcription factor LBP-1c/CP2/LSF polymorphism with late-onset Alzheimer's disease.

    Luedecking-Zimmer E, DeKosky ST, Nebes R and Kamboh MI

    Department of Human Genetics, University of Pittsburgh, Pittsburgh, Pennsylvania 15261, USA.

    Alzheimer's disease (AD) is a genetically heterogeneous neurodegenerative disorder. To date, apolipoprotein E (apoE) is the only established susceptibility gene for late-onset AD. ApoE accounts for less than 50% of the risk of AD, indicating the presence of other unknown susceptibility loci. Linkage studies have indicated chromosome 12 as the most likely location for another late-onset AD locus. We examined seven polymorphisms in five candidate genes located in and around the linkage peaks on chromosome 12 in 564 cases and 523 controls. The genes included complement component 1R (C1R), vitamin D receptor (VDR), scavenger-receptor B1 (SR-B1), low-density lipoprotein receptor related protein 1 (LRP1), and transcription factor LBP-1c/CP2/LSF. We found no association with C1R, VDR, SR-B1, and LRP1 polymorphisms. However, the frequency of the A allele of the 3' (untranslated region) UTR LBP-1c/CP2/LSF polymorphism was higher in controls than cases (0.071 vs. 0.051; P = 0.042) with an adjusted odds ratio (OR) of 0.65 (95% confidence interval [CI]: 0.43-0.96; P = 0.0498). Our data suggest that the LBP-1c/CP2/LSF polymorphism may have a moderate protective effect against the risk of AD.

    Funded by: NIA NIH HHS: AG 05133, AG 07562, AG 13672, AG 14051

    American journal of medical genetics. Part B, Neuropsychiatric genetics : the official publication of the International Society of Psychiatric Genetics 2003;117B;1;114-7

  • C766T low-density lipoprotein receptor-related protein 1 (LRP1) gene polymorphism and susceptibility to breast cancer.

    Benes P, Jurajda M, Zaloudík J, Izakovicová-Hollá L and Vácha J

    Department of Molecular Biology and Genetics, Faculty of Science, Masaryk University, Brno, Czech Republic.

    Background: Low-density lipoprotein receptor-related protein 1 (LRP1) is a multifunctional endocytic receptor with an important role in regulating the activity of proteinases in extracellular matrix. Several studies have also described its role in intracellular signaling. Previous studies showed that the expression of LRP1 is related to invasiveness of cancer cells. However, recent data on LRP1 suggest that this receptor can also be involved in tumor establishment and progression.

    Methods: We investigated an association between the C766T polymorphism of the third exon of the LRP1 gene and breast cancer in a sample of women of Caucasian origin. Allele and genotype frequencies of this polymorphism were assessed in 164 women with breast cancer and in 183 age-compatible women without a history of any cancer disease.

    Results: An increase in LRP1 T allele frequency in subjects with breast cancer was observed compared with controls (0.21 versus 0.15, P = 0.01963). A significant excess of genotypes with the T allele (homozygotes plus heterozygotes) was also observed (odds ratio 1.743, 95% confidence interval 1.112-2.732).

    Conclusion: The T allele of the C766T polymorphism in the LRP1 gene is associated with an increased risk of breast cancer development in women of Caucasian origin.

    Breast cancer research : BCR 2003;5;3;R77-81

  • Expression and regulation by insulin of low-density lipoprotein receptor-related protein mRNA in human skeletal muscle.

    Boucher P, Ducluzeau PH, Davelu P, Andreelli F, Vallier P, Riou JP, Laville M and Vidal H

    Department of Biochemistry, E. Herriot Hospital, Lyons, France.

    Evidence suggests that increased hydrolysis and/or uptake of triglyceride-rich lipoprotein particles in skeletal muscle can be involved in insulin resistance. We determined the steady state mRNA levels of the low-density lipoprotein-related receptor (LRP) and lipoprotein lipase (LPL) in skeletal muscle of eight healthy lean control subjects, eight type 2 diabetic patients and eight nondiabetic obese individuals. The regulation by insulin of LRP and LPL mRNA expression was also investigated in biopsies taken before and at the end of a 3 h euglycemic hyperinsulinemic clamp (insulinemia of about 1 nM). LRP mRNA was expressed in human skeletal muscle (1.3+/-0.1 amol/microg total RNA in control subjects). Type 2 diabetic patients, but not nondiabetic obese subjects, were characterized by a reduced expression of LRP (0.8+/-0.2 and 1.3+/-0.3 amol/microg total RNA in diabetic and obese patients, respectively; P<0.05 in diabetic vs. control subjects). Insulin infusion decreased LRP mRNA levels in muscle of the control subjects but not in muscle of type 2 diabetic and nondiabetic obese patients. Similar results were found when investigating the regulation of the expression of LPL. Taken together, these results did not support the hypothesis that a higher capacity for clearance or hydrolysis of circulating triglycerides in skeletal muscle is present during obesity- or type 2 diabetes-associated insulin resistance.

    Biochimica et biophysica acta 2002;1588;3;226-31

  • Low-density lipoprotein upregulates low-density lipoprotein receptor-related protein expression in vascular smooth muscle cells: possible involvement of sterol regulatory element binding protein-2-dependent mechanism.

    Llorente-Cortés V, Otero-Viñas M, Sánchez S, Rodríguez C and Badimon L

    Cardiovascular Research Center, IICB (CSIC)-ICCC, Barcelona. Spain.

    Background: Low-density lipoprotein (LDL) receptor-related protein (LRP) is highly expressed in vascular smooth muscle cells (VSMCs) of both normal and atherosclerotic lesions. However, little is known about LRP regulation in the vascular wall.

    We analyzed the regulation of LRP expression in vitro in human VSMCs cultured with native LDL (nLDL) or aggregated LDL (agLDL) by semiquantitative reverse transcriptase-polymerase chain reaction, real-time polymerase chain reaction, and Western blot and in vivo during diet-induced hypercholesterolemia by in situ hybridization. LRP expression in human VSMCs is increased by nLDL and agLDL in a time- and dose-dependent manner. Maximal induction of LRP mRNA expression was observed after 24 hours of exposure to LDL. However, agLDL induced higher LRP mRNA expression (3.0-fold) than nLDL (1.76-fold). LRP mRNA upregulation was associated with an increase on LRP protein expression with the greatest induction by agLDL. VSMC-LRP upregulation induced by nLDL or agLDL was reduced by an inhibitor of sterol regulatory element binding protein (SREBP) catabolism (N-acetyl-leucyl-leucyl-norleucinal). In situ hybridization analysis indicates that there is a higher VSMC-LRP expression in hypercholesterolemic than in normocholesterolemic pig aortas.

    Conclusions: These results indicate that LRP expression in VSMCs is upregulated by intravascular and systemic LDL.

    Circulation 2002;106;24;3104-10

  • The role of Grp 78 in alpha 2-macroglobulin-induced signal transduction. Evidence from RNA interference that the low density lipoprotein receptor-related protein is associated with, but not necessary for, GRP 78-mediated signal transduction.

    Misra UK, Gonzalez-Gronow M, Gawdi G, Hart JP, Johnson CE and Pizzo SV

    Department of Pathology, Duke University Medical Center, Durham, North Carolina 27710, USA.

    The low density lipoprotein receptor-related protein (LRP) is a scavenger receptor that binds to many proteins, some of which trigger signal transduction. Receptor-recognized forms of alpha(2)-Macroglobulin (alpha(2)M*) bind to LRP, but the pattern of signal transduction differs significantly from that observed with other LRP ligands. For example, neither Ni(2+) nor the receptor-associated protein, which blocks binding of all known ligands to LRP, block alpha(2)M*-induced signal transduction. In the current study, we employed alpha(2)-macroglobulin (alpha(2)M)-agarose column chromatography to purify cell surface membrane binding proteins from 1-LN human prostate cancer cells and murine macrophages. The predominant binding protein purified from 1-LN prostate cancer cells was Grp 78 with small amounts of LRP, a fact that is consistent with our previous observations that there is little LRP present on the surface of these cells. The ratio of LRP:Grp 78 is much higher in macrophages. Flow cytometry was employed to demonstrate the presence of Grp 78 on the cell surface of 1-LN cells. Purified Grp 78 binds to alpha(2)M* with high affinity (K(d) approximately 150 pm). A monoclonal antibody directed against Grp 78 both abolished alpha(2)M*-induced signal transduction and co-precipitated LRP. Ligand blotting with alpha(2)M* showed binding to both Grp 78 and LRP heavy chains in these preparations. Use of RNA interference to silence LRP expression had no effect on alpha(2)M*-mediated signaling. We conclude that Grp 78 is essential for alpha(2)M*-induced signal transduction and that a "co-receptor" relationship exists with LRP like that seen with several other ligands and receptors such as the uPA/uPAR (urinary type plasminogen activator or urokinase/uPA receptor) system.

    The Journal of biological chemistry 2002;277;44;42082-7

  • The LDL receptor-related protein (LRP1/A2MR) and coronary atherosclerosis--novel genomic variants and functional consequences.

    Schulz S, Schagdarsurengin U, Greiser P, Birkenmeier G, Müller-Werdan U, Hagemann M, Riemann D, Werdan K and Gläser C

    Institute of Human Genetics and Medical Biology, University of Halle, Halle, Germany.

    The LDL receptor-related protein/alpha 2-macroglobulin receptor (LRP1/A2MR) is a multifunctional cell-surface glycoprotein that endocytoses several structurally and functionally distinct ligands. In clinical studies different genomic variants of the LRP1/A2MR and its role in the development of degenerative diseases like atherosclerosis or Alzheimer's disease were studied. We screened for novel genomic variants of LRP1/A2MR and investigated the importance of these variants in 214 coronary patients suffering from myocardial infarction as well as in 224 healthy controls. We detected a novel C>G polymorphism at position -25 in the functionally important promoter region of LRP1/A2MR. This polymorphism (c.1-25C>G) leads to the creation of a new GC-box, recognized by the constitutively expressed SP 1 transcription factor. Investigating the LRP1/A2MR gene expression with respect to this polymorphism, carriers of the mutant G-allele were found to have a higher mRNA expression level. A novel polymorphism in exon 22 (c.4012C>T), and two novel polymorphisms in intron 24 (IVS24+123C>A and IVS24+690G>A) associated with a previously described polymorphism in exon 61 (c.10249G>A), were related to the development of myocardial infarction. Two novel rare genetic variants of exon 88 (c.13933C>T) and intron 88 (IVS88+15G>A) were identified in four patients with severe coronary symptoms. However, the LRP1/A2MR gene expression was found to be independent of all identified novel genomic variants as well as other previously described changes (A217V, A775P, D2080N, D2632E, G4379S) except the promoter polymorphism.

    Human mutation 2002;20;5;404

  • Cellular catabolism of lipid poor apolipoprotein E via cell surface LDL receptor-related protein.

    Narita M, Holtzman DM, Fagan AM, LaDu MJ, Yu L, Han X, Gross RW, Bu G and Schwartz AL

    Department of Pediatrics, Washington University School of Medicine and St. Louis Children's Hospital, St. Louis, Missouri 63110, USA. narita_m@md.tsukuba.ac.jp

    Apolipoprotein E (apoE), an apoprotein involved in lipid transport in both the plasma and within the brain, mediates the binding of lipoproteins to members of the low density lipoprotein (LDL) receptor family including the LDL receptor and the LDL receptor-related protein (LRP). ApoE/LRP interactions may be particularly important in brain where both are expressed at high levels, and polymorphisms in the apoE and LRP genes have been linked to AD. To date, only apoE-enriched lipoproteins have been shown to be LRP ligands. To investigate further whether other, more lipid-poor forms of apoE interact with LRP, we tested whether lipid-free apoE in the absence of lipoprotein particles interacts with its cell-surface receptors. No detectable lipid was found associated with bacterially expressed and purified apoE either prior to or following incubation with cells when analyzed by electrospray ionization mass spectrometry. We found that the degradation of lipid-poor (125)I-apoE was significantly higher in wild type as compared to LRP-deficient cells, and was inhibited by receptor-associated protein (RAP). In contrast, (125)I-apoE-enriched beta-VLDL was degraded by both LRP and the LDL receptor. When analyzed via a single cycle of endocytosis, (125)I-apoE was internalized prior to its subsequent intracellular degradation with kinetics typical of receptor-mediated endocytosis. Thus, we conclude that a very lipid-poor form of apoE can be catabolized via cell surface LRP, suggesting that the conformation of apoE necessary for recognition by LRP can be imposed by situations other than an apoE-enriched lipoprotein.

    Funded by: NIA NIH HHS: AG05681, AG13956; NINDS NIH HHS: NS37525

    Journal of biochemistry 2002;132;5;743-9

  • The PX-domain protein SNX17 interacts with members of the LDL receptor family and modulates endocytosis of the LDL receptor.

    Stockinger W, Sailler B, Strasser V, Recheis B, Fasching D, Kahr L, Schneider WJ and Nimpf J

    The Institute of Medical Biochemistry, Department of Molecular Genetics, BioCenter and University of Vienna, A-1030 Vienna, Austria.

    Sorting nexins (SNXs) comprise a family of proteins characterized by the presence of a phox-homology domain, which mediates the association of these proteins with phosphoinositides and recruits them to specific membranes or vesicular structures within cells. Although only limited information about SNXs and their functions is available, they seem to be involved in membrane trafficking and sorting processes by directly binding to target proteins such as certain growth factor receptors. We show that SNX17 binds to the intracellular domain of some members of the low-density lipoprotein receptor (LDLR) family such as LDLR, VLDLR, ApoER2 and LDLR-related protein. SNX17 resides on distinct vesicular structures partially overlapping with endosomal compartments characterized by the presence of EEA1 and rab4. Using rhodamine-labeled LDL, it was possible to demonstrate that during endocytosis, LDL passes through SNX17-positive compartments. Functional studies on the LDLR pathway showed that SNX17 enhances the endocytosis rate of this receptor. Our results identify SNX17 as a novel adaptor protein for LDLR family members and define a novel mechanism for modulation of their endocytic activity.

    The EMBO journal 2002;21;16;4259-67

  • The role of the low-density lipoprotein receptor-related protein (LRP1) in Alzheimer's A beta generation: development of a cell-based model system.

    Goto JJ and Tanzi RE

    Genetics and Aging Research Unit, Center for Aging, Genetics and Neurodegeneration, Boston, MA 02129, USA.

    The clearance and degradation of extracellular A beta is critical for regulating beta-amyloid deposition, a major hallmark of brains of patients with A beta in Alzheimer's Disease. The low-density lipoprotein receptor-related protein, LRP1, is a large endocytic receptor that significantly contributes to the balance between degradation and production of A beta. An extracellular portion of the LRP, known as the cluster II region can bind to the secreted form of APP (sAPP-KPI). We show here that a GST fusion protein containing the cluster II region of LRP can be used as a 'mini-receptor' that specifically binds to sAPP-KPI from conditioned cultured medium. The binding between the GST-LRP-cluster II fusion protein and sAPP-KPI can be inhibited with the strong binding ligand of LRP1, called receptor-associated protein (RAP). Furthermore, a cell-based in vitro assay system has been developed to monitor the production of total A beta and A beta(1-42) in the presence and absence of RAP in Chinese hamster ovary (CHO) cell lines both deficient in LRP and expressing LRP. A 3-day treatment of the L2 (CHO cells deficient in LRP and overexpressing APP751) and L3 (CHO cells expressing LRP and overexpressing APP751) cell lines with RAP showed a decrease in total A beta and, interestingly, also a decrease in the ratio of A beta42/A beta(total). This cell-based model system and LRP-cluster II mini-receptor will be very useful for screening novel compounds that can reduce A beta accumulation by inhibiting binding of APP-KPI to LRP1.

    Journal of molecular neuroscience : MN 2002;19;1-2;37-41

  • Variation in the lipoprotein receptor-related protein, alpha2-macroglobulin and lipoprotein receptor-associated protein genes in relation to plasma lipid levels and risk of early myocardial infarction.

    González P, Alvarez R, Reguero JR, Batalla A, Alvarez V, Cortina A, Cubero GI, García-Castro M and Coto E

    Laboratorio de Genética Molecular-Instituto de Investigación Nefrológica (IRSIN-FRIAT), Hospital Central Asturias, Oviedo, Spain.

    Background: The lipoprotein receptor-related protein (LRP) is an endocytic receptor for several ligands, such as alpha2-macroglobulin (alpha2 M) and apolipoprotein E. LRP is involved in the clearance of lipids from the bloodstream and is expressed in the atherosclerotic plaque. The LRP-associated protein (LRPAP in humans, RAP in mice) acts as a chaperone protein, stabilizing the nascent LRP peptide in the endoplasmic reticulum and Golgi complex. In mice, the amount of LRP activity was modulated by RAP, and RAP-null mice showed higher levels of total cholesterol.

    Objective: To evaluate the association between DNA polymorphisms at the LRP, LRPAP and alpha2 M genes and early myocardial infarction (MI).

    Methods: We genotyped 210 patients with early MI (<55 years) and 200 healthy control participants for three polymorphisms in the LRP, LRPAP and alpha2 M genes.

    Results: No association was found between these polymorphisms and plasma lipid levels in patients and control participants. Only the LRPAP-intron 1 polymorphism (a 21 bp insertion/deletion) was associated with MI (P = 0.0065; odds ratio = 2.18, 95% confidence intervals = 1.22-3.90).

    Conclusions: According to our data, the variation at the LRPAP1 gene could contribute to the risk of developing an early episode of MI.

    Coronary artery disease 2002;13;5;251-4

  • Proteolytic processing of low density lipoprotein receptor-related protein mediates regulated release of its intracellular domain.

    May P, Reddy YK and Herz J

    Department of Molecular Genetics, University of Texas Southwestern Medical Center, Dallas, Texas 75390, USA.

    The low density lipoprotein (LDL) receptor-related protein (LRP) is a multifunctional cell surface receptor that interacts through its cytoplasmic tail with adaptor and scaffold proteins that participate in cellular signaling. Its extracellular domain, like that of the signaling receptor Notch and of amyloid precursor protein (APP), is proteolytically processed at multiple positions. This similarity led us to investigate whether LRP, like APP and Notch, might also be cleaved at a third, intramembranous or cytoplasmic site, resulting in the release of its intracellular domain. Using independent experimental approaches we demonstrate that the cytoplasmic domain is released by a gamma-secretase-like activity and that this event is modulated by protein kinase C. Furthermore, cytoplasmic adaptor proteins that bind to the LRP tail affect the subcellular localization of the free intracellular domain and may regulate putative signaling functions. Finally, we show that the degradation of the free tail fragment is mediated by the proteasome. These findings suggest a novel role for the intracellular domain of LRP that may involve the subcellular translocation of preassembled signaling complexes from the plasma membrane.

    Funded by: NHLBI NIH HHS: HL20948, HL63762; NINDS NIH HHS: NS43408

    The Journal of biological chemistry 2002;277;21;18736-43

  • The low density lipoprotein receptor-related protein mediates fibronectin catabolism and inhibits fibronectin accumulation on cell surfaces.

    Salicioni AM, Mizelle KS, Loukinova E, Mikhailenko I, Strickland DK and Gonias SL

    Department of Pathology, University of Virginia School of Medicine, Charlottesville, Virginia 22908, USA.

    Low density lipoprotein receptor-related protein (LRP) is a member of the low density lipoprotein receptor family, which functions as an endocytic receptor for diverse ligands. In this study, we demonstrate that murine embryonic fibroblasts (MEF-2 cells) and 13-5-1 Chinese hamster ovary cells, which are LRP-deficient, accumulate greatly increased levels of cell-surface fibronectin (Fn), compared with LRP-expressing MEF-1 and CHO-K1 cells. Increased Fn was also detected in conditioned medium from LRP-deficient MEF-2 cells; however, biosynthesis of Fn by MEF-1 and MEF-2 cells was not significantly different. When LRP-deficient cells were dissociated from monolayer culture, increased levels of Fn remained with the cells, as determined by cell-surface protein biotinylation, suggesting an intimate relationship with cell surface-binding sites. The LRP antagonist, receptor-associated protein (RAP), promoted Fn accumulation in association with MEF-1 cells, whereas expression of full-length LRP in MEF-2 cells substantially decreased Fn accumulation, confirming the role of LRP in this process. Purified LRP bound directly to immobilized Fn, and this interaction was inhibited by RAP. Furthermore, MEF-1 cells degraded (125)I-Fn at an increased rate, compared with MEF-2 cells. (125)I-Fn degradation by MEF-1 cells was inhibited by RAP. These results demonstrate that LRP functions as a catabolic receptor for Fn. The function of LRP in Fn degradation and the ability of LRP to regulate levels of other plasma membrane proteins represent possible mechanisms whereby LRP prevents Fn accumulation on cell surfaces.

    Funded by: NHLBI NIH HHS: HL50784, HL54710, HL60551

    The Journal of biological chemistry 2002;277;18;16160-6

  • Platelet-derived growth factor (PDGF)-induced tyrosine phosphorylation of the low density lipoprotein receptor-related protein (LRP). Evidence for integrated co-receptor function betwenn LRP and the PDGF.

    Loukinova E, Ranganathan S, Kuznetsov S, Gorlatova N, Migliorini MM, Loukinov D, Ulery PG, Mikhailenko I, Lawrence DA and Strickland DK

    Department of Vascular Biology, Holland Laboratory, American Red Cross, Rockville, Maryland 20855, USA.

    The low density lipoprotein receptor-related protein (LRP) functions in the catabolism of numerous ligands including proteinases, proteinase inhibitor complexes, and lipoproteins. In the current study we provide evidence indicating an expanded role for LRP in modulating cellular signaling events. Our results show that platelet-derived growth factor (PDGF) BB induces a transient tyrosine phosphorylation of the LRP cytoplasmic domain in a process dependent on PDGF receptor activation and c-Src family kinase activity. Other growth factors, including basic fibroblast growth factor, epidermal growth factor, insulin-like growth factor-1, were unable to mediate tyrosine phosphorylation of LRP. The basis for this selectivity may result from the ability of LRP to bind PDGFBB, because surface plasmon resonance experiments demonstrated that only PDGF, and not basic fibroblast growth factor, epidermal growth factor, or insulin-like growth factor-1, bound to purified LRP immobilized on a sensor chip. The use of LRP mini-receptor mutants as well as in vitro phosphorylation studies demonstrated that the tyrosine located within the second NPXY motif found in the LRP cytoplasmic domain is the primary site of tyrosine phosphorylation by Src and Src family kinases. Co-immunoprecipitation experiments revealed that PDGF-mediated tyrosine phosphorylation of LRPs cytoplasmic domain results in increased association of the adaptor protein Shc with LRP and that Shc recognizes the second NPXY motif within LRPs cytoplasmic domain. In the accompanying paper, Boucher et al. (Boucher, P., Liu, P. V., Gotthardt, M., Hiesberger, T., Anderson, R. G. W., and Herz, J. (2002) J. Biol. Chem. 275, 15507-15513) reveal that LRP is found in caveolae along with the PDGF receptor. Together, these studies suggest that LRP functions as a co-receptor that modulates signal transduction pathways initiated by the PDGF receptor.

    Funded by: NHLBI NIH HHS: HL50784, HL54710

    The Journal of biological chemistry 2002;277;18;15499-506

  • Phosphorylation of LRP1: regulation of transport and signal transduction.

    van der Geer P

    Department of Chemistry and Biochemistry, University of California San Diego, La Jolla 92093-0601, USA. geer@ucsd.edu

    Low-density lipoprotein receptor-related protein 1 (LRP1) is a member of the low-density lipoprotein receptor family. Members of this family were once thought to be involved exclusively in receptor-mediated uptake of extracellular molecules, including lipoproteins and proteases. This article reviews recent work that indicates that LRP1 is phosphorylated on both serine and tyrosine residues. Tyrosine-phosphorylated LRP1 is specifically associated with the cellular docking protein Shc. The results suggest that ligand internalization by LRP1 is regulated by phosphorylation. In addition, LRP1 is now, like several of its close relatives, implicated in signal transduction.

    Funded by: NCI NIH HHS: 1R29 CA78629

    Trends in cardiovascular medicine 2002;12;4;160-5

  • Distinct binding sites in the structure of alpha 2-macroglobulin mediate the interaction with beta-amyloid peptide and growth factors.

    Mettenburg JM, Webb DJ and Gonias SL

    Department of Biochemistry and Molecular Genetics, University of Virginia School of Medicine, Charlottesville, VA 22908, USA.

    Alpha(2)-macroglobulin (alpha(2)M) and its receptor, low density lipoprotein receptor-related protein (LRP), function together to facilitate the cellular uptake and degradation of beta-amyloid peptide (Abeta). In this study, we demonstrate that Abeta binds selectively to alpha(2)M that has been induced to undergo conformational change by reaction with methylamine. Denatured alpha(2)M subunits, which were immobilized on polyvinylidene difluoride membranes, bound Abeta, suggesting that alpha(2)M tertiary and quaternary structure are not necessary. To determine whether a specific sequence in alpha(2)M is responsible for Abeta binding, we prepared and analyzed defined alpha(2)M fragments and glutathione S-transferase-alpha(2)M peptide fusion proteins. A single sequence, centered at amino acids (aa) 1314-1365, was identified as the only major Abeta-binding site. Importantly, Abeta did not bind to the previously characterized growth factor-binding site (aa 718-734). Although the Abeta binding sequence is adjacent to the binding site for LRP, the results of experiments with mutated fusion proteins indicate that the two sites are distinct. Furthermore, a saturating concentration of Abeta did not inhibit LRP-mediated clearance of alpha(2)M-MA in mice. Using various methods, we determined that the K(D) for the interaction of Abeta with its binding site in the individual alpha(2)M subunit is 0.7-2.4 microm. The capacity of alpha(2)M to bind Abeta and deliver it to LRP may be greater than that predicted by the K(D), because each alpha(2)M subunit may bind Abeta and the bound Abeta may multimerize. These studies suggest a model in which alpha(2)M has three protein interaction sites with distinct specificities, mediating the interaction with Abeta, growth factors, and LRP.

    Funded by: NCI NIH HHS: CA-53462

    The Journal of biological chemistry 2002;277;15;13338-45

  • Interaction of CED-6/GULP, an adapter protein involved in engulfment of apoptotic cells with CED-1 and CD91/low density lipoprotein receptor-related protein (LRP).

    Su HP, Nakada-Tsukui K, Tosello-Trampont AC, Li Y, Bu G, Henson PM and Ravichandran KS

    Beirne Carter Center for Immunology Research and the Department of Microbiology, University of Virginia, Charlottesville, Virginia 22908, USA.

    The prompt clearance of cells undergoing apoptosis is critical during embryonic development, normal tissue turnover, as well as inflammation and autoimmunity. The molecular details of the engulfment of apoptotic cells are not fully understood. ced-6 and its human homologue gulp, encode an adapter protein, whose function in engulfment is highly evolutionarily conserved; however, the upstream and downstream components of CED-6 mediated signaling are not known. Recently, ced-1 has been shown to encode a transmembrane protein on phagocytic cells, with two functional sequence motifs in its cytoplasmic tail that are important for engulfment. In this study, using a combination of biochemical approaches and yeast two-hybrid analysis, we present evidence for a physical interaction between GULP/CED-6 and one of the two motifs (NPXY motif) in the cytoplasmic tail of CED-1. The phosphotyrosine binding domain of GULP was necessary and sufficient for this interaction. Since the precise mammalian homologue of CED-1 is not known, we undertook a database search for human proteins that contain the motifs shown to be important for CED-1 function and identified CD91/LRP (low density lipoprotein receptor-related protein) as one candidate. Interestingly, recent studies have also identified CD91/LRP as a receptor involved in the phagocytosis of apoptotic cells in mammals. The GULP phosphotyrosine binding domain was able to specifically interact with one specific NPXY motif in the CD91 cytoplasmic tail. During these studies we have also identified the mouse GULP sequence. These studies suggest a physical link between CED-1 or CD91/LRP and the adapter protein CED-6/GULP during engulfment of apoptotic cells and further elucidate the pathway suggested by the genetic studies.

    The Journal of biological chemistry 2002;277;14;11772-9

  • [Properties and expression of the human activated alpha2-macroglobulin receptor].

    Zhloba AA and Ivanova SIu

    A multi-step purification procedure has been used for isolation of alpha 2M/LRP receptor from human placenta. Enzymatic properties of complexes of purified alpha 2M trypsin with alpha 2M/LRP receptor were studied. This complex is characterized by enzymatic activity measured by a kinetic procedure based on the trypsin reaction with synthetic substrate alpha 2N-benzoyl-D,L-arginine nitroparaanilide. Purified triple complexes of alpha 2M trypsin with alpha 2M/LRP had 22-28% residual trypsin activity, while that of trypsin complex with alpha 2-macroglobulin was 52-53%. Patients' blood samples were characterized by a high level of residual activity of the studied double and triple complexes only initially. A decrease in the activities of these complexes was observed in patients with high levels of malonic dialdehyde (by TBA-reactive products) combined with decreased level of superoxidedismutase-like activity.

    Klinicheskaia laboratornaia diagnostika 2002;4;7-11

  • Recombinant von Willebrand factor-insight into structure and function through infusion studies in animals with severe von Willebrand disease.

    Schwarz HP, Schlokat U, Mitterer A, Váradi K, Gritsch H, Muchitsch EM, Auer W, Pichler L, Dorner F and Turecek PL

    Baxter BioScience, Vienna, Austria. schwarh@baxter.com

    We used a canine and a murine model of von Willebrand disease (vWD) to study the in vivo effects of recombinant von Willebrand factor (vWF). Two preparations were used: (1) a fully processed mature vWF; this was achieved by coexpression of furin. (2) A preparation containing unprocessed pro-vWF, the propeptide still covalently linked to mature vWF. Both preparations induced an increase in canine and murine factor VIII:C (FVIII), which was sustained even when vWF antigen had been removed from the circulation. vWF multimers were analyzed in the plasma samples after infusion using ultra high-resolution 3% agarose gels to allow the separation of homoforms and heteroforms of the vWF polymers. Administration of pro-vWF to dogs with severe vWD resulted in the removal of the propeptide and maturation of vWF in the circulation, indicating that the propeptide cleavage from unprocessed vWF can occur extracellularly. This suggests that the vWF propeptide, besides being derived from the Weibel-Palade bodies of endothelial cells after stimulation, can also be cleaved by pro-vWF in plasma. Using a murine model of vWD, the involvement of the low-density lipoprotein receptor-related protein (LRP) in the clearance of FVIII was established. The low levels of FVIII observed in the absence of vWF are due to an enhanced clearance of FVIII by binding to LRP and removal from the circulation through endocytosis. Administration of the receptor-associated protein (RAP) as a recombinant fusion protein to vWF knockout mice significantly improved the in vivo recovery of recombinant FVIII and the survival time of otherwise rapidly cleared FVIII.

    Seminars in thrombosis and hemostasis 2002;28;2;215-26

  • An integrated view of the roles and mechanisms of heat shock protein gp96-peptide complex in eliciting immune response.

    Li Z, Dai J, Zheng H, Liu B and Caudill M

    Center for Immunotherapy of Cancer and Infectious Diseases, University of Connecticut School of Medicine, Farmington, CT 06030-1601, USA. zli@up.uchc.edu

    Heat shock protein (HSP) gp96, or grp94 is an endoplasmic reticular (ER) paralog of the cytosolic HSP90. Being abundant and non-polymorphic, gp96 plays significant roles in maintaining protein homeostasis in the secretory pathway. This "house-keeping" role of gp96 has now been overshadowed by the intriguing findings that gp96 modulates both the innate and adaptive components of the immune system. It has been found that, (i) gp96 is one of the major peptide binding proteins in the ER, (ii) gp96 interacts specifically with receptors including CD91 and possibly toll-like receptors (TLRs), on the surface of professional antigen presenting cells (APCs), (iii) interaction with APCs leads to re-presentation of gp96-chaperoned peptides to the major histocompatibility complex (MHC) molecules of APCs, (iv) direct access of gp96 to APCs triggers functional activation of APCs. In this review, we will examine each of these immunological attributes of gp96 critically. As experimentalists, we will also propose specific experiments to examine the argument that gp96, perhaps along with other members of HSP family, is the antigenic carrier for mediating cross priming of antigen-specific T lymphocytes in vertebrates.

    Frontiers in bioscience : a journal and virtual library 2002;7;d731-51

  • The relationship among apolipoprotein(a) polymorphisms, the low-density lipoprotein receptor-related protein, and the very low density lipoprotein receptor genes, and plasma lipoprotein(A) concentration in the Czech population.

    Benes P, Muzík J, Benedík J, Znojil V and Vácha J

    Department of Pathological Physiology, Faculty of Medicine, Masaryk University, Brno, Czech Republic.

    Increased plasma concentration of lipoprotein(a) [Lp(a)] is an established independent risk factor for coronary artery disease (CAD), which is strongly genetically determined. This study was designed to investigate the relationship between the K-IV and (TTTTA)n apolipoprotein(a) [apo(a), protein; APOA, gene] polymorphisms, as well as the C766T low-density lipoprotein receptor-related protein (LRP) and the (CGG)n very low density lipoprotein receptor (VLDLR) polymorphisms on the one hand, and plasma Lp(a) levels in Czech subjects who underwent coronary angiography on the other hand. The lengths of the alleles of the APOA K-IV and (TTTTA)n polymorphisms were strongly inversely correlated with plasma Lp(a) levels in univariate analysis (r = -0.41, p < 10(-4) and r = -0.20, p < 0.01, respectively). Multivariate analysis revealed significant associations between the APOA polymorphisms studied and plasma Lp(a) levels in subjects expressing only one APOA K-IV allele (p < 10(-6) for K-IV and p < 0.001 for TTTTA). In subjects expressing both APOA K-IV alleles, the multivariate analysis revealed that only the APOA K-IV alleles were inversely correlated with plasma Lp(a) levels (p < 0.001). Associations between both the LRP and VLDLR gene polymorphisms and plasma Lp(a) levels were only of borderline significance (p < 0.06 and p < 0.07, respectively) and were not confirmed in multivariate analysis. In conclusion, both APOA length polymorphisms significantly influenced plasma Lp(a) concentration in the Czech population studied, and this circumstance could explain the association in this population observed earlier between APOA (TTTTA)n polymorphism and CAD (Benes et al. 2000). Only a minor role in the regulation of plasma Lp(a) levels is suggested for the C766T LRP and the (CGG)n VLDLR polymorphisms.

    Human biology 2002;74;1;129-36

  • The alpha -chains of C4b-binding protein mediate complex formation with low density lipoprotein receptor-related protein.

    Westein E, Denis CV, Bouma BN and Lenting PJ

    Laboratory for Thrombosis and Haemostasis, Department of Haematology, University Medical Center Utrecht, 3584 CX Utrecht, The Netherlands.

    C4b-binding protein (C4BP) is a heparin-binding protein that participates in both the complement and hemostatic system. We investigated the interaction between C4BP and low density lipoprotein receptor-related protein (LRP), an endocytic receptor involved in the catabolism of various heparin-binding proteins. Both plasma-derived C4BP and recombinant C4BP consisting of only its alpha-chains (rC4BPalpha) bound efficiently to immobilized LRP, as determined by surface plasmon resonance analysis. Complementary, two distinct fragments of LRP, i.e. clusters II and IV, both associated to immobilized rC4BPalpha, and binding could be inhibited by the LRP antagonist receptor-associated protein. Further analysis showed that association of rC4BPalpha to LRP was inhibited by heparin or by anti-C4BP antibody RU-3B9, which recognizes the heparin-binding region of the C4BP alpha-chains. In cellular degradation experiments, LRP-expressing fibroblasts effectively degraded (125)I-labeled rC4BPalpha, whereas their LRP-deficient counterparts displayed a 4-fold diminished capacity of degrading (125)I-rC4BPalpha. Finally, initial clearance of C4BP in mice was significantly delayed upon co-injection with receptor-associated protein. In conclusion, our data demonstrate that the alpha-chains of C4BP comprise a binding site for LRP. We propose that LRP mediates at least in part the catabolism of C4BP and, as such, may regulate C4BP participation in complement and hemostatic processes.

    The Journal of biological chemistry 2002;277;4;2511-6

  • Case-control study and meta-analysis of low density lipoprotein receptor-related protein gene exon 3 polymorphism in Alzheimer's disease.

    Sánchez-Guerra M, Combarros O, Infante J, Llorca J, Berciano J, Fontalba A, Fernández-Luna JL, Peña N and Fernández-Viadero C

    Service of Neurology, University Hospital Marqués de Valdecilla, 39008 Santander, Spain.

    The low density lipoprotein receptor-related protein (LRP) may influence both the clearance and the production of beta-amyloid peptide and thus plays a role in Alzheimer's disease (AD) pathogenesis. Previous studies, although inconsistent, have suggested that the LRP exon 3 CC genotype contributes to the risk of AD. A case-control study utilizing a clinically well-defined group of 305 sporadic AD patients and 304 control subjects was performed to test this association in an ethnically homogeneous population from Spain. In the current study, the LRP CC genotype was not over-represented in AD patients compared to non-demented controls. A meta-analysis of previous studies revealed a weak correlation of LRP CC genotype with AD (odds ratio of 1.35, P=0.01).

    Neuroscience letters 2001;316;1;17-20

  • Genetic diagnosis of familial hypercholesterolemia in a South European outbreed population: influence of low-density lipoprotein (LDL) receptor gene mutations on treatment response to simvastatin in total, LDL, and high-density lipoprotein cholesterol.

    Chaves FJ, Real JT, García-García AB, Civera M, Armengod ME, Ascaso JF and Carmena R

    Institute of Cytological Research, Service of Endocrinology and Nutrition, Hospital Clínico Universitario, University of Valencia, Avda. Blasco Ibáñez 17, E-46010 Valencia, Spain.

    The aims of this study were to examine the presence of mutations in the low-density lipoprotein receptor gene among subjects clinically diagnosed with familial hypercholesterolemia and to analyze whether the molecular diagnosis helps to predict the response to simvastatin treatment in our familial hypercholesterolemia population. Fifty-five probands and 128 related subjects with familial hypercholesterolemia were studied. Genetic diagnosis was carried out following a three-step protocol based on Southern blot and PCR-single strand conformational polymorphism analysis. A randomized clinical trial with simvastatin was conducted in 42 genetically diagnosed subjects with familial hypercholesterolemia classified as carriers of null mutations (n = 22) and of defective mutations (n = 20). A mutation-causing familial hypercholesterolemia was identified in 46 probands (84%). In 41 of them (89%), a total of 28 point mutations were detected, 13 of which have not been previously described. The remaining five probands (11%) were carriers of large rearrangements. Familial hypercholesterolemia with null mutations showed a poor response to simvastatin treatment. The mean percentage reduction of plasma total and low-density lipoprotein cholesterol levels in these subjects were significantly lower (24.8 +/- 10.3 vs. 34.8 +/- 10.9, P = 0.04 and 30.0 +/- 39.8 vs. 46.1 +/- 18.2, P = 0.02, respectively) than in subjects with defective mutations. Baseline and posttreatment high-density lipoprotein cholesterol plasma values were significantly lower in subjects with familial hypercholesterolemia with null mutations (P < 0.001). In an outbreed Caucasian population, a three-step protocol for genetic screening detected a mutation in the low-density lipoprotein receptor gene in a high percentage (84%) of subjects with familial hypercholesterolemia. Subjects with familial hypercholesterolemia with null mutations (class I) showed lower plasma high-density lipoprotein cholesterol values and a poor low-density lipoprotein cholesterol response to simvastatin treatment.

    The Journal of clinical endocrinology and metabolism 2001;86;10;4926-32

  • Low density lipoprotein receptor-related protein is required for macrophage-mediated oxidation of low density lipoprotein by 12/15-lipoxygenase.

    Xu W, Takahashi Y, Sakashita T, Iwasaki T, Hattori H and Yoshimoto T

    Department of Molecular Pharmacology, Kanazawa University Graduate School of Medicine, Kanazawa 920-8640, Japan.

    The oxidative modification of low density lipoprotein (LDL) has been implicated in the early stage of atherosclerosis through multiple potential pathways, and 12/15-lipoxygenase is suggested to be involved in this oxidation process. We demonstrated previously that the 12/15-lipoxygenase overexpressed in mouse macrophage-like J774A.1 cells was required for the cell-mediated LDL oxidation. However, the mechanism of the oxidation of extracellular LDL by the intracellular 12/15-lipoxygenase has not yet been elucidated. In the present study, we found that not only the LDL receptor but also LDL receptor-related protein (LRP), both of which are cell surface native LDL-binding receptors, were down-regulated by the preincubation of the cells with cholesterol or LDL and up-regulated by lipoprotein-deficient serum. Moreover, 12/15-lipoxygenase-expressing cell-mediated LDL oxidation was decreased by the preincubation of the cells with LDL or cholesterol and increased by the preincubation with lipoprotein-deficient serum. Heparin-binding protein 44, an antagonist of the LDL receptor family, also suppressed the cell-mediated LDL oxidation in a dose-dependent manner. The cell-mediated LDL oxidation was dose-dependently blocked by an anti-LRP antibody but not by an anti-LDL receptor antibody. Furthermore, antisense oligodeoxyribonucleotides against LRP reduced the cell-mediated LDL oxidation under the conditions in which the expression of LRP was decreased. The results taken together indicate that LRP was involved essentially for the cell-mediated LDL oxidation by 12/15-lipoxygenase expressed in J774A.1 cells, suggesting an important pathophysiological role of this receptor-enzyme system as the initial trigger of the progression of atherosclerosis.

    The Journal of biological chemistry 2001;276;39;36454-9

  • C1q and mannose binding lectin engagement of cell surface calreticulin and CD91 initiates macropinocytosis and uptake of apoptotic cells.

    Ogden CA, deCathelineau A, Hoffmann PR, Bratton D, Ghebrehiwet B, Fadok VA and Henson PM

    Department of Pediatrics, National Jewish Medical and Research Center, Denver, CO 80206, USA.

    Removal of apoptotic cells is essential for maintenance of tissue homeostasis, organogenesis, remodeling, development, and maintenance of the immune system, protection against neoplasia, and resolution of inflammation. The mechanisms of this removal involve recognition of the apoptotic cell surface and initiation of phagocytic uptake into a variety of cell types. Here we provide evidence that C1q and mannose binding lectin (MBL), a member of the collectin family of proteins, bind to apoptotic cells and stimulate ingestion of these by ligation on the phagocyte surface of the multifunctional protein, calreticulin (also known as the cC1qR), which in turn is bound to the endocytic receptor protein CD91, also known as the alpha-2-macroglobulin receptor. Use of these proteins provides another example of apoptotic cell clearance mediated by pattern recognition molecules of the innate immune system. Ingestion of the apoptotic cells through calreticulin/CD91 stimulation is further shown to involve the process of macropinocytosis, implicated as a primitive and relatively nonselective uptake mechanism for C1q- and MBL-enhanced engulfment of whole, intact apoptotic cells, as well as cell debris and foreign organisms to which these molecules may bind.

    Funded by: NIGMS NIH HHS: GM 61031, GM48211

    The Journal of experimental medicine 2001;194;6;781-95

  • Role of apolipoprotein E receptors in regulating the differential in vivo neurotrophic effects of apolipoprotein E.

    Veinbergs I, Van Uden E, Mallory M, Alford M, McGiffert C, DeTeresa R, Orlando R and Masliah E

    Department of Neurosciences, University of California-San Diego, La Jolla, CA 92093-0624, USA.

    Apolipoprotein E (apoE) is known to bind to at least five receptors, including the low-density lipoprotein (LDL) receptor-related protein (LRP), very low density LDL receptor (VLDL-R), LDL-R, apoE receptor 2 (apoER2), and megalin/gp330. In this context, the main objective of the present study was to better understand the contributions of LRP and LDL-R to the in vivo neurotrophic effects of apoE. For this purpose, apoE-deficient and receptor-associated protein (RAP)-deficient mice were infused with recombinant apoE3, RAP, or saline. Infusion of apoE3 into apoE-deficient mice resulted in amelioration of degenerative alterations of pyramidal neurons, but had no effect on somatostatin-producing interneurons. In contrast, infusion of apoE3 into RAP-deficient mice resulted in amelioration of degenerative alterations of somatostatin-producing interneurons. LRP and LDL-R levels were significantly reduced in RAP-deficient mice, but significantly increased in the apoE-deficient mice. In contrast, levels of apoE were reduced in the RAP-deficient mice compared to wildtype controls, suggesting that neurotrophic effects of apoE3 in the RAP-deficient mice were related to a combined deficit in endogenous apoE and selected apoE receptors. Furthermore, in apoE-deficient mice, infusion of apoE3 had a neurotrophic effect on somatostatin-producing interneurons only when combined with RAP, suggesting that increased expression of apoE receptors in apoE-deficient mice prevented apoE from rescuing somatostatin-producing neurons. This study supports the contention that some of the in vivo neurotrophic effects of apoE are mediated by LRP and LDL-R and that a critical balance between levels of apoE and its receptors is necessary for the differential neurotrophic effects to appear.

    Funded by: NIA NIH HHS: AG10869, AG5131

    Experimental neurology 2001;170;1;15-26

  • Lack of replication of association findings in complex disease: an analysis of 15 polymorphisms in prior candidate genes for sporadic Alzheimer's disease.

    Prince JA, Feuk L, Sawyer SL, Gottfries J, Ricksten A, Nägga K, Bogdanovic N, Blennow K and Brookes AJ

    Center for Genomics Research, Karolinska Institute, Stockholm, Sweden. Jonathan.Prince@cgr.ki.se

    There is considerable enthusiasm for the prospect of using common polymorphisms (primarily single nucleotide polymorphisms; SNPs) in candidate genes to unravel the genetics of complex disease. This approach has generated a number of findings of loci which are significantly associated with sporadic Alzheimer's disease (AD). In the present study, a total of 15 genes of interest were chosen from among the previously published reports of significant association in AD. Genotyping was performed on polymorphisms within those genes (14 SNPs and one deletion) using Dynamic Allele Specific Hybridization (DASH) in 204 Swedish patients with sporadic late-onset AD and 186 Swedish control subjects. The genes chosen for analysis were; low-density lipoprotein receptor-related protein (LRP1), angiotensin converting enzyme (DCP1), alpha-2-macroglobulin (A2M), bleomycin hydrolase (BLMH), dihydrolipoyl S-succinyltransferase (DLST), tumour necrosis factor receptor superfamily member 6 (TNFRSF6), nitric oxide synthase (NOS3), presenilin 1 (PSEN1), presenilin 2 (PSEN2), butyrylcholinesterase (BCHE), Fe65 (APBB1), oestrogen receptor alpha (ESR1), cathepsin D (CTSD), methylenetetrahydrofolate reductase (MTHFR), and interleukin 1A (IL1A). We found no strong evidence of association for any of these loci with AD in this population. While the possibility exists that the genes analysed are involved in AD (ie they have weak effects and/or are population specific), results reinforce the need for extensive replication studies if we are to be successful in defining true risk factors in complex diseases.

    European journal of human genetics : EJHG 2001;9;6;437-44

  • The low density lipoprotein receptor-related protein modulates levels of matrix metalloproteinase 9 (MMP-9) by mediating its cellular catabolism.

    Hahn-Dantona E, Ruiz JF, Bornstein P and Strickland DK

    Department of Vascular Biology, Holland Laboratory, American Red Cross, Rockville, Maryland 20855, USA.

    Members of the matrix metalloproteinase (MMP) family of enzymes participate in matrix remodeling and share a number of structural and functional features. The activity of this family of proteinases is carefully regulated at the level of zymogen activation and by a family of specific inhibitors termed tissue inhibitors of metalloproteinases (TIMP). It is now becoming clear that levels of certain MMPs are modulated by their association with cellular receptors that mediate their rapid internalization and degradation. In the current investigation we report that the amount of MMP-9 in conditioned cell culture medium is significantly increased when mouse embryonic fibroblasts are grown in the presence of the 39-kDa receptor-associated protein (RAP), an antagonist of ligand binding to low density lipoprotein receptor-related protein (LRP). In vitro assays reveal that the MMP-9.TIMP-1 complex binds to LRP with high affinity and that the binding determinant for LRP appears to reside on MMP-9. Cell lines expressing LRP mediate the internalization of 125I-labeled MMP-9.TIMP-1 complexes, whereas cell lines genetically deficient in LRP show a diminished capacity to mediate the cellular catabolism of MMP-9.TIMP-1 complexes. The results demonstrate that LRP is a functional receptor for MMP-9 and suggest a major role for LRP in modulating remodeling of the extracellular matrix by regulating extracellular proteinase activity.

    Funded by: NHLBI NIH HHS: HL50784, HL54710; NIAMS NIH HHS: AR 45418

    The Journal of biological chemistry 2001;276;18;15498-503

  • Direct binding of occupied urokinase receptor (uPAR) to LDL receptor-related protein is required for endocytosis of uPAR and regulation of cell surface urokinase activity.

    Czekay RP, Kuemmel TA, Orlando RA and Farquhar MG

    Department of Cellular and Molecular Medicine, San Diego, La Jolla, California 92093-0651, USA. rpczekay@scripps.edu

    Low-density lipoprotein receptor-related protein (LRP) mediates internalization of urokinase:plasminogen activator inhibitor complexes (uPA:PAI-1) and the urokinase receptor (uPAR). Here we investigated whether direct interaction between uPAR, a glycosyl-phosphatidylinositol-anchored protein, and LRP, a transmembrane receptor, is required for clearance of uPA:PAI-1, regeneration of unoccupied uPAR, activation of plasminogen, and the ability of HT1080 cells to invade extracellular matrix. We found that in the absence of uPA:PAI-1, uPAR is randomly distributed along the plasma membrane, whereas uPA:PAI-1 promotes formation of uPAR-LRP complexes and initiates redistribution of occupied uPAR to clathrin-coated pits. uPAR-LRP complexes are endocytosed via clathrin-coated vesicles and traffic together to early endosomes (EE) because they can be coimmunoprecipitated from immunoisolated EE, and internalization is blocked by depletion of intracellular K(+). Direct binding of domain 3 (D3) of uPAR to LRP is required for clearance of uPA-PAI-1-occupied uPAR because internalization is blocked by incubation with recombinant D3. Moreover, uPA-dependent plasmin generation and the ability of HT1080 cells to migrate through Matrigel-coated invasion chambers are also inhibited in the presence of D3. These results demonstrate that GPI-anchored uPAR is endocytosed by piggybacking on LRP and that direct binding of occupied uPAR to LRP is essential for internalization of occupied uPAR, regeneration of unoccupied uPAR, plasmin generation, and invasion and migration through extracellular matrix.

    Molecular biology of the cell 2001;12;5;1467-79

  • Disabled-2 colocalizes with the LDLR in clathrin-coated pits and interacts with AP-2.

    Morris SM and Cooper JA

    Fred Hutchinson Cancer Research Center, Division of Basic Sciences, 1100 Fairview Avenue North, Seattle, WA 98109-1024, USA.

    Disabled-2 (Dab2) is a widely expressed relative of Disabled-1, a neuron-specific signal-transduction protein that binds to and receives signals from members of the low-density lipoprotein receptor (LDLR) family. Members of the LDLR family internalize through clathrin-coated pits and vesicles to endosomes, from where they return to the cell surface through the secretory pathway. In this study, we show that the Dab2 phosphotyrosine-binding domain binds peptides containing the sequence FXN-PXY. This core sequence is found in the intracellular domains of LDLR family members and is important for receptor internalization. Dab2 transiently colocalizes with the LDLR in clathrin-coated pits, but is absent from endosomes and lysosomes. Dab2 is alternatively spliced and its localization depends on a region of the protein that contains two DPF motifs that are present in the p96 Dab2 protein and absent in the p67 splice variant. This region is sufficient to confer Dab2 binding to the alpha-adaptin subunit of the clathrin adaptor protein, AP-2. Overexpression of p96 but not of p67 Dab2 disrupts the localization of AP-2. These findings suggest that in addition to previously reported signal-transduction functions, Dab2 could also act as an adaptor protein that may regulate protein trafficking.

    Funded by: NCI NIH HHS: R37-CA41072

    Traffic (Copenhagen, Denmark) 2001;2;2;111-23

  • Identification of a major cyclic AMP-dependent protein kinase A phosphorylation site within the cytoplasmic tail of the low-density lipoprotein receptor-related protein: implication for receptor-mediated endocytosis.

    Li Y, van Kerkhof P, Marzolo MP, Strous GJ and Bu G

    Department of Pediatrics, Washington University School of Medicine, St. Louis, Missouri 63110, USA.

    The low-density lipoprotein (LDL) receptor-related protein (LRP) is a multiligand endocytic receptor that belongs to the LDL receptor family. Recently, studies have revealed new roles of LDL receptor family members as transducers of extracellular signals. Our previous studies have demonstrated LRP phosphorylation within its cytoplasmic tail, but the nature of LRP phosphorylation and its potential function was unknown. In the present study using both in vivo and in vitro analysis, we found that LRP phosphorylation is mediated by the cAMP-dependent protein kinase A (PKA). Using site-directed mutagenesis and LRP minireceptor constructs, we further identified the predominant LRP phosphorylation site at serine 76 of its cytoplasmic tail. Finally, we demonstrated that mutations of serine 76, which abolish LRP phosphorylation by PKA, result in a decrease in the initial endocytosis rate of LRP and a lower efficiency in delivery of ligand for degradation. Thus, the role of PKA phosphorylation of LRP in receptor-mediated endocytosis may provide a mechanism by which the endocytic function of LRP can be regulated by external signals.

    Funded by: NHLBI NIH HHS: HL59150; NINDS NIH HHS: NS37525

    Molecular and cellular biology 2001;21;4;1185-95

  • Uptake of HIV-1 tat protein mediated by low-density lipoprotein receptor-related protein disrupts the neuronal metabolic balance of the receptor ligands.

    Liu Y, Jones M, Hingtgen CM, Bu G, Laribee N, Tanzi RE, Moir RD, Nath A and He JJ

    Department of Microbiology and Immunology, Indiana University School of Medicine, Indianapolis, Indiana 46202, USA.

    Neurological disorders develop in most people infected with human immunodeficiency virus type 1 (HIV-1). However, the underlying mechanisms remain largely unknown. Here we report that binding of HIV-1 transactivator (Tat) protein to low-density lipoprotein receptor-related protein (LRP) promoted efficient uptake of Tat into neurons. LRP-mediated uptake of Tat was followed by translocation to the neuronal nucleus. Furthermore, the binding of Tat to LRP resulted in substantial inhibition of neuronal binding, uptake and degradation of physiological ligands for LRP, including alpha2-macroglobulin, apolipoprotein E4, amyloid precursor protein and amyloid beta-protein. In a model of macaques infected with a chimeric strain of simian-human immunodeficiency virus, increased staining of amyloid precursor protein was associated with Tat expression in the brains of simian-human immunodeficiency virus-infected macaques with encephalitis. These results indicate that HIV-1 Tat may mediate HIV-1-induced neuropathology through a pathway involving disruption of the metabolic balance of LRP ligands and direct activation of neuronal genes.

    Funded by: NINDS NIH HHS: NS39253

    Nature medicine 2000;6;12;1380-7

  • Differential expression of the alpha(2)-macroglobulin receptor and the receptor associated protein in normal human endometrium and endometrial carcinoma.

    Foca C, Moses EK, Quinn MA and Rice GE

    Perinatal Research Centre, Department of Perinatal Medicine, Royal Women's Hospital, Carlton, Victoria 3053, Australia.

    Extracellular matrix degradation, mediated by the activation of receptor-bound proteolytic enzymes, is essential to the process of cellular invasion. Many normal physiological functions such as endometrial remodelling are reliant on the activation of these surface associated proteolytic enzymes, as are pathological functions such as cancer-cell invasion. The internalization of proteolytic complexes is mediated by the multi-functional clearance receptor, alpha(2)-macroglobulin receptor/LRP. The role of LRP and its ligand binding inhibitor, the receptor-associated protein (RAP), in the advancement of invasive endometrial carcinoma is unknown. The aim of this study was to compare the expression of LRP and RAP mRNA in normal endometrium (n = 14) and endometrial carcinoma (n = 33) by semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR). Expression of LRP mRNA in normal endometrium was significantly increased in the secretory phase when compared with proliferative phase endometrium (P: < 0.05). The expression of LRP in all carcinomas examined was significantly reduced to about 20% of the amount in normal endometrium (P: < 0.05), whereas RAP expression was not significantly different between endometrium and carcinoma. No significant difference in the level of LRP or RAP expression was observed between carcinoma grades or stages. In conclusion, we have shown that LRP expression is differentially regulated in the normal endometrium during the menstrual cycle and is decreased in invasive endometrial carcinomas.

    Molecular human reproduction 2000;6;10;921-7

  • Low density lipoprotein receptor-related protein gene polymorphisms and risk for late-onset Alzheimer's disease in a Japanese population.

    Hatanaka Y, Kamino K, Fukuo K, Mitsuda N, Nishiwaki-Ueda Y, Sato N, Satoh T, Yamamoto H, Yoneda H, Imagawa M, Miki T, Ohta S and Ogihara T

    Department of Geriatric Medicine, Osaka University Medical School, Suita, Japan.

    Low density lipoprotein (LDL) receptor-related protein (LRP) gene polymorphisms located in the 5' region and in exon 3, and the apolipoprotein E (APOE) genotype were determined in 100 Japanese patients affected by late-onset Alzheimer's disease (AD). We matched 246 controls for age and found no association between the polymorphism located in the 5' region of the LRP gene. The distribution of LRP exon 3 genotypes and alleles did not differ between AD and the control groups. However, the frequency of T allele in the Alzheimer's group having APOE-epsilon4 was lower than that in the control group having APOE-epsilon4, but it was only marginally significant (p = 0.022). Age of onset was significantly younger in the patients with CC genotype than those carrying the T allele (p = 0.03), and this trend was more evident among non-APOE-epsilon4 carriers (p = 0.008). These results support the possibility that ApoE and LRP may contribute to the development of AD.

    Clinical genetics 2000;58;4;319-23

  • Interactions of the low density lipoprotein receptor gene family with cytosolic adaptor and scaffold proteins suggest diverse biological functions in cellular communication and signal transduction.

    Gotthardt M, Trommsdorff M, Nevitt MF, Shelton J, Richardson JA, Stockinger W, Nimpf J and Herz J

    Department of Molecular Genetics and Pathology, University of Texas Southwestern Medical Center, Dallas 75390-9046, USA.

    The members of the low density lipoprotein (LDL) receptor gene family bind a broad spectrum of extracellular ligands. Traditionally, they had been regarded as mere cargo receptors that promote the endocytosis and lysosomal delivery of these ligands. However, recent genetic experiments in mice have revealed critical functions for two LDL receptor family members, the very low density lipoprotein receptor and the apoE receptor-2, in the transmission of extracellular signals and the activation of intracellular tyrosine kinases. This process regulates neuronal migration and is crucial for brain development. Signaling through these receptors requires the interaction of their cytoplasmic tails with the intracellular adaptor protein Disabled-1 (DAB1). Here, we identify an extended set of cytoplasmic proteins that might also participate in signal transmission by the LDL receptor gene family. Most of these novel proteins are adaptor or scaffold proteins that contain PID or PDZ domains and function in the regulation of mitogen-activated protein kinases, cell adhesion, vesicle trafficking, or neurotransmission. We show that binding of DAB1 interferes with receptor internalization suggesting a mechanism by which signaling through this class of receptors might be regulated. Taken together, these findings imply much broader physiological functions for the LDL receptor family than had previously been appreciated. They form the basis for the elucidation of the molecular pathways by which cells respond to the diversity of ligands that bind to these multifunctional receptors on the cell surface.

    Funded by: NHLBI NIH HHS: HL20948, HL63762, R37 HL063762

    The Journal of biological chemistry 2000;275;33;25616-24

  • CD91: a receptor for heat shock protein gp96.

    Binder RJ, Han DK and Srivastava PK

    Center for Immunotherapy of Cancer and Infectious Diseases, University of Connecticut School of Medicine, Farmington, CT 06030, USA.

    Antigen presenting cells (APCs) can take up exogenous antigenic peptides chaperoned by heat shock protein gp96 and re-present them through the endogenous pathway on their major histocompatibility class I molecules. The high efficiency of this process has been attributed previously to a receptor for gp96 on APCs. The CD91 molecule (also called alpha 2-macroglobulin receptor or the low density lipoprotein-related protein) is shown here to be a cell surface receptor for the heat shock protein gp96. CD91 binds gp96 directly, rather than through another ligand for CD91. The previously known CD91 ligand, alpha 2-macroglobulin, inhibits re-presentation of gp96-chaperoned antigenic peptides by macrophages, as do antibodies to CD91. As gp96 is exclusively intracellular and is released as a result of necrotic but not apoptotic cell death, we propose that CD91 acts as a sensor for necrotic cell death.

    Funded by: NCI NIH HHS: CA64394, CA84479

    Nature immunology 2000;1;2;151-5

  • Involvement of alpha-2-macroglobulin receptor in clearance of interleukin 8-alpha-2-macroglobulin complexes by human alveolar macrophages.

    Kurdowska A, Alden SM, Noble JM, Stevens MD and Carr FK

    Department of Biochemistry, University of Texas Health Center at Tyler, 75708, USA. ANNA@uthct.edu

    The purpose of this study was to determine if interleukin 8 (IL-8) in complex with alpha2-macroglobulin (alpha-2-M) can be taken up by human alveolar macrophages. First, we demonstrated that human alveolar macrophages have receptors for alpha-2-M but not IL-8. The binding of(125)I-labeled alpha-2-M to the cells was specific and saturable, whereas(125)I-labeled recombinant human IL-8 (rhIL-8) did not bind to macrophages. However,(125)I-rhIL-8-alpha-2-M complexes bound to macrophages, and unlabeled alpha-2-M competed for the binding. We then cultured the cells in the presence of(125)I-rhIL-8-alpha-2-M complexes,(125)I-rhIL-8 alone or buffer for 24 h. Macrophages were lysed, and the released radioactivity measured. IL-8 concentrations in supernatants and cells were also measured using an IL-8 ELISA. When the macrophages were incubated with(125)I-rhIL-8-alpha-2-M complexes there was a significant amount of IL-8 associated with the cells. However, this was not the case when the cells were incubated with(125)I- rhIL-8 alone suggesting that only these complexes were taken-up by human alveolar macrophages. Furthermore, the clearance of complexes was specifically inhibited by a monoclonal antibody against the 515-kDa subunit of the alpha-2-M receptor (alpha-2-MR) but not by an isotopic mouse IgG1. The study shows an important clearance mechanism for IL-8 in the lung.

    Funded by: NHLBI NIH HHS: R29-HL56768

    Cytokine 2000;12;7;1046-53

  • The YXXL motif, but not the two NPXY motifs, serves as the dominant endocytosis signal for low density lipoprotein receptor-related protein.

    Li Y, Marzolo MP, van Kerkhof P, Strous GJ and Bu G

    Departments of Pediatrics, and Cell Biology and Physiology, Washington University School of Medicine and St. Louis Children's Hospital, St. Louis, Missouri 63110, USA.

    All members of the low density lipoprotein (LDL) receptor family contain at least one copy of the NPXY sequence within their cytoplasmic tails. For the LDL receptor, it has been demonstrated that the NPXY motif serves as a signal for rapid endocytosis through coated pits. Thus, it is generally believed that the NPXY sequences function as endocytosis signals for all the LDL receptor family members. The primary aim of this study is to define the endocytosis signal(s) within the cytoplasmic tail of LDL receptor-related protein (LRP). By using LRP minireceptors, which mimic the function and trafficking of full-length endogenous LRP, we demonstrate that the YXXL motif, but not the two NPXY motifs, serves as the dominant signal for LRP endocytosis. We also found that the distal di-leucine motif within the LRP tail contributes to its endocytosis, and its function is independent of the YXXL motif. Although the proximal NPXY motif and the proximal di-leucine motif each play a limited role in LRP endocytosis in the context of the full-length tail, these motifs were functional within the truncated receptor tail. In addition, we show that LRP minireceptor mutants defective in endocytosis signal(s) accumulate at the cell surface and are less efficient in delivery of ligand for degradation.

    Funded by: NHLBI NIH HHS: HL59150; NINDS NIH HHS: NS37525

    The Journal of biological chemistry 2000;275;22;17187-94

  • LDL receptor-related protein as a component of the midkine receptor.

    Muramatsu H, Zou K, Sakaguchi N, Ikematsu S, Sakuma S and Muramatsu T

    Department of Biochemistry, Nagoya University School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya, 466-8550, Japan.

    Midkine (MK) is a heparin-binding growth factor with migration-promoting and survival-promoting activities. To identify signaling receptor(s) of MK, membrane glycoproteins with MK-binding activity were isolated from day 13 mouse embryos by lectin- and MK-affinity chromatography. SDS-PAGE followed by protein sequence analysis revealed the presence of LDL receptor-related protein (LRP) and NCAM in the fraction. The dissociation constant of binding between LRP and MK was 3.5 nM. Receptor-associated protein (RAP), which interfered with the binding, inhibited MK-dependent survival of embryonic neurons. Brushin/megalin, which is also a high molecular weight protein belonging to the LDL receptor family, bound to MK less strongly than LRP. These findings suggest that LRP is a component of the receptor complex for MK.

    Biochemical and biophysical research communications 2000;270;3;936-41

  • NMR solution structure of complement-like repeat CR3 from the low density lipoprotein receptor-related protein. Evidence for specific binding to the receptor binding domain of human alpha(2)-macroglobulin.

    Dolmer K, Huang W and Gettins PG

    Department of Biochemistry, College of Medicine, University of Illinois at Chicago, Chicago, Illinois 60612-4316, USA.

    We have used NMR methods to determine the structure of the calcium complex of complement-like repeat 3 (CR3) from the low density lipoprotein receptor-related protein (LRP) and to examine its specific interaction with the receptor binding domain of human alpha(2)-macroglobulin. CR3 is one of eight related repeats that constitute a major ligand binding region of LRP. The structure is very similar in overall fold to homologous complement-like repeat CR8 from LRP and complement-like repeats LB1, LB2, and LB5 from the low density lipoprotein receptor and contains a short two-strand antiparallel beta-sheet, a one turn alpha-helix, and a high affinity calcium site with coordination from four carboxyls and two backbone carbonyls. The surface electrostatics and topography are, however, quite distinct from each of these other repeats. Two-dimensional (1)H,(15)N-heteronuclear single quantum coherence spectra provide evidence for a specific, though relatively weak (K(d) approximately 140 microM), interaction between CR3 and human alpha2-macroglobulin receptor binding domain that involves a contiguous patch of surface residues in the central region of CR3. This specific interaction is consistent with a mode of LRP binding to ligands that uses contributions from more than one domain to generate a wide array of different binding sites, each with overall high affinity.

    Funded by: NIGMS NIH HHS: GM54414

    The Journal of biological chemistry 2000;275;5;3264-9

  • Role of tissue plasminogen activator receptor LRP in hippocampal long-term potentiation.

    Zhuo M, Holtzman DM, Li Y, Osaka H, DeMaro J, Jacquin M and Bu G

    Department of Anesthesiology, Washington University School of Medicine, St. Louis, Missouri 63110, USA.

    The low-density lipoprotein (LDL) receptor-related protein (LRP) is a multifunctional endocytic receptor that is expressed abundantly in neurons of the CNS. Both LRP and several of its ligands, including tissue plasminogen activator (tPA), apolipoprotein E/lipoproteins, alpha(2)-macroglobulin, and the beta-amyloid precursor protein, have been implicated in various neuronal functions and in the pathogenesis of Alzheimer's disease. It has been reported that induction of tPA expression may contribute to activity-dependent synaptic plasticity in the hippocampus and cerebellum. In addition, long-term potentiation (LTP) is significantly decreased in mice lacking tPA. Here we demonstrate that tPA receptor LRP is abundantly expressed in hippocampal neurons and participates in hippocampal LTP. Perfusion of hippocampal slices with receptor-associated protein (RAP), an antagonist for ligand interactions with LRP, significantly reduced late-phase LTP (L-LTP). In addition, RAP also blocked the enhancing effect of synaptic potentiation by exogenous tPA in hippocampal slices prepared from tPA knock-out mice. Metabolic labeling and ligand binding analyses showed that both tPA and LRP are synthesized by hippocampal neurons and that LRP is the major cell surface receptor that binds tPA. Finally, we found that tPA binding to LRP in hippocampal neurons enhances the activity of cyclic AMP-dependent protein kinase, a key molecule that is known to be involved in L-LTP. Taken together, our results demonstrate that interactions between tPA and cell surface LRP are important for hippocampal L-LTP.

    Funded by: NIA NIH HHS: AG13956; NINDS NIH HHS: NS37525

    The Journal of neuroscience : the official journal of the Society for Neuroscience 2000;20;2;542-9

  • Metabolism of activated complement component C3 is mediated by the low density lipoprotein receptor-related protein/alpha(2)-macroglobulin receptor.

    Meilinger M, Gschwentner C, Burger I, Haumer M, Wahrmann M, Szollar L, Nimpf J and Huettinger M

    Department of Medical Chemistry, University of Vienna, Währingerstrasse 10, A-1090 Vienna, Austria.

    Complement component 3 (C3) and alpha(2)-macroglobulin evolved from a common, evolutionarily old, ancestor gene. Low density lipoprotein-receptor-related protein/alpha(2)-macroglobulin receptor (LRP/alpha(2)MR), a member of the low density lipoprotein receptor family, is responsible for the clearance of alpha(2)-macroglobulin-protease complexes. In this study, we examined whether C3 has conserved affinity for LRP/alpha(2)MR. Ligand blot experiments with human (125)I-C3 on endosomal proteins show binding to a 600-kDa protein, indistinguishable from LRP/alpha(2)MR by the following criteria: it is competed by receptor-associated protein (the 39-kDa receptor-associated protein that impairs binding of all ligands to LRP/alpha(2)MR) and by lactoferrin and Pseudomonas exotoxin, other well known ligands of the multifunctional receptor. Binding of C3 is sensitive to reduction of the receptor and is Ca(2+)-dependent. All these features are typical for cysteine-rich binding repeats of the low density lipoprotein receptor family. In LRP/alpha(2)MR, they are found in four cassettes (2, 8, 10, and 11 repeats). Ligand blotting to chicken LR8 demonstrates that a single 8-fold repeat is sufficient for binding. Confocal microscopy visualizes initial surface labeling of human fibroblasts incubated with fluorescent labeled C3, which changes after 5 min to an intracellular vesicular staining pattern that is abolished in the presence of receptor-associated protein. Cell uptake is abolished in mouse fibroblasts deficient in LRP/alpha(2)MR. Native plasma C3 is not internalized. We demonstrate that the capacity to internalize C3 is saturable and exhibits a K(D) value of 17 nM. After intravenous injection, rat hepatocytes accumulate C3 in sedimentable vesicles with a density typical for endosomes. In conclusion, our ligand blot and uptake studies demonstrate the competence of the LRP/alpha(2)MR to bind and endocytose C3 and provide evidence for an LRP/alpha(2)MR-mediated system participating in C3 metabolism.

    The Journal of biological chemistry 1999;274;53;38091-6

  • Collagenase-3 binds to a specific receptor and requires the low density lipoprotein receptor-related protein for internalization.

    Barmina OY, Walling HW, Fiacco GJ, Freije JM, López-Otín C, Jeffrey JJ and Partridge NC

    Department of Pharmacological Science, St. Louis University School of Medicine, St. Louis, Missouri 63104, USA.

    We have previously identified a specific receptor for collagenase-3 that mediates the binding, internalization, and degradation of this ligand in UMR 106-01 rat osteoblastic osteosarcoma cells. In the present study, we show that collagenase-3 binding is calcium-dependent and occurs in a variety of cell types, including osteoblastic and fibroblastic cells. We also present evidence supporting a two-step mechanism of collagenase-3 binding and internalization involving both a specific collagenase-3 receptor and the low density lipoprotein receptor-related protein. Ligand blot analysis shows that (125)I-collagenase-3 binds specifically to two proteins ( approximately 170 kDa and approximately 600 kDa) present in UMR 106-01 cells. Western blotting identified the 600-kDa protein as the low density lipoprotein receptor-related protein. Our data suggest that the 170-kDa protein is a specific collagenase-3 receptor. Low density lipoprotein receptor-related protein-null mouse embryo fibroblasts bind but fail to internalize collagenase-3, whereas UMR 106-01 and wild-type mouse embryo fibroblasts bind and internalize collagenase-3. Internalization, but not binding, is inhibited by the 39-kDa receptor-associated protein. We conclude that the internalization of collagenase-3 requires the participation of the low density lipoprotein receptor-related protein and propose a model in which the cell surface interaction of this ligand requires a sequential contribution from two receptors, with the collagenase-3 receptor acting as a high affinity primary binding site and the low density lipoprotein receptor-related protein mediating internalization.

    Funded by: NIAMS NIH HHS: AR 40661; NICHD NIH HHS: HD 05291

    The Journal of biological chemistry 1999;274;42;30087-93

  • Characterization of the soluble form of the low density lipoprotein receptor-related protein (LRP).

    Quinn KA, Pye VJ, Dai YP, Chesterman CN and Owensby DA

    Centre for Thrombosis and Vascular Research, School of Pathology, Kensington, New South Wales, 2052, Australia. k.quinn@unsw.edu.au

    We report characterization of the soluble form of the low density lipoprotein receptor-related protein (sLRP) which circulates in human plasma. Amino acid sequence analysis confirmed that sLRP isolated from human plasma contains the alpha-chain of LRP1. In addition, Western blot analysis identified a truncated beta-chain noncovalently associated with the purified alpha-chain. The molecular size (M(r) 55K) of the peptide portion of the truncated beta-chain indicates that the subunit comprises the extracellular portion of the beta-chain and terminates in a membrane-proximal region. We investigated the mechanism by which sLRP may be generated using the trophoblast cell line, BeWo, which releases sLRP in culture. Cell surface labeling experiments indicate that LRP is released from BeWo cells following expression at the cell surface. Incubation of BeWo cells in the presence of a metalloproteinase inhibitor, INH-3855-PI, results in a dose-dependent inhibition of LRP shedding. The metalloproteinase responsible for the shedding of LRP by BeWo cells is not up-regulated by phorbol ester and is not dependent on serine proteases, such as plasmin, for activity. The BeWo cell line is derived from a human gestational choriocarcinoma and preliminary studies suggest that LRP may be shed within the placenta during gestation. Increased levels of sLRP were detected in cord blood. In term placenta, LRP is expressed in the syncytium, which comprises the maternal-fetal interface. Increased levels of sLRP in cord blood may reflect cellular dysfunction and increased metalloproteinase activity at this important interface.

    Experimental cell research 1999;251;2;433-41

  • NMR solution structure of complement-like repeat CR8 from the low density lipoprotein receptor-related protein.

    Huang W, Dolmer K and Gettins PG

    Department of Biochemistry and Molecular Biology, College of Medicine, University of Illinois at Chicago, Chicago, Illinois 60612-4316, USA.

    The low density lipoprotein receptor-related protein is a member of the low density lipoprotein receptor family and contains clusters of cysteine-rich complement-like repeats of about 42 residues that are present in all members of this family of receptors. These clusters are thought to be the principal binding sites for protein ligands. We have expressed one complement-like repeat, CR8, from the cluster in lipoprotein receptor-related protein that binds certain proteinase inhibitor-proteinase complexes and used three-dimensional NMR on the 13C/15N-labeled protein to determine the structure in solution of the calcium-bound form. The structure is very similar in overall fold to repeat 5 from the low density lipoprotein receptor (LB5), with backbone root mean square deviation of 1.5 A. The calcium-binding site also appears to be homologous, with four carboxyl and two backbone carbonyl ligands. However, differences in primary structure are such that equivalent surfaces that might represent the binding interfaces are very different from one another, indicating that different domains will have very different ligand specificities.

    Funded by: NIGMS NIH HHS: GM54414

    The Journal of biological chemistry 1999;274;20;14130-6

  • Interaction of cytosolic adaptor proteins with neuronal apolipoprotein E receptors and the amyloid precursor protein.

    Trommsdorff M, Borg JP, Margolis B and Herz J

    Department of Molecular Genetics, University of Texas Southwestern Medical Center, Dallas, Texas 75235-9046, USA.

    Apolipoprotein E, alpha2-macroglobulin, and amyloid precursor protein (APP) are involved in the development of Alzheimer's disease. All three proteins are ligands for the low density lipoprotein (LDL) receptor-related protein (LRP), an abundant neuronal surface receptor that has also been genetically linked to Alzheimer's disease. The cytoplasmic tails of LRP and other members of the LDL receptor gene family contain NPxY motifs that are required for receptor endocytosis. To investigate whether these receptors may have functions that go beyond ligand internalization, e.g. possible roles in cellular signaling, we searched for proteins that might interact with the cytoplasmic tails of the receptors. A family of adaptor proteins containing protein interaction domains that can interact with NPxY motifs has previously been described. Using yeast 2-hybrid and protein coprecipitation approaches in vitro, we show that the neuronal adaptor proteins FE65 and mammalian Disabled bind to the cytoplasmic tails of LRP, LDL receptor, and APP, where they can potentially serve as molecular scaffolds for the assembly of cytosolic multiprotein complexes. FE65 contains two distinct protein interaction domains that interact with LRP and APP, respectively, raising the possibility that LRP can modulate the intracellular trafficking of APP. Tyrosine-phosphorylated mammalian Disabled can recruit nonreceptor tyrosine kinases, such as src and abl, to the cytoplasmic tails of the receptors to which it binds, suggesting a molecular pathway by which receptor/ligand interaction on the cell surface could generate an intracellular signal.

    Funded by: NHLBI NIH HHS: HL20948, R37 HL063762

    The Journal of biological chemistry 1998;273;50;33556-60

  • Low-density-lipoprotein-receptor-related protein (LRP) interacts with a GTP-binding protein.

    Goretzki L and Mueller BM

    The Scripps Research Institute, Department of Immunology, 10550 N. Torrey Pines Road, IMM13, La Jolla, CA 92037, USA.

    The low-density-lipoprotein-receptor-related protein (LRP) binds and internalizes numerous ligands, including lipoproteins, proteinase-inhibitor complexes and others. We have shown previously that LRP-mediated ligand internalization is dependent on cAMP-dependent protein kinase (PKA) activity. Here, we investigated whether ligation of LRP increases the intracellular cAMP level and PKA activity via a stimulatory GTP-binding protein. Treatment of LRP-expressing cell lines with the LRP ligands lactoferrin or urokinase-type plasminogen activator caused a significant elevation in cAMP and stimulated PKA activity in a dose-dependent manner. Addition of the 39 kDa receptor-associated protein (RAP), an antagonist for ligand interactions with LRP, blocked the lactoferrin-induced increase in PKA activity, demonstrating a requirement for ligand binding to LRP. Incubation of cell membrane fractions with lactoferrin increased GTPase activity in a time- and dose-dependent manner, and treatment with LRP ligands suppressed cholera-toxin-mediated ADP-ribosylation of the Gsalpha subunit of a heterotrimeric G-protein. Affinity precipitation of LRP with RAP resulted in co-precipitation of two isoforms of Gsalpha from detergent extracts. We thus conclude that LRP is a signalling receptor that associates directly with a stimulatory heterotrimeric G-protein and activates a downstream PKA-dependent pathway.

    Funded by: NCI NIH HHS: CA59692

    The Biochemical journal 1998;336 ( Pt 2);381-6

  • CD19 is linked to the integrin-associated tetraspans CD9, CD81, and CD82.

    Horváth G, Serru V, Clay D, Billard M, Boucheix C and Rubinstein E

    INSERM U268, Hôpital Paul Brousse, 94807 Villejuif Cedex, France.

    The CD19-CD21-CD81 complex regulates signal transduction events critical for B lymphocyte development and humoral immunity. CD81, a molecule with 4 transmembrane domains, member of the tetraspan superfamily, is engaged, together with other tetraspans such as CD9, CD53, CD63, and CD82, in multimolecular complexes containing beta1 integrins and major histocompatibility complex antigens. Here we demonstrate that two other tetraspans, CD82 and the early B cell marker CD9, are coimmunoprecipitated with CD19 from Brij97 lysates of B cell lines. Moreover, CD9 was coprecipitated from lysates of purified CD10(+) early B cells. These associations were confirmed by the cocapping of CD19 with CD9 or CD82. The CD9/CD19 association was disrupted in the presence of digitonin, contrary to the CD81/CD19 association, indicating that CD9 and CD81 interact with CD19 in different ways. The CD9/CD81 association is also disrupted in the presence of digitonin, suggesting that CD9 associates with CD19 only through CD81. To characterize the regions involved in the CD81/CD19 association, two reciprocal CD9/CD81 chimeric molecules were tested for the association with CD19, but none of them could be coprecipitated with CD19 in digitonin, indicating that the domain of CD81 responsible for its association with CD19 is complex. Finally, engagement of CD9 could induce the tyrosine phosphorylation of different proteins, including CD19 itself, suggesting that the CD9/CD19 association is functionally relevant. Thus, a physical and functional link is formed between the CD19-CD21-CD81 complex and the integrin-tetraspan complexes, which is dynamically modulated in the process of B cell differentiation.

    The Journal of biological chemistry 1998;273;46;30537-43

  • Strategy to sequence the 89 exons of the human LRP1 gene coding for the lipoprotein receptor related protein: identification of one expressed mutation among 48 polymorphisms.

    Van Leuven F, Stas L, Thiry E, Nelissen B and Miyake Y

    Experimental Genetics Group (EGG), Center for Human Genetics (CME), Flemish Institute for Biotechnology (VIB), Campus Gasthuisberg, Leuven, Belgium. fredvl@med.kuleuven.ac.be/legtegg/

    The human lipoprotein receptor related protein (LRP) binds and internalizes a diverse set of ligands, making LRP the most multifunctional endocytic receptor known. This is possible due to the large size, i.e., 600 kDa, of the receptor protein containing three clusters of putative ligand binding domains, each structurally comparable to the classical LDL receptor. Based on previous structural analysis of the human LRP1 gene (Van Leuven et al., 1994, Genomics, 24: 78-89), a strategy was developed to sequence the 89 exons of the LRP1 gene, including partial intron sequences. The gene was amplified from individual genomic DNA by long-range PCR, in 14 amplicons sized between 0.4 and 11 kb that were used as templates for 110 sequencing primers. In total, 48 mutations and intronic polymorphisms were identified. Two previously reported polymorphisms, i.e., in the promoter region and in exon 3, were precisely defined by sequencing. The first expressed mutation, i.e., an alanine to valine transition at position 217 of the LRP precursor protein, was detected on one allele in 2 of 33 individuals. Although the strategy is still subject to refinement, this approach is reported to allow others to analyze genetic differences in the human LRP1 gene, with particular reference to the recently reported association with late-onset Alzheimer disease.

    Genomics 1998;52;2;138-44

  • Soluble low density lipoprotein receptor-related protein (LRP) circulates in human plasma.

    Quinn KA, Grimsley PG, Dai YP, Tapner M, Chesterman CN and Owensby DA

    Center for Thrombosis and Vascular Research, University of New South Wales, Sydney 2052, Australia.

    Our studies have identified a soluble molecule in normal human plasma and serum with the characteristics of the alpha-chain of the low density lipoprotein receptor-related protein (LRP). LRP is a large multifunctional receptor mediating the clearance of diverse ligands, including selected lipoproteins, various protease inhibitor complexes, and thrombospondin. A soluble molecule (sLRP) has been isolated from plasma using an affinity matrix coupled with methylamine-activated alpha2-macroglobulin, the ligand uniquely recognized by LRP, and eluted with EDTA. This eluate contains a protein that co-migrates on SDS-polyacrylamide gel electrophoresis with authentic human placental LRP alpha-chain, is recognized by anti-LRP alpha-chain monoclonal antibodies, and binds the 39-kDa receptor-associated protein (RAP) and tissue plasminogen activator-inhibitor complexes. A similar RAP-binding molecule was detected in medium conditioned for 24 h by primary cultures of rat hepatocytes, suggesting that the liver may be the in vivo source of sLRP. In contrast, immunoprecipitation experiments failed to detect the production of sLRP by cultured HepG2 hepatoma and primary human fibroblast cells. Addition of a soluble form of LRP to cultured HepG2 cells resulted in a significant inhibition of capacity of these cells to degrade tPA, a process that has been demonstrated to be mediated by cell surface LRP. Preliminary data indicate that the concentration of sLRP is altered in the plasma of patients with liver disease. Increased levels of sLRP may antagonize the clearance of ligands by cell bound LRP perturbing diverse processes including lipid metabolism, cell migration and extracellular proteinase activity.

    The Journal of biological chemistry 1997;272;38;23946-51

  • The type V transforming growth factor beta receptor is the putative insulin-like growth factor-binding protein 3 receptor.

    Leal SM, Liu Q, Huang SS and Huang JS

    Department of Biochemistry and Molecular Biology, St. Louis University School of Medicine, St. Louis, Missouri 63104, USA.

    Insulin-like growth factor-binding protein 3 (IGFBP-3) has been shown to inhibit cell growth by IGF-dependent and -independent mechanisms. The putative cell-surface IGFBP-3 receptor that mediates the IGF-independent growth inhibition has not been identified. Here we show that recombinant human IGFBP-3 inhibits 125I-transforming growth factor (TGF)-beta1 binding to the type V TGF-beta receptor (Mr 400,000) in mink lung epithelial cells. We also demonstrate that the approximately 400-kDa 125I-IGFBP-3 affinity-labeled putative IGFBP-3 receptor is immunoprecipitated by specific antiserum to the type V TGF-beta receptor. The 125I-IGFBP-3 affinity labeling of the putative receptor and IGFBP-3-induced growth inhibition as measured by DNA synthesis in these cells is blocked by a TGF-beta1 peptide antagonist. The 125I-IGFBP-3 affinity-labeled putative receptor can only be detected in cells expressing the type V TGF-beta receptor, but not in cells lacking the type V TGF-beta receptor. These results indicate that the type V TGF-beta receptor is the putative IGFBP-3 receptor and that IGFBP-3 is a functional ligand for the type V TGF-beta receptor.

    Funded by: NCI NIH HHS: CA 38808

    The Journal of biological chemistry 1997;272;33;20572-6

  • Cellular internalization and degradation of thrombospondin-1 is mediated by the amino-terminal heparin binding domain (HBD). High affinity interaction of dimeric HBD with the low density lipoprotein receptor-related protein.

    Mikhailenko I, Krylov D, Argraves KM, Roberts DD, Liau G and Strickland DK

    Holland Laboratory, American Red Cross, Rockville, Maryland 20855, USA.

    Thrombospondin-1 (TSP-1) is a large modular trimeric protein that has been proposed to play a diverse role in biological processes. Newly synthesized TSP-1 either is incorporated into the matrix or binds to the cell surface where it is rapidly internalized and degraded. TSP-1 catabolism is mediated by the low density lipoprotein receptor-related protein (LRP), a large endocytic receptor that is a member of the low density lipoprotein receptor family. Using adenovirus-mediated gene transfer experiments, we demonstrate that the very low density lipoprotein receptor can also bind and internalize TSP-1. An objective of the current investigation was to identify the portion of TSP-1 that binds to these endocytic receptors. The current studies found that the amino-terminal heparin binding domain (HBD, residues 1-214) of mouse TSP-1, when prepared as a fusion protein with glutathione S-transferase (GST), bound to purified LRP with an apparent KD ranging from 10 to 25 nM. Recombinant HBD (rHBD) purified following proteolytic cleavage of GST-HBD, also bound to purified LRP, but with an apparent KD of 830 nM. The difference in affinity was attributed to the fact that GST-HBD exists in solution as a dimer, whereas rHBD is a monomer. Like TSP-1, 125I-labeled GST-HBD or 125I-labeled rHBD were internalized and degraded by wild type fibroblasts that express LRP, but not by fibroblasts that are genetically deficient in LRP. The catabolism of both 125I-labeled GST-HBD and rHBD in wild type fibroblast was blocked by the 39-kDa receptor-associated protein, an inhibitor of LRP function. GST-HBD and rHBD both completely blocked catabolism of 125I-labeled TSP-1 in a dose-dependent manner, as did antibodies prepared against the HBD. Taken together, these data provide compelling evidence that the amino-terminal domain of TSP-1 binds to LRP and thus the recognition determinants on TSP-1 for both LRP and for cell surface proteoglycans reside within the same TSP-1 domain. Further, high affinity binding of TSP-1 to LRP likely results from the trimeric structure of TSP-1.

    Funded by: NHLBI NIH HHS: HL37510, HL50787; NIGMS NIH HHS: GM42581

    The Journal of biological chemistry 1997;272;10;6784-91

  • Low density lipoprotein receptor-related protein modulates the expression of tissue-type plasminogen activator in human colon fibroblasts.

    Hardy MM, Feder J, Wolfe RA and Bu G

    Department of Cell Culture and Biochemistry, Monsanto Company, St. Louis, Missouri 63167, USA. MMHard@MONSANTO.COM

    Human colon fibroblasts (HCF) produce tissue-type plasminogen activator (t-PA) in culture, but after 24-48 h, t-PA ceases to accumulate in the medium. Here, we report negative feedback regulation of t-PA expression, exerted by t-PA or complexes of t-PA with its physiological inhibitor, plasminogen activator inhibitor type 1 (PAI-1). Inhibition of t-PA expression could be induced by addition of exogenous t-PA or t-PA.PAI-1 complexes and reversed by monoclonal antibody directed against the active site of t-PA. Analysis of metabolically radiolabeled protein and cellular mRNA showed that both t-PA protein and mRNA levels declined considerably after 24 h. When 125I-labeled t-PA or t-PA.PAI-1 complexes were incubated with HCF, monensin-inhibitable endocytosis and catabolism were observed. The low density lipoprotein receptor-related protein (LRP) was found to be expressed by HCF and to mediate these events. Addition of the 39-kDa receptor-associated protein (RAP), an antagonist for ligand interactions with LRP, removed the block to t-PA expression and restored its accumulation in the medium. Moreover, RAP completely prevented the degradation of exogenous 125I-labeled t-PA by HCF, suggesting that LRP is the endocytic receptor for t-PA in these cells. These results demonstrate that cellular modulation of t-PA expression in HCF involves LRP receptor-mediated clearance of t-PA. This LRP receptor-mediated event results in down-regulation of t-PA expression at the mRNA level.

    The Journal of biological chemistry 1997;272;10;6812-7

  • Immunocytochemical demonstration of alpha 2-M-R/LRP on Müller (glial) cells isolated from rabbit and human retina.

    Birkenmeier G, Grosche J and Reichenbach A

    Institute of Biochemistry, University of Leipzig, Germany.

    The alpha 2-macroglobulin receptor/low-density lipoprotein receptor-related protein (alpha 2-M-R/LRP) is a multifunctional receptor which has been implicated in lipoprotein metabolism, clearance of proteinase-proteinase inhibitor complexes and regulation of growth factor/cytokine metabolism. This receptor is abundantly present in numerous tissues and organs such as liver, lung, placenta and brain. In brain it is expressed in neurones but not in normal macroglia. Using immunocytochemistry and monoclonal antibodies against the large extracellular receptor subunit we have detected alpha 2-M-R/LRP on enzymatically isolated retinal Müller (glial) cells. This receptor may be involved in vital functions of Müller cells.

    Neuroreport 1996;8;1;149-51

  • Receptor-associated protein is a folding chaperone for low density lipoprotein receptor-related protein.

    Bu G and Rennke S

    Edward Mallinkrodt Department of Pediatrics, Washington University School of Medicine, St. Louis, Missouri 63110, USA.

    The 39-kDa receptor-associated protein (RAP) is a receptor antagonist that inhibits ligand interactions with the receptors that belong to the low density lipoprotein receptor gene family. Our previous studies have demonstrated that RAP interacts with the low density lipoprotein receptor-related protein (LRP) within the endoplasmic reticulum and prevents premature interaction of ligands with the receptor. To analyze whether RAP is also involved in the folding of LRP during receptor biosynthesis, we generated anchor-free, soluble minireceptors that represent each of the four putative ligand-binding domains of LRP (SLRP1, -2, -3, and -4, corresponding to the clusters with 2, 8, 10, and 11 cysteine-rich complement-type repeats, respectively). When these SLRPs were overexpressed by cell transfection, only SLRP1 was secreted. Little or no secretion was observed for SLRP2, -3, and -4. However, when RAP cDNA was cotransfected with SLRP2, -3, and -4 cDNAs, each of these SLRPs was secreted. The cellular retention of SLRPs in the absence of RAP coexpression appeared to be a result of the formation of SDS-resistant, oligomeric aggregates observed under nonreducing conditions. Such oligomers of the SLRPs likely resulted from formation of intermolecular disulfide bonds since they were reduced to monomers when analyzed under reducing conditions. The oligomers were formed not only among molecules of a given SLRP, but also between different SLRPs. The role of RAP in the process of LRP folding was shown by the reduction in aggregated SLRP oligomers upon RAP coexpression. A similar role of RAP in preventing the aggregation of newly synthesized receptor was also observed using membrane-containing minireceptor of LRP. Coimmunoprecipitation and ligand binding studies demonstrated that RAP binds avidly to SLRP2, -3, and -4, but not to SLRP1. These results suggest that these interactions may be important for proper folding of LRP by ensuring the formation of proper intradomain, but not intermolecular or interdomain, disulfide bonds. Thus, our results strongly suggest that, in addition to the prevention of premature binding of ligands to LRP, RAP also plays an important role in receptor folding.

    The Journal of biological chemistry 1996;271;36;22218-24

  • Characterization of alpha 2-macroglobulin binding to human trabecular meshwork cells: presence of the alpha 2-macroglobulin signaling receptor.

    Howard GC, Roberts BC, Epstein DL and Pizzo SV

    Department of Pathology, Duke University Medical Center, Durham, North Carolina 27710, USA.

    Direct binding of receptor-recognized alpha 2-macroglobulin (alpha 2M*) or a cloned receptor binding fragment from rat alpha 1-macroglobulin (RBF) to human trabecular meshwork cells demonstrated two classes of cell surface binding sites. One class has an apparent Kd of 5.0 nM and a receptor number of 31,800 receptors/cell. The other class has an apparent Kd of 20 pM and a receptor number of 1600 receptors/cell. Binding studies of alpha 2M* or RBF in the presence of a competitor for binding to low-density-lipoprotein receptor-related protein/alpha 2M* receptor (LRP/alpha 2MR) called receptor-associated protein (RAP) show that only the lower affinity class of binding sites is susceptible to competition with RAP. Uptake studies demonstrate specific internalization and degradation of alpha 2M* which is inhibitable by RAP. Exposure of the cells to alpha 2M* and RBF (40 nM) is associated with mean increases of 171 and 210%, respectively, in the intracellular calcium concentration, which is not inhibitable by RAP or pertussis toxin. These studies present the first characterization of alpha 2M* and RBF signaling in a primary human cell type and suggest a role for alpha 2M* in the physiology of the eye.

    Funded by: NCI NIH HHS: CA-29589; NEI NIH HHS: EY01894; NHLBI NIH HHS: HL-24066

    Archives of biochemistry and biophysics 1996;333;1;19-26

  • The low-density-lipoprotein receptor-related protein (LRP) is processed by furin in vivo and in vitro.

    Willnow TE, Moehring JM, Inocencio NM, Moehring TJ and Herz J

    Department of Molecular Genetics, University of Texas Southwestern Medical Center, Dallas 75235, USA.

    The low-density-lipoprotein receptor-related protein (LRP) is a multifunctional receptor involved in the clearance of a large number of diverse ligands, including proteases, protease-inhibitor complexes and lipoproteins. The mature receptor is composed of a 515 kDa and a 85 kDa subunit generated by proteolytic cleavage from a 600 kDa precursor polypeptide in a trans-Golgi compartment. Proteolytic processing occurs C-terminal to the tetrabasic amino acid sequence RHRR, a consensus recognition site for precursor processing endoproteases or convertases. In this study we have identified furin, a subtilisin-type protease, to be necessary for efficient processing of LRP in cells. Furin-deficient RPE.40 cells exhibited an impaired processing of endogenous LRP and of a recombinant soluble form of the receptor containing the processing site. The processing defect in RPE.40 cells could be complemented by expression of furin from a transfected cDNA in cultured cells and by purified furin in vitro. The impaired maturation of LRP in RPE.40 cells did not affect its intracellular transport, and correlated with a slight but consistent reduction in the endocytosis of LRP-specific ligands. These data suggest that proteolytic processing of LRP by furin is not necessary for intracellular trafficking but might be required for normal receptor activity.

    Funded by: NHLBI NIH HHS: HL 20948; NIAID NIH HHS: AI09100

    The Biochemical journal 1996;313 ( Pt 1);71-6

  • LDL receptor-related protein, a multifunctional ApoE receptor, binds secreted beta-amyloid precursor protein and mediates its degradation.

    Kounnas MZ, Moir RD, Rebeck GW, Bush AI, Argraves WS, Tanzi RE, Hyman BT and Strickland DK

    Department of Biochemistry, Holland Laboratories, American Red Cross, Rockville, Maryland 20855, USA.

    The secreted form of beta-amyloid precursor protein (APP) containing the Kunitz proteinase inhibitor (KPI) domain, also called protease nexin II, is internalized and degraded by cells. We show that the low density lipoprotein (LDL) receptor-related protein (LRP) is responsible for the endocytosis of secreted APP. APPs770 degradation is inhibited by an LRP antagonist called the receptor-associated protein (RAP) and by LRP antibodies and is greatly diminished in fibroblasts genetically deficient in LRP. APPs695, which lacks the KPI domain, is a poor LRP ligand. Since LRP also binds apolipoprotein E (apoE)-enriched lipoproteins and inheritance of the epsilon 4 allele of the apoE gene is a risk factor for Alzheimer's disease (AD), these data link in a single metabolic pathway two molecules strongly implicated in the pathophysiology of AD.

    Funded by: NHLBI NIH HHS: HL50787; NIDDK NIH HHS: DK45598; NIGMS NIH HHS: GM42581; ...

    Cell 1995;82;2;331-40

  • Identification of the low density lipoprotein receptor-related protein (LRP) as an endocytic receptor for thrombospondin-1.

    Godyna S, Liau G, Popa I, Stefansson S and Argraves WS

    J. H. Holland Laboratory, Biochemistry Department, American Red Cross, Rockville, Maryland 20855, USA.

    Thrombospondin-1 (TSP1) has potent biological effects on vasculature smooth muscle cells (SMCs) and endothelial cells. The regulation of extracellular accumulation of TSP1 is mediated by a previously obscure process of endocytosis which leads to its lysosomal degradation. Since members of the low density lipoprotein receptor (LDLR) family have been found to mediate endocytosis which leads to degradation of a diverse array of ligands, we evaluated their possible role in the uptake and degradation of TSP1 by vascular SMCs, endothelial-cells and fibroblasts. 125I-TSP1 was found to be internalized and degraded lysosomally by all these cell types. Both the internalization and degradation of 125I-TSP1 could be inhibited by a specific antagonist of the LDLR family, the 39-kD receptor-associated protein (RAP). Antibodies to the LDLR-related protein (LRP) completely blocked the uptake and degradation of 125I-TSP1 in SMCs and fibroblasts but not endothelial cells. Solid-phase binding assays confirmed that LRP bound to TSP1 and that the interaction was of high affinity (Kd = 5 nM). Neither RAP nor LRP antibodies inhibited the binding of 125I-TSP1 to surfaces of SMCs. However, cell surface binding, as well as, endocytosis and degradation could be blocked by heparin or by pre-treatment of the cells with either heparitinase, chondroitinase or beta-D-xyloside. The data indicates that cell surface proteoglycans are involved in the LRP-mediated clearance of TSP1. A model for the clearance of TSP1 by these cells is that TSP1 bound to proteoglycans is presented to LRP for endocytosis. In endothelial cells, however, the internalization of TSP1 was not mediated by LRP but since RAP inhibited TSP1 uptake and degradation, we postulate that another member of the LDLR family is likely to be involved.

    Funded by: NHLBI NIH HHS: HL02449, HL37510; NIDDK NIH HHS: DK45598

    The Journal of cell biology 1995;129;5;1403-10

  • The cellular internalization and degradation of hepatic lipase is mediated by low density lipoprotein receptor-related protein and requires cell surface proteoglycans.

    Kounnas MZ, Chappell DA, Wong H, Argraves WS and Strickland DK

    Holland Laboratory, Department of Biochemistry, American Red Cross, Rockville, Maryland 20855, USA.

    Hepatic lipase (HL) and lipoprotein lipase (LpL) are structurally related lipolytic enzymes that have distinct functions in lipoprotein catabolism. In addition to its lipolytic activity, LpL binds to very low density lipoproteins and promotes their interaction with the low density lipoprotein receptor-related protein (LRP) (Chappell, D. A., Fry, G. L., Waknitz, M. A., Muhonen, L. E., Pladet M. W., Iverius, P. H., and Strickland, D. K. (1993) J. Biol. Chem. 268, 14168-14175). In vitro binding assays revealed that HL also binds to purified LRP with a KD of 52 nM. Its binding to LRP is inhibited by the 39-kDa receptor-associated protein (RAP), a known LRP antagonist, and by heparin. 125I-Labeled HL is rapidly internalized and degraded by HepG2 cell lines, and approximately 70% of the cellular internalization and degradation is blocked by either exogenously added RAP or anti-LRP IgG. Mouse fibroblasts that lack LRP display a greatly diminished capacity to internalize and degrade HL when compared to control fibroblasts. These data indicate that LRP-mediated cellular uptake of HL accounts for a substantial portion of the internalization of this molecule. Proteoglycans have been shown to participate in the clearance of LpL, and consequently a role for proteoglycans in HL clearance pathway was also investigated. Chinese hamster ovary cell lines that are deficient in proteoglycan biosynthesis were unable to internalize or degrade 125I-HL despite the fact that these cells express LRP. Thus, the initial binding of HL to cell surface proteoglycans is an obligatory step for the delivery of the enzyme to LRP for endocytosis. A small, but significant, amount of 125I-HL was internalized in LRP deficient cells indicating that an LRP-independent pathway for HL internalization does exist. This pathway could involve cell surface proteoglycans, the LDL receptor, or some other unidentified surface protein.

    Funded by: NHLBI NIH HHS: HL49264, HL50787; NIGMS NIH HHS: GM42581; ...

    The Journal of biological chemistry 1995;270;16;9307-12

  • A carboxyl-terminal fragment of lipoprotein lipase binds to the low density lipoprotein receptor-related protein and inhibits lipase-mediated uptake of lipoprotein in cells.

    Nykjaer A, Nielsen M, Lookene A, Meyer N, Røigaard H, Etzerodt M, Beisiegel U, Olivecrona G and Gliemann J

    Department of Medical Biochemistry, University of Aarhus, Denmark.

    It has previously been shown that lipoprotein lipase can mediate uptake of remnant lipoprotein particles via binding to the low density lipoprotein receptor-related protein/alpha 2-macroglobulin receptor (LRP). Binding of lipoprotein lipase, and of triglyceride-rich lipoproteins associated with the lipase, to LRP depends on an intact carboxyl-terminal folding domain of the lipase (Nykjaer, A., Bengtsson-Olivecrona, G., Lookene, A., Moestrup, S. K., Petersen, C. M., Weber, W., Beisiegel, W., and Gliemann, J. (1993) J. Biol. Chem. 268, 15048-15055). Here we show that the site for binding to the receptor is within residues 380-425 of the bovine and residues 378-423 of the human lipoprotein lipase. We demonstrate that a carboxyl-terminal fragment of human lipoprotein lipase (residues 378-448), expressed as fusion protein in Escherichia coli, binds to purified and cellular LRP but not to lipoproteins. Binding of the fragment to purified LRP was blocked by heparin. In addition, the fragment inhibited the binding of lipase and the lipase-mediated binding of lipoproteins to the purified receptor. The fragment exhibited reduced binding to proteoglycan-deficient cells. Moreover, the fragment inhibited the uptake of lipoproteins in cells mediated by the lipase via binding to heparan sulfate proteoglycans and LRP. We conclude that the fragment contains the site for binding to LRP and a candidate site for interaction with heparan sulfate proteoglycans, whereas binding to lipoproteins is inefficient. The fragment can therefore inhibit the lipase-mediated lipoprotein uptake, a process that may promote the development of atherosclerosis when occurring in cells of the arterial wall.

    The Journal of biological chemistry 1994;269;50;31747-55

  • Subcellular localization and endocytic function of low density lipoprotein receptor-related protein in human glioblastoma cells.

    Bu G, Maksymovitch EA, Geuze H and Schwartz AL

    Edward Mallinckrodt Department of Pediatrics, Washington University School of Medicine, St. Louis, Missouri 63110.

    The low density lipoprotein receptor-related protein (LRP) is a multifunctional cell surface receptor that binds and endocytoses several structurally and functionally distinct ligands. Several of the ligands for LRP participate in both normal physiology and pathophysiology of the central nervous system. To begin to gain insights into the role of LRP in the central nervous system, we have analyzed the expression, subcellular distribution, and endocytic function of LRP in human glioblastoma U87 cells. These cells express an abundance of LRP at both the mRNA and protein levels. A 39-kDa protein, which copurifies with LRP and regulates its ligand binding activity, is also highly expressed in U87 cells. The subcellular localization of LRP and the 39-kDa protein was analyzed using scanning laser confocal and electron microscopy combined with immunolabeled U87 cells. At the plasma membrane, LRP was largely confined to clathrin-coated pits. Within cells, LRP and the 39-kDa protein partially colocalized within rough endoplasmic reticulum and the Golgi complex, suggesting a potential intracellular interaction between the two proteins. Little 39-kDa protein was found in endosomes in which LRP occurred abundantly. In examining the functional role of LRP in U87 cells, we found that LRP at the cell surface and along the cellular processes was functional in the binding and endocytosis of its ligands, and its activity therein was regulated by the 39-kDa protein. Using truncated recombinant 39-kDa protein constructs, we also demonstrated that distinct regions of the 39-kDa protein were responsible for inhibiting the binding of different LRP ligands on U87 cells. Our results thus strongly suggest several potential roles for LRP in brain protein and lipoprotein metabolism, as well as control of extracellular protease activity.

    Funded by: NHLBI NIH HHS: HL07275, HL52040

    The Journal of biological chemistry 1994;269;47;29874-82

  • Structure of the gene (LRP1) coding for the human alpha 2-macroglobulin receptor lipoprotein receptor-related protein.

    Van Leuven F, Stas L, Hilliker C, Lorent K, Umans L, Serneels L, Overbergh L, Torrekens S, Moechars D, De Strooper B et al.

    Experimental Genetics Group, Center for Human Genetics, K. U. Leuven, Belgium.

    The alpha 2-macroglobulin receptor or lipoprotein receptor-related protein (A2MR/LRP) is an amazingly large and multifunctional receptor. The active receptor protein is derived from a 600-kDa precursor, encoded by a 15-kb mRNA, cloned and sequenced in human, mouse, and chicken. We report here the cloning of the entire human gene (LRP1) coding for A2MR/LRP. The gene covered about 92 kb and a total of 89 exons were identified, varying in size from 65 bases (exon 86) to 925 bases (exon 89). The introns varied from 82 bases (intron 53) to about 8 kb (intron 6). In the introns, 3 complete and 4 partial Alu sequences were identified. In intron 44 a complex repetitive sequence posed a cloning problem since it was not retrieved from any genomic library screened. Interexon PCR from exon 43 to 45 yielded a fragment of 2.5 kb. Attempts to subclone this fragment yielded inserts ranging between 0.8 and 1.6 kb. Sequencing of 3 subclones with different-size inserts revealed a complex repetitive element with a different size in each subclone. In the mouse LRP gene this intron was much smaller, and no repetitive sequence was observed. In 18 unrelated individuals no difference in size was observed when analyzed by interexon PCR.

    Genomics 1994;24;1;78-89

  • The carboxyl-terminal domain of lipoprotein lipase binds to the low density lipoprotein receptor-related protein/alpha 2-macroglobulin receptor (LRP) and mediates binding of normal very low density lipoproteins to LRP.

    Williams SE, Inoue I, Tran H, Fry GL, Pladet MW, Iverius PH, Lalouel JM, Chappell DA and Strickland DK

    Biochemistry Laboratory, American Red Cross, Rockville, Maryland 20855.

    Lipoprotein lipase (LPL) binds with high affinity to the low density lipoprotein receptor-related protein/alpha 2-macroglobulin receptor (LRP) and promotes binding, uptake, and degradation of normal triglyceride-rich lipoproteins in a process mediated by LRP (Chappell, D. A., Fry, G. L., Naknitx, M.A., Muhonen, L. E., Pladet, M. W., Iverius, P-H., and Strickland, D. K. (1993) J. Biol. Chem. 268, 14168-14175). To localize the portion of LPL that is responsible for interacting with LRP, fragments of LPL were expressed in bacteria. A fragment of human LPL containing the COOH-terminal domain (residues 313-448, designated LPLC) which lacks the catalytic site was able to bind to LRP. Purified LRP bound specifically to microtiter wells coated with LPL or LPLC with KD values of 2.8 and 5 nM, respectively. The effects of several mutations of LPLC were tested. Mutation of Lys407 to Ala reduced the affinity of LPLC for LRP by approximately 10-fold. Like native LPL, LPLC prevented the binding of activated alpha 2-macroglobulin and the 39-kDa receptor-associated protein to LRP and inhibited the internalization and degradation of activated alpha 2-macroglobulin and receptor-associated protein in cultured fibroblasts. LPLC also bound to 125I-labeled human normal triglyceride-rich lipoproteins and promoted their binding to purified LRP and to cultured cells. Mutation of Trp393 and Trp394 to Ala completely abolished the ability of LPLC to bind to lipoproteins, but had little effect on its interaction with LRP. These data indicate that the COOH-terminal domain of LPL may function both in binding lipoproteins and mediating their interaction with LRP.

    Funded by: NHLBI NIH HHS: HL30200, HL49264; NIGMS NIH HHS: GM42581; ...

    The Journal of biological chemistry 1994;269;12;8653-8

  • Expression of alpha 2-macroglobulin receptor/low density lipoprotein receptor-related protein is increased in reactive and neoplastic glial cells.

    Lopes MB, Bogaev CA, Gonias SL and VandenBerg SR

    Department of Pathology, University of Virginia Health Sciences Center, Charlottesville 22908.

    alpha 2-Macroglobulin receptor/low density lipoprotein receptor-related protein (alpha 2M-R/LRP) is a multi-functional cell-surface receptor that has been implicated in diverse physiologic processes. In normal human brain, alpha 2M-R/LRP is expressed principally by pyramidal neurons with localization to cell bodies and proximal processes. By contrast, alpha 2M-R/LRP is not present in either the cell bodies or processes of most normal macroglia (including astrocytes). In this investigation, we studied the expression of alpha 2M-R/LRP in the brain, following tissue injury or neoplastic transformation, by immunohistochemistry. Significantly increased alpha 2M-R/LRP immunoreactivity was identified in reactive astrocytes, indicating the expression of this receptor is regulated in vivo in response to brain injury. alpha 2M-R/LRP immunoreactivity was also detected in glial cell tumors; this finding is novel since malignant transformation is typically thought to turn off expression of this receptor.

    Funded by: NCI NIH HHS: CA53462; NHLBI NIH HHS: HL-02272; NINDS NIH HHS: NS7236

    FEBS letters 1994;338;3;301-5

  • Expression of alpha 2-macroglobulin receptor/low density lipoprotein receptor-related protein and the 39-kd receptor-associated protein in human trophoblasts.

    Coukos G, Gåfvels ME, Wisel S, Ruelaz EA, Strickland DK, Strauss JF and Coutifaris C

    Department of Obstetrics and Gynecology, University of Pennsylvania School of Medicine, Philadelphia.

    The alpha 2-macroglobulin receptor/low density lipoprotein receptor-related protein (alpha 2MR/LRP) and its 39-kd receptor-associated protein (RAP) were identified by indirect immunofluorescence in human extravillous and villous trophoblast cells at different stages of pregnancy. The alpha 2MR/LRP was detected in invading trophoblast cells and in some instances these invading cells did not express RAP. In chorionic villi of first and second trimester placenta, alpha 2MR/LRP and RAP were found in cytotrophoblast and syncytiotrophoblast. With advancing pregnancy alpha 2MR/LRP became primarily localized to the apical surface of the syncytiotrophoblast, while RAP was present in the cytoplasm. Villous cytotrophoblast cells lost both proteins by the third trimester. Isolated cytotrophoblast cells that undergo spontaneous differentiation into syncytiotrophoblast in culture increased expression of both alpha 2MR/LRP and RAP. With syncytium formation, alpha 2MR/LRP became localized to the plasma membrane in cup-like structures. Changes in the mRNAs for alpha 2MR/LRP and RAP paralleled the changes in relative abundance of the proteins assessed by immunofluorescence. cAMP treatment suppressed both alpha 2MR/LRP and RAP in the cultured trophoblasts, but alpha 2MR/LRP was reduced to a greater extent than RAP. We conclude that alpha 2MR/LRP and RAP are developmentally regulated in human trophoblast cells, that the temporal and spatial patterns of expression of these proteins can be dissociated, and that cAMP modulates both alpha 2MR/LRP and RAP in human trophoblast. The patterns of alpha 2MR/LRP and RAP expression in trophoblast cells are consistent with roles for the receptor in trophoblast invasion and transport of molecules across the syncytiotrophoblast.

    Funded by: NICHD NIH HHS: HD-29946; NIGMS NIH HHS: GM-42581

    The American journal of pathology 1994;144;2;383-92

  • The low density lipoprotein receptor-related protein/alpha 2-macroglobulin receptor binds and mediates catabolism of bovine milk lipoprotein lipase.

    Chappell DA, Fry GL, Waknitz MA, Iverius PH, Williams SE and Strickland DK

    Department of Internal Medicine, University of Iowa College of Medicine, Iowa City 52242.

    Lipoprotein lipase (LPL), the major lipolytic enzyme involved in the conversion of triglyceride-rich lipoproteins to remnants, was found to compete with binding of activated alpha 2-macroglobulin (alpha 2M*) to the low density lipoprotein receptor-related protein (LRP)/alpha 2-macroglobulin receptor. Bovine milk LPL displaced both 125I-labeled alpha 2M* and 39-kDa alpha 2M receptor-associated protein (RAP) from the surface of cultured mutant fibroblasts lacking LDL receptors with apparent KI values at 4 degrees C of 6.8 and 30 nM, respectively. Furthermore, LPL inhibited the cellular degradation of 125I-alpha 2M* at 37 degrees C. Because both alpha 2M* and RAP interact with LRP, these data suggest that LPL binds specifically to this receptor. This was further supported by observing that an immunoaffinity-isolated polyclonal antibody against LRP blocked cellular degradation of 125I-LPL in a dose-dependent manner. In addition, 125I-LPL bound to highly purified LRP in a solid-phase assay with a KD of 18 nM, and this binding could be partially displaced with alpha 2M* (KI = 7 nM) and RAP (KI = 3 nM). Taken together, these data establish that LPL binds with high affinity to LRP and undergoes LRP-mediated cellular uptake. The implication of these findings for lipoprotein catabolism in vivo may be important if LRP binding is preserved when LPL is attached to lipoproteins. If so, LPL might facilitate LRP-mediated clearance of lipoproteins.

    Funded by: NHLBI NIH HHS: HL02024, HL39595; NIGMS NIH HHS: GM42581; ...

    The Journal of biological chemistry 1992;267;36;25764-7

  • Low density lipoprotein receptor-related protein and gp330 bind similar ligands, including plasminogen activator-inhibitor complexes and lactoferrin, an inhibitor of chylomicron remnant clearance.

    Willnow TE, Goldstein JL, Orth K, Brown MS and Herz J

    Department of Molecular Genetics, University of Texas Southwestern Medical Center, Dallas 75235.

    The low density lipoprotein receptor-related protein/alpha 2-macroglobulin receptor (LRP) and gp330, two members of the low density lipoprotein receptor gene family, share a multitude of cysteine-rich repeats. LRP has been shown to act as an endocytosis-mediating receptor for several ligands, including protease-antiprotease complexes and plasma lipoproteins. The former include alpha 2-macroglobulin-protease complexes and plasminogen activator inhibitor-activator complexes. The latter include chylomicron remnant-like particles designated beta-very low density lipoproteins (beta-VLDL) complexed with apoprotein E or lipoprotein lipase. The binding specificity of gp330 is unknown. In the current studies we show that gp330 from rat kidney membranes binds several of these ligands on nitrocellulose blots. We also show that both LRP and gp330 bind an additional ligand, bovine lactoferrin, which is known to inhibit the hepatic clearance of chylomicron remnants. Lactoferrin blocked the LRP-dependent stimulation of cholesteryl ester synthesis in cultured human fibroblasts elicited by apoprotein E-beta-VLDL or lipoprotein lipase-beta-VLDL complexes. Cross-competition experiments in fibroblasts showed that the multiple ligands recognize at least three distinct, but partially overlapping sites on the LRP molecule. Binding of all ligands to LRP and gp330 was inhibited by the 39-kDa protein, which co-purifies with the two receptors, suggesting that the 39-kDa protein is a universal regulator of ligand binding to both receptors. The correlation of the inhibitory effects of lactoferrin in vivo and in vitro support the notion that LRP functions as a chylomicron remnant receptor in liver. LRP and gp330 share a multiplicity of binding sites, and both may function as endocytosis-mediating receptors for a large number of ligands in different organs.

    Funded by: NHLBI NIH HHS: HL 20948

    The Journal of biological chemistry 1992;267;36;26172-80

  • Distribution of the alpha 2-macroglobulin receptor/low density lipoprotein receptor-related protein in human tissues.

    Moestrup SK, Gliemann J and Pallesen G

    Institute of Medical Biochemistry, Aarhus University, Denmark.

    The hepatic alpha 2-macroglobulin receptor (alpha 2MR)/low density lipoprotein receptor-related protein (LRP) binds and endocytoses alpha 2-macroglobulin-proteinase complexes in plasma. In addition, it binds lipoproteins, a novel 40 kDa protein, and complexes between plasminogen activators and plasminogen activator inhibitor type-1. This study shows, for the first time, the tissue distribution of alpha 2MR/LRP as determined by immunohistochemistry with specific monoclonal antibodies. The analysis revealed alpha 2MR/LRP-expression in a restricted spectrum of cell types, including neurons and astrocytes in the central nervous system, epithelial cells of the gastrointestinal tract, smooth muscle cells, fibroblasts, Leydig cells in testis, granulosa cells in ovary, and dendritic interstitial cells of kidney. Monocyte-derived cells displayed marked alpha 2MR/LRP expression in the phagocytes of liver, lung and lymphoid tissues, but no or low expression in antigen-presenting cells including Langerhans' cells of the skin. The high abundance of alpha 2MR/LRP in certain cell types of most organs suggests two main routes for alpha 2MR/LRP-mediated ligand clearance: (1) systemic removal in liver of circulating ligands, and (2) non-hepatic interstitial removal in different organs, including the brain.

    Cell and tissue research 1992;269;3;375-82

  • Complexes of tissue-type plasminogen activator and its serpin inhibitor plasminogen-activator inhibitor type 1 are internalized by means of the low density lipoprotein receptor-related protein/alpha 2-macroglobulin receptor.

    Orth K, Madison EL, Gething MJ, Sambrook JF and Herz J

    Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas 75235.

    Tissue-type plasminogen activator and urokinase are serine proteases secreted by many cell types that participate in biological processes, such as tissue restructuring, cell migration, and tumor metastasis. Clinically, these proteases are used to dissolve coronary fibrin clots that are the proximal causes of acute myocardial infarction. In vivo, the activity of these enzymes is controlled by plasminogen-activator inhibitors, members of the serpin family of protease inhibitors. This study shows that tissue-type plasminogen activator-inhibitor complexes bind in solution to low density lipoprotein receptor-related protein (LRP), a large heterodimeric ubiquitous membrane receptor. In cultured cells, endocytosis and degradation of these complexes is reduced by polyclonal antibodies directed against LRP and inhibited by a M(r) 39,000 protein that binds to LRP and inhibits its interaction with previously known ligands, including apolipoprotein E and alpha 2-macroglobulin. We propose a role for LRP in the clearance of plasminogen activator-inhibitor complexes that is analogous to its function in the endocytosis of alpha 2-macroglobulin-protease complexes.

    Proceedings of the National Academy of Sciences of the United States of America 1992;89;16;7422-6

  • Assignment of the gene coding for the alpha 2-macroglobulin receptor to mouse chromosome 15 and to human chromosome 12q13-q14 by isotopic and nonisotopic in situ hybridization.

    Hilliker C, Van Leuven F and Van den Berghe H

    Center for Human Genetics, University of Leuven, Belgium.

    The assignment of the gene encoding the alpha 2-macroglobulin receptor (A2MR), which was first described as the low-density lipoprotein receptor-related protein, was confirmed by nonisotopic and isotopic in situ hybridizations on normal human metaphases to the region 12q13-q14. The same human cDNA, which has 95% sequence identity with the mouse A2mr, was hybridized to metaphases containing the Robertsonian translocation Rb(6;15)1Ald. The mouse A2mr gene was assigned to chromosome 15 in the region B2-D1. This locus and other loci on mouse chromosome 15 have been shown to be homologous with loci on human chromosome 12q.

    Genomics 1992;13;2;472-4

  • A novel mechanism for controlling the activity of alpha 2-macroglobulin receptor/low density lipoprotein receptor-related protein. Multiple regulatory sites for 39-kDa receptor-associated protein.

    Williams SE, Ashcom JD, Argraves WS and Strickland DK

    Biochemistry Laboratory, American Red Cross, Rockville, Maryland 20855.

    The alpha 2-macroglobulin receptor/low density lipoprotein receptor-related protein (alpha 2MR/LRP) consists of two polypeptides, 515 and 85 kDa, that are noncovalently associated. A 39-kDa polypeptide, termed the receptor-associated protein (RAP), interacts with the 515-kDa subunit after biosynthesis of these molecules and remains associated on the cell surface. This molecule regulates ligand binding of alpha 2MR/LRP (Herz, J., Goldstein, J. L., Strickland, D. K., Ho, Y. K., and Brown, M. S. (1991) J. Biol. Chem. 266, 21232-21238). Titration and binding studies indicate that RAP binds to two equivalent binding sites on alpha 2MR/LRP, with a KD of 14 nM. Heterologous ligand displacement experiments demonstrated that RAP completely inhibits the binding of 125I-activated alpha 2M to human fibroblasts and to the purified alpha 2MR/LRP, with a Ki of 23 and 26 nM, respectively. A direct correlation between the degree of binding of RAP to the receptor and the degree of ligand inhibition was observed, indicating that as the RAP binding sites are saturated, alpha 2MR/LRP loses its ability to bind ligands. Thus, the amount of RAP bound to alpha 2MR/LRP dictates the level of receptor activity. A model is proposed in which alpha 2MR/LRP contains multiple ligand binding sites, each regulated by a separate RAP site.

    Funded by: NHLBI NIH HHS: HL-02113, HL30200; NIGMS NIH HHS: GM42581; ...

    The Journal of biological chemistry 1992;267;13;9035-40

  • 39-kDa protein modulates binding of ligands to low density lipoprotein receptor-related protein/alpha 2-macroglobulin receptor.

    Herz J, Goldstein JL, Strickland DK, Ho YK and Brown MS

    Department of Molecular Genetics, University of Texas, Southwestern Medical Center, Dallas 75235.

    A 39-kDa protein of unknown function has previously been reported to copurify with the low density lipoprotein receptor-related protein (LRP)/alpha 2-macroglobulin receptor. In this study we demonstrate that a recombinant 39-kDa fusion protein can reversibly bind to the 515-kDa subunit of the LRP/alpha 2-macroglobulin receptor. This interaction inhibits the binding and uptake of the receptor's two known ligands: 1) beta-migrating very low density lipoproteins activated by enrichment with apoprotein E and 2) alpha 2-macroglobulin activated by incubation with plasma proteases or methylamine. A potential in vivo role of the 39-kDa protein is to modulate the uptake of apoE-enriched lipoproteins and activated alpha 2-macroglobulin in hepatic and extrahepatic tissues.

    Funded by: NHLBI NIH HHS: HL20948, HL30200; NIGMS NIH HHS: GM42581; ...

    The Journal of biological chemistry 1991;266;31;21232-8

  • Evidence that the newly cloned low-density-lipoprotein receptor related protein (LRP) is the alpha 2-macroglobulin receptor.

    Kristensen T, Moestrup SK, Gliemann J, Bendtsen L, Sand O and Sottrup-Jensen L

    Department of Molecular Biology, University of Aarhus, Denmark.

    The human placental receptor (alpha 2MR) for alpha 2-macroglobulin-proteinase complexes contains 3 polypeptides of approx. 500 kDa, 85 kDa, and 40 kDa. N-terminal sequence analysis of the 500 kDa and 85 kDa polypeptides, analysis of a random selection of peptides convering 536 residues from these polypeptides, and analysis of a 1772 bp cDNA encoding part of the 500 kDa polypeptide provide evidence that the 500 kDa and 85 kDa chains are the alpha- and beta-subunits, respectively, of a recently cloned hepatic membrane protein, termed the low density lipoprotein receptor related protein (LRP) (Herz, J., Hamann, U., Rogne, S., Myklebost, O., Gausepohl, H. and Stanley, K.K. (1988) EMBO J. 7, 4119-4127; Herz, J., Kowal, R.C., Goldstein, J.L. and Brown, M.S. (1990) EMBO J. 9, 1769-1776). N-terminal sequence analysis of the 40 kDa polypeptide shows that it is of distinct genetic origin. It is suggested that LRP is the functional receptor for alpha 2-macroglobulin-proteinase complexes (alpha 2MR) and in addition may have as yet unsettled functions in lipoprotein metabolism.

    FEBS letters 1990;276;1-2;151-5

  • Sequence identity between the alpha 2-macroglobulin receptor and low density lipoprotein receptor-related protein suggests that this molecule is a multifunctional receptor.

    Strickland DK, Ashcom JD, Williams S, Burgess WH, Migliorini M and Argraves WS

    Biochemistry Laboratory, American Red Cross, Rockville, Maryland 20855.

    Ten peptides, derived from human alpha 2-macroglobulin (alpha 2M) receptor by chemical or proteolytic digestion, were sequenced. Comparative analysis revealed that all of the resulting sequences were present within the cDNA-deduced structure of low density lipoprotein receptor-related protein (LRP) (Herz, J., Hamann, U., Rogne, S., Myklebost, O., Gausepohl, H., and Stanley, K. K. (1988) EMBO J. 7, 4119-4127). The findings provide evidence that the alpha 2M receptor and LRP are the same molecule. Further evidence comes from immunoprecipitation experiments using a monoclonal antibody specific for the alpha 2M receptor that show this molecule, like LRP, to contain two polypeptides of approximately 420 and 85 kDa that are noncovalently associated. An additional component of this receptor system is a 39-kDa polypeptide that co-purifies with the alpha 2M receptor during affinity chromatography. Solid phase binding studies reveal that the 39-kDa polypeptide binds with high affinity (Kd = 18 nM) to the 420-kDa component of the alpha 2M receptor. The apparent identity of LRP and the alpha 2M receptor suggests that this molecule is a multifunctional receptor with the capacity to bind diverse biological ligands and highlights a possible relationship between two previously unrelated biological processes, lipid metabolism and proteinase regulation.

    Funded by: NHLBI NIH HHS: HL-02113, HL30200; NIGMS NIH HHS: GM42581

    The Journal of biological chemistry 1990;265;29;17401-4

  • Proteolytic processing of the 600 kd low density lipoprotein receptor-related protein (LRP) occurs in a trans-Golgi compartment.

    Herz J, Kowal RC, Goldstein JL and Brown MS

    Department of Molecular Genetics, University of Texas Southwestern Medical Center, Dallas 75235.

    The low density lipoprotein receptor-related protein (LRP) is a cell surface glycoprotein that binds and transports plasma lipoproteins enriched in apolipoprotein E. It is synthesized in the endoplasmic reticulum as a transmembrane glycosylated precursor that migrates with an apparent molecular mass of about 600 kd on SDS-polyacrylamide gels. After it reaches the Golgi complex, the protein is cleaved to generate two subunits with apparent molecular masses of approximately 515 and 85 kd respectively. The larger NH2-terminal alpha-subunit lacks a membrane-spanning region. It remains attached to the membrane through noncovalent association with the smaller COOH-terminal beta-subunit. Proteolysis occurs at the sequence RHRR, which resembles the sequence RKRR at the proteolytic site in the receptors for insulin and insulin-like growth factor-1 (IGF-1), the only other cell surface receptors known to undergo proteolytic processing. Proteolysis of LRP occurs coincident with the conversion of the N-linked carbohydrates to the mature endoglycosidase H-resistant, neuraminidase-sensitive form. Proteolysis is prevented by brefeldin A, which blocks transport to the Golgi complex. These data raise the possibility that LRP and the receptors for insulin and IGF-1 are processed by a specific endoprotease that recognizes protein with extended basic sequences and resides in the trans-Golgi complex or in post-Golgi vesicles of the constitutive secretory pathway.

    Funded by: NHLBI NIH HHS: HL 20948; NIGMS NIH HHS: GM 08014

    The EMBO journal 1990;9;6;1769-76

  • Structure of the low-density lipoprotein receptor-related protein (LRP) promoter.

    Kütt H, Herz J and Stanley KK

    European Molecular Biology Laboratory, Heidelberg, F.R.G.

    The low-density Lipoprotein receptor-related protein (LRP) is a 4544-amino-acid membrane protein which closely resembles the LDL receptor in its arrangement of cysteine-rich motifs. Binding studies have suggested that one function of the molecule is as a receptor for ligands containing apolipoprotein E. We present here the sequence and structure of the promoter region of the LRP. These data show that the LRP contains no sterol regulatory element, and is not down-regulated by sterols like the LDL receptor. This lends further support to the identity of the LRP as a chylomicron remnant receptor.

    Biochimica et biophysica acta 1989;1009;3;229-36

  • The LDL-receptor-related protein, LRP, is an apolipoprotein E-binding protein.

    Beisiegel U, Weber W, Ihrke G, Herz J and Stanley KK

    Medical Clinic, University Hospital Eppendorf, Hamburg.

    The low-density-lipoprotein (LDL) receptor is a cell-surface protein that plays an important part in the metabolism of cholesterol by mediating the uptake of LDL from plasma into cells. Although LDL particles bind to the LDL receptor through their apolipoprotein B (apo B) and apolipoprotein E (apo E) moieties, other apo E-containing particles, like chylomicron remnants, are not dependent on the LDL receptor for uptake into cells. Chylomicrons formed in the intestinal mucosa during the absorption of the products of digestion, are processed by the peripheral circulation by lipoprotein lipase, which catalyses the breakdown of triglycerides in chylomicrons to free fatty acids and glycerol. The resulting chylomicron remnants, which are cholesterol-rich lipoproteins, are subsequently taken up in the liver. A second distinct protein that binds to apo E-containing lipoproteins, but not to LDL, has been proposed to be the receptor mediating the clearance of chylomicron remnants from the plasma. This protein has a relative molecular mass (Mr) of 56,000 (56K). More recent studies have failed, however, to establish whether this protein is a cell-surface receptor. Here we describe crosslinking experiments in which apo E liposomes were found to bind specifically to the cell surface of hepG2 cells and to human liver membranes. The size and immunological cross-reactivity of the protein to which the liposomes bound was indistinguishable from that of the recently cloned and sequenced LDL-receptor-related protein, LRP. We therefore conclude that the LRP might function as an apo E receptor.

    Nature 1989;341;6238;162-4

  • The gene for the human putative apoE receptor is on chromosome 12 in the segment q13-14.

    Myklebost O, Arheden K, Rogne S, Geurts van Kessel A, Mandahl N, Herz J, Stanley K, Heim S and Mitelman F

    Biochemistry Department, Institute for Cancer Research, Montebello, Norway.

    We have previously described the cDNA coding for a new lipoprotein receptor that contains domains closely related to the ligand-binding domain of the LDL receptor. We have now investigated the localization of the gene for this new receptor by hybridization of the cDNA to panels of rodent cells containing subsets of human chromosomes and by in situ hybridization of the cDNA to chromosomes. The gene maps to 12q13-14, a known hot spot for chromosomal rearrangements in human neoplasia. Of particular interest is the frequent involvement of the 12q13-14 segment in clonal abnormalities in lipomas and myxoid liposarcomas, and it is possible that LRP may play a role in the pathogenesis of such tumors.

    Genomics 1989;5;1;65-9

  • Surface location and high affinity for calcium of a 500-kd liver membrane protein closely related to the LDL-receptor suggest a physiological role as lipoprotein receptor.

    Herz J, Hamann U, Rogne S, Myklebost O, Gausepohl H and Stanley KK

    European Molecular Biology Laboratory, Heidelberg, FRG.

    We describe a cell surface protein that is abundant in liver and has close structural and biochemical similarities to the low density lipoprotein (LDL) receptor. The complete sequence of the protein containing 4544 amino acids is presented. From the sequence a remarkable resemblance to the LDL-receptor and epidermal growth factor (EGF) precursor is apparent. Three types of repeating sequence motifs entirely account for the extracellular domain of the molecule. These are arranged in a manner resembling four copies of the ligand binding and the EGF-precursor homologous region of the LDL-receptor. Following a proline-rich segment of 17 amino acids are found six consecutive repeats with close homology to EGF. A single membrane-spanning segment precedes a carboxy-terminal 'tail' of 100 amino acids. This contains two seven-amino acid sequences with striking homology to the cytoplasmic tail of the LDL-receptor in the region that contains the signal for clustering into coated pits. The mRNA for this protein is most abundant in liver, brain and lung. By using an antibody raised against a 13-amino acid peptide corresponding to the deduced amino acid sequence of the carboxy-terminus of the protein we have demonstrated its existence on the cell surface and its abundance in liver. Like the LDL-receptor this protein also strongly binds calcium, a cation absolutely required for binding of apolipoproteins B and E to their receptors. We propose that this LDL-receptor related protein (LRP) is a recycling lipoprotein receptor with possible growth-modulating effects.

    The EMBO journal 1988;7;13;4119-27

Gene lists (4)

Gene List Source Species Name Description Gene count
L00000009 G2C Homo sapiens Human PSD Human orthologues of mouse PSD adapted from Collins et al (2006) 1080
L00000016 G2C Homo sapiens Human PSP Human orthologues of mouse PSP adapted from Collins et al (2006) 1121
L00000069 G2C Homo sapiens BAYES-COLLINS-HUMAN-PSD-FULL Human cortex biopsy PSD full list 1461
L00000071 G2C Homo sapiens BAYES-COLLINS-MOUSE-PSD-FULL Mouse cortex PSD full list (ortho) 1556
© G2C 2014. The Genes to Cognition Programme received funding from The Wellcome Trust and the EU FP7 Framework Programmes:
EUROSPIN (FP7-HEALTH-241498), SynSys (FP7-HEALTH-242167) and GENCODYS (FP7-HEALTH-241995).

Cookies Policy | Terms and Conditions. This site is hosted by Edinburgh University and the Genes to Cognition Programme.