G2Cdb::Gene report

Gene id
G00002112
Gene symbol
DLG1 (HGNC)
Species
Homo sapiens
Description
discs, large homolog 1 (Drosophila)
Orthologue
G00000863 (Mus musculus)

Databases (7)

Gene
ENSG00000075711 (Ensembl human gene)
1739 (Entrez Gene)
12 (G2Cdb plasticity & disease)
DLG1 (GeneCards)
Literature
601014 (OMIM)
Marker Symbol
HGNC:2900 (HGNC)
Protein Sequence
Q12959 (UniProt)

Synonyms (5)

  • DLGH1
  • SAP-97
  • SAP97
  • dJ1061C18.1.1
  • hdlg

Diseases (2)

Disease Nervous effect Mutations Found Literature Mutations Type Genetic association?
D00000108: Mammary ductal carcinoma N Y (14871824) Single nucleotide polymorphism (SNP) Y
D00000318: 3q29 microdeletion syndrome Y Y (15918153) Microdeletion (MD) Y

References

  • 3q29 microdeletion syndrome: clinical and molecular characterization of a new syndrome.

    Willatt L, Cox J, Barber J, Cabanas ED, Collins A, Donnai D, FitzPatrick DR, Maher E, Martin H, Parnau J, Pindar L, Ramsay J, Shaw-Smith C, Sistermans EA, Tettenborn M, Trump D, de Vries BB, Walker K and Raymond FL

    Department of Medical Genetics, Addenbrooke's Hospital, University of Cambridge, Cambridge, United Kingdom.

    We report the identification of six patients with 3q29 microdeletion syndrome. The clinical phenotype is variable despite an almost identical deletion size. The phenotype includes mild-to-moderate mental retardation, with only slightly dysmorphic facial features that are similar in most patients: a long and narrow face, short philtrum, and high nasal bridge. Autism, gait ataxia, chest-wall deformity, and long and tapering fingers were noted in at least two of six patients. Additional features--including microcephaly, cleft lip and palate, horseshoe kidney and hypospadias, ligamentous laxity, recurrent middle ear infections, and abnormal pigmentation--were observed, but each feature was only found once, in a single patient. The microdeletion is approximately 1.5 Mb in length, with molecular boundaries mapping within the same or adjacent bacterial artificial chromosome (BAC) clones at either end of the deletion in all patients. The deletion encompasses 22 genes, including PAK2 and DLG1, which are autosomal homologues of two known X-linked mental retardation genes, PAK3 and DLG3. The presence of two nearly identical low-copy repeat sequences in BAC clones on each side of the deletion breakpoint suggests that nonallelic homologous recombination is the likely mechanism of disease causation in this syndrome.

    Funded by: Wellcome Trust

    American journal of human genetics 2005;77;1;154-60

  • Somatic mutations and altered expression of the candidate tumor suppressors CSNK1 epsilon, DLG1, and EDD/hHYD in mammary ductal carcinoma.

    Fuja TJ, Lin F, Osann KE and Bryant PJ

    Developmental Biology Center and Department of Medicine, University of California-Irvine, Irvine, California, USA.

    We report somatic mutations in three genes (CSNK1 epsilon, encoding the Ser/Thr kinase casein kinase I epsilon; DLG1, encoding a membrane-associated putative scaffolding protein; and EDD/hHYD, encoding a progestin induced putative ubiquitin-protein ligase) in mammary ductal carcinoma. These genes were suspected of playing a role in cancer because loss-of-function mutations in their Drosophila homologues cause excess tissue growth. Using DNA from 82 laser-microdissected tumor samples, followed by microsatellite analysis, denaturing HPLC and direct sequencing, we found multiple somatic point mutations in all three genes, and these mutations showed significant association with loss of heterozygosity of closely linked polymorphic microsatellite markers. For CSNK1 epsilon and DLG1, most of the mutations affected highly conserved residues, some were found repetitively in different patients, and no synonymous mutations were found, indicating that the observed mutations were selected in tumors and may be functionally significant. Immunohistochemical reactivity of each protein was reduced in poorly differentiated tumors, and there was a positive association between altered protein reactivity, loss of heterozygosity, and somatic mutations. There was a statistically significant association of hDlg staining with p53 and Ki67 reactivity, whereas CSK1 epsilon and EDD/hHYD staining levels were associated with progesterone receptor status. The results provide strong indications for a role of all three genes in mammary ductal carcinoma. They also justify additional studies of the functional significance of the changes, as well as a search for additional changes in these and other genes identified from studies on model systems.

    Funded by: NCI NIH HHS: CA91043

    Cancer research 2004;64;3;942-51

Literature (107)

Pubmed - human_disease

  • 3q29 microdeletion syndrome: clinical and molecular characterization of a new syndrome.

    Willatt L, Cox J, Barber J, Cabanas ED, Collins A, Donnai D, FitzPatrick DR, Maher E, Martin H, Parnau J, Pindar L, Ramsay J, Shaw-Smith C, Sistermans EA, Tettenborn M, Trump D, de Vries BB, Walker K and Raymond FL

    Department of Medical Genetics, Addenbrooke's Hospital, University of Cambridge, Cambridge, United Kingdom.

    We report the identification of six patients with 3q29 microdeletion syndrome. The clinical phenotype is variable despite an almost identical deletion size. The phenotype includes mild-to-moderate mental retardation, with only slightly dysmorphic facial features that are similar in most patients: a long and narrow face, short philtrum, and high nasal bridge. Autism, gait ataxia, chest-wall deformity, and long and tapering fingers were noted in at least two of six patients. Additional features--including microcephaly, cleft lip and palate, horseshoe kidney and hypospadias, ligamentous laxity, recurrent middle ear infections, and abnormal pigmentation--were observed, but each feature was only found once, in a single patient. The microdeletion is approximately 1.5 Mb in length, with molecular boundaries mapping within the same or adjacent bacterial artificial chromosome (BAC) clones at either end of the deletion in all patients. The deletion encompasses 22 genes, including PAK2 and DLG1, which are autosomal homologues of two known X-linked mental retardation genes, PAK3 and DLG3. The presence of two nearly identical low-copy repeat sequences in BAC clones on each side of the deletion breakpoint suggests that nonallelic homologous recombination is the likely mechanism of disease causation in this syndrome.

    Funded by: Wellcome Trust

    American journal of human genetics 2005;77;1;154-60

  • Somatic mutations and altered expression of the candidate tumor suppressors CSNK1 epsilon, DLG1, and EDD/hHYD in mammary ductal carcinoma.

    Fuja TJ, Lin F, Osann KE and Bryant PJ

    Developmental Biology Center and Department of Medicine, University of California-Irvine, Irvine, California, USA.

    We report somatic mutations in three genes (CSNK1 epsilon, encoding the Ser/Thr kinase casein kinase I epsilon; DLG1, encoding a membrane-associated putative scaffolding protein; and EDD/hHYD, encoding a progestin induced putative ubiquitin-protein ligase) in mammary ductal carcinoma. These genes were suspected of playing a role in cancer because loss-of-function mutations in their Drosophila homologues cause excess tissue growth. Using DNA from 82 laser-microdissected tumor samples, followed by microsatellite analysis, denaturing HPLC and direct sequencing, we found multiple somatic point mutations in all three genes, and these mutations showed significant association with loss of heterozygosity of closely linked polymorphic microsatellite markers. For CSNK1 epsilon and DLG1, most of the mutations affected highly conserved residues, some were found repetitively in different patients, and no synonymous mutations were found, indicating that the observed mutations were selected in tumors and may be functionally significant. Immunohistochemical reactivity of each protein was reduced in poorly differentiated tumors, and there was a positive association between altered protein reactivity, loss of heterozygosity, and somatic mutations. There was a statistically significant association of hDlg staining with p53 and Ki67 reactivity, whereas CSK1 epsilon and EDD/hHYD staining levels were associated with progesterone receptor status. The results provide strong indications for a role of all three genes in mammary ductal carcinoma. They also justify additional studies of the functional significance of the changes, as well as a search for additional changes in these and other genes identified from studies on model systems.

    Funded by: NCI NIH HHS: CA91043

    Cancer research 2004;64;3;942-51

Pubmed - other

  • Identification of new putative susceptibility genes for several psychiatric disorders by association analysis of regulatory and non-synonymous SNPs of 306 genes involved in neurotransmission and neurodevelopment.

    Gratacòs M, Costas J, de Cid R, Bayés M, González JR, Baca-García E, de Diego Y, Fernández-Aranda F, Fernández-Piqueras J, Guitart M, Martín-Santos R, Martorell L, Menchón JM, Roca M, Sáiz-Ruiz J, Sanjuán J, Torrens M, Urretavizcaya M, Valero J, Vilella E, Estivill X, Carracedo A and Psychiatric Genetics Network Group

    CIBER en Epidemiología y Salud Pública (CIBERESP), Instituto de Salud Carlos III, Madrid, Spain.

    A fundamental difficulty in human genetics research is the identification of the spectrum of genetic variants that contribute to the susceptibility to common/complex disorders. We tested here the hypothesis that functional genetic variants may confer susceptibility to several related common disorders. We analyzed five main psychiatric diagnostic categories (substance-abuse, anxiety, eating, psychotic, and mood disorders) and two different control groups, representing a total of 3,214 samples, for 748 promoter and non-synonymous single nucleotide polymorphisms (SNPs) at 306 genes involved in neurotransmission and/or neurodevelopment. We identified strong associations to individual disorders, such as growth hormone releasing hormone (GHRH) with anxiety disorders, prolactin regulatory element (PREB) with eating disorders, ionotropic kainate glutamate receptor 5 (GRIK5) with bipolar disorder and several SNPs associated to several disorders, that may represent individual and related disease susceptibility factors. Remarkably, a functional SNP, rs945032, located in the promoter region of the bradykinin receptor B2 gene (BDKRB2) was associated to three disorders (panic disorder, substance abuse, and bipolar disorder), and two additional BDKRB2 SNPs to obsessive-compulsive disorder and major depression, providing evidence for common variants of susceptibility to several related psychiatric disorders. The association of BDKRB2 (odd ratios between 1.65 and 3.06) to several psychiatric disorders supports the view that a common genetic variant could confer susceptibility to clinically related phenotypes, and defines a new functional hint in the pathophysiology of psychiatric diseases.

    American journal of medical genetics. Part B, Neuropsychiatric genetics : the official publication of the International Society of Psychiatric Genetics 2009;150B;6;808-16

  • Interaction of the RhoA exchange factor Net1 with discs large homolog 1 protects it from proteasome-mediated degradation and potentiates Net1 activity.

    Carr HS, Cai C, Keinänen K and Frost JA

    Department of Integrative Biology and Pharmacology, University of Texas Health Science Center at Houston, Houston, Texas 77030, USA.

    Net1 is a nuclear Rho guanine nucleotide exchange factor that is specific for the RhoA subfamily of small G proteins. Truncated forms of Net1 are transforming in NIH3T3 cells, and this activity requires cytoplasmic localization of Net1 as well as the presence of a COOH-terminal PDZ binding site. We have previously shown that Net1 interacts with PDZ domain-containing proteins within the Discs Large (Dlg) family and relocalizes them to the nucleus. In the present work, we demonstrate that Net1 binds directly to the first two PDZ domains of Dlg1 and that both PDZ domains are required for maximal interaction in cells. Furthermore, we show that Net1 is an unstable protein in MCF7 breast epithelial cells and that interaction with Dlg1 significantly enhances Net1 stability. Stabilization by Dlg1 significantly increases the ability of Net1 to stimulate RhoA activation in cells. The stability of endogenous Net1 is strongly enhanced by cell-cell contact, and this correlates with a dramatic increase in the interaction between Net1 and Dlg1. Importantly, disruption of E-cadherin-mediated cell contacts, either by depletion of external calcium or by treatment with transforming growth factor beta, leads to a rapid loss of the interaction between Net1 and Dlg1 and a subsequent increase in the ubiquitylation of Net1. These results indicate that Net1 requires interaction with PDZ domain proteins, such as Dlg1, to protect it from proteasome-mediated degradation and to maximally stimulate RhoA and that this interaction is regulated by cell-cell contact.

    Funded by: NCI NIH HHS: 5R01CA116356-02, R01 CA116356

    The Journal of biological chemistry 2009;284;36;24269-80

  • 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; NIGMS NIH HHS: GM054137, GM67945, R01 GM054137, R01 GM054137-14, R01 GM067945

    Cell 2009;138;2;389-403

  • The high-risk HPV E6 oncoprotein preferentially targets phosphorylated nuclear forms of hDlg.

    Narayan N, Subbaiah VK and Banks L

    Tumour Virology Laboratory, International Centre for Genetic Engineering and Biotechnology (ICGEB), Padriciano 99, Trieste, TS 34012, Italy.

    High-risk mucosal HPV E6 oncoproteins target a number of PDZ domain-containing substrates for proteasome mediated degradation. One of these, Discs Large (Dlg), is involved in the regulation of cell polarity and proliferation control. Previous studies had suggested that Dlg when hyperphosphorylated by osmotic shock, or when present in the nucleus could be preferentially targeted by E6. In this study we use phospho-specific antibodies directed against Dlg phosphorylated at residues S158 and S442 to show that these two observations are, in fact, linked. Dlg, when phosphorylated on S158 and S442 by CDK1 or CDK2, shows a preferential nuclear accumulation. However, these forms of Dlg are absent in cells derived from HPV-induced cervical cancers. Upon either proteasome inhibition or siRNA ablation of E6 expression, we see specific rescue of these phosphorylated forms of Dlg. These results demonstrate that nuclear forms of Dlg phosphorylated on its CDK phospho-acceptor sites has enhanced susceptibility to E6-induced degradation and place previous studies on the stress-induced phosphorylation of Dlg into a relevant biological context.

    Virology 2009;387;1;1-4

  • Kv4 potassium channels form a tripartite complex with the anchoring protein SAP97 and CaMKII in cardiac myocytes.

    El-Haou S, Balse E, Neyroud N, Dilanian G, Gavillet B, Abriel H, Coulombe A, Jeromin A and Hatem SN

    UMRS-956, Faculté de Médecine Pierre-Marie Curie, 91 Boulevard de l'Hôpital, 75013 Paris, France.

    Membrane-associated guanylate kinase (MAGUK) proteins are major determinants of the organization of ion channels in the plasma membrane in various cell types. Here, we investigated the interaction between the MAGUK protein SAP97 and cardiac Kv4.2/3 channels, which account for a large part of the outward potassium current, I(to), in heart. We found that the Kv4.2 and Kv4.3 channels C termini interacted with SAP97 via a SAL amino acid sequence. SAP97 and Kv4.3 channels were colocalized in the sarcolemma of cardiomyocytes. In CHO cells, SAP97 clustered Kv4.3 channels in the plasma membrane and increased the current independently of the presence of KChIP and dipeptidyl peptidase-like protein-6. Suppression of SAP97 by using short hairpin RNA inhibited I(to) in cardiac myocytes, whereas its overexpression by using an adenovirus increased I(to). Kv4.3 channels without the SAL sequence were no longer regulated by Ca2+/calmodulin kinase (CaMK)II inhibitors. In cardiac myocytes, pull-down and coimmunoprecipitation assays showed that the Kv4 channel C terminus, SAP97, and CaMKII interact together, an interaction suppressed by SAP97 silencing and enhanced by SAP97 overexpression. In HEK293 cells, SAP97 silencing reproduced the effects of CaMKII inhibition on current kinetics and suppressed Kv4/CaMKII interactions. In conclusion, SAP97 is a major partner for surface expression and CaMKII-dependent regulation of cardiac Kv4 channels.

    Circulation research 2009;104;6;758-69

  • DLG1/SAP97 modulates transforming growth factor alpha bioavailability.

    Surena AL, de Faria GP, Studler JM, Peiretti F, Pidoux M, Camonis J, Chneiweiss H, Formstecher E and Junier MP

    Inserm, UMR894, University of Paris 5, 75014 Paris, France.

    TGFalpha and its receptor EGFR participate in the development of a wide range of tumors including gliomas, the main adult primary brain tumors. TGFalpha soluble form results from the cleavage by the metalloprotease TACE/ADAM17 of the extracellular part of its transmembrane precursor, pro-TGFalpha. To gain insights into the mechanisms underlying TGFalpha bioavailability, a yeast two-hybrid screen was performed to identify proteins interacting with pro-TGFalpha intracellular domain (ICD). DLG1/SAP97 (Discs Large Gene 1 or Synapse Associated Protein 97) was found to interact with both pro-TGFalpha and TACE ICDs through distinct PDZ domains. An in vivo pro-TGFalpha-DLG1-TACE complex was detected in U251 glioma cells and in gliomas-derived tumor initiating cells. Interaction between DLG1 and TACE diminished in response to stimulations promoting pro-TGFalpha shedding. Manipulation of DLG1 levels revealed dual actions of DLG1 on pro-TGFalpha shedding, favoring approximation of pro-TGFalpha and TACE, while limiting TACE full shedding activity. These results show that DLG1 participates in the control of TGFalpha bioavailability through its dynamic interaction with the growth factor precursor and TACE.

    Biochimica et biophysica acta 2009;1793;2;264-72

  • Muscarinic-induced recruitment of plasma membrane Ca2+-ATPase involves PSD-95/Dlg/Zo-1-mediated interactions.

    Kruger WA, Yun CC, Monteith GR and Poronnik P

    School of Biomedical Sciences and School of Pharmacy, The University of Queensland, Brisbane QLD 4072, Australia.

    Efflux of cytosolic Ca2+ mediated by plasma membrane Ca2+-ATPases (PMCA) plays a key role in fine tuning the magnitude and duration of Ca2+ signaling following activation of G-protein-coupled receptors. However, the molecular mechanisms that underpin the trafficking of PMCA to the membrane during Ca2+ signaling remain largely unexplored in native cell models. One potential mechanism for the recruitment of proteins to the plasma membrane involves PDZ interactions. In this context, we investigated the role of PMCA interactions with the Na+/H+ exchanger regulatory factor 2 (NHERF-2) during muscarinic-induced Ca2+ mobilization in the HT-29 epithelial cell line. GST pull-downs in HT-29 cell lysates showed that the PDZ2 module of NHERF-2 bound to the PDZ binding motif on the C terminus of PMCA. Co-immunoprecipitations confirmed that PMCA1b and NHERF-2 associated under normal conditions in HT-29 cells. Cell surface biotinylations revealed significant increases in membrane-associated NHERF-2 and PMCA within 60 s following muscarinic activation, accompanied by increased association of the two proteins as seen by confocal microscopy. The recruitment of NHERF-2 to the membrane preceded that of PMCA, suggesting that NHERF-2 was involved in nucleating an efflux complex at the membrane. The muscarinic-mediated translocation of PMCA was abolished when NHERF-2 was silenced, and the rate of relative Ca2+ efflux was also reduced. These experiments also uncovered a NHERF-2-independent PMCA retrieval mechanism. Our findings describe rapid agonist-induced translocation of PMCA in a native cell model and suggest that NHERF-2 plays a key role in scaffolding and maintaining PMCA at the cell membrane.

    Funded by: NIDDK NIH HHS: DK061418, R01 DK061418, R01 DK061418-05, R01 DK061418-06A1

    The Journal of biological chemistry 2009;284;3;1820-30

  • CDK phosphorylation of the discs large tumour suppressor controls its localisation and stability.

    Narayan N, Massimi P and Banks L

    Tumour Virology Laboratory, International Centre for Genetic Engineering and Biotechnology, Trieste, Italy.

    The Discs Large (Dlg) protein is known to be involved in the regulation of cellular proliferation and polarity in a variety of tissues. The human homologue DLG1 is thought to be a tumour suppressor, through formation of a complex with the APC (adenomatous polyposis coli) protein, causing negative regulation of the cell cycle. An alternative oncogenic role has also been proposed, in which the PI3-kinase pathway is activated under the influence of the adenovirus E4 ORF1 protein. The differing roles seem to be related to differences in the precise pattern of expression. However, the biochemical pathways involved in regulating DLG1 function during different phases of the cell cycle remain unclear. In this study we show that phosphorylation is a major post-translational modification of the protein and it affects both location and function. DLG1 lies at the cellular junctions in G1, is enriched in the cytoplasm in S phase and locates to the mitotic spindle in M phase. We also show that DLG1 is phosphorylated by both CDK1 and CDK2 on Ser158 and Ser442. These phosphorylated sites together affect the nuclear localisation of the protein, and implicate the role of phosphorylation on Ser158 and Ser442 in its putative nuclear functions as a tumour suppressor. In addition, the mutants at these sites demonstrate different half-lives as well as different susceptibilities to ubiquitylation, suggesting a role for these phosphorylation events in controlling DLG1 protein stability. These findings establish phosphorylation events as key regulators of DLG1 localisation and function.

    Journal of cell science 2009;122;Pt 1;65-74

  • Preso, a novel PSD-95-interacting FERM and PDZ domain protein that regulates dendritic spine morphogenesis.

    Lee HW, Choi J, Shin H, Kim K, Yang J, Na M, Choi SY, Kang GB, Eom SH, Kim H and Kim E

    National Creative Research Initiative Center for Synaptogenesis and Department of Biological Sciences, Korea Advanced Institute of Science and Technology, Daejeon 305-701, Korea.

    PSD-95 is an abundant postsynaptic density (PSD) protein involved in the formation and regulation of excitatory synapses and dendritic spines, but the underlying mechanisms are not comprehensively understood. Here we report a novel PSD-95-interacting protein Preso that regulates spine morphogenesis. Preso is mainly expressed in the brain and contains WW (domain with two conserved Trp residues), PDZ (PSD-95/Dlg/ZO-1), FERM (4.1, ezrin, radixin, and moesin), and C-terminal PDZ-binding domains. These domains associate with actin filaments, the Rac1/Cdc42 guanine nucleotide exchange factor betaPix, phosphatidylinositol-4,5-bisphosphate, and the postsynaptic scaffolding protein PSD-95, respectively. Preso overexpression increases the density of dendritic spines in a manner requiring WW, PDZ, FERM, and PDZ-binding domains. Conversely, knockdown or dominant-negative inhibition of Preso decreases spine density, excitatory synaptic transmission, and the spine level of filamentous actin. These results suggest that Preso positively regulates spine density through its interaction with the synaptic plasma membrane, actin filaments, PSD-95, and the betaPix-based Rac1 signaling pathway.

    The Journal of neuroscience : the official journal of the Society for Neuroscience 2008;28;53;14546-56

  • Cloning and functional analysis of the promoter region of the human Disc large gene.

    Cavatorta AL, Giri AA, Banks L and Gardiol D

    Area Virología, Facultad de Ciencias Bioquímicas y Farmacéuticas, Instituto de Biología Molecular y Celular de Rosario-CONICET, Universidad Nacional de Rosario, Rosario, Argentina.

    A number of studies have demonstrated the involvement of human Disc large (DLG1) in the control of both cell polarity and maintenance of tissue architecture. However, the mechanisms controlling DLG1 transcription are not fully understood. This is relevant since DLG1 is lost in many tumours during the later stages of malignant progression. Therefore, we performed the cloning and functional analysis of a genomic 5' flanking region of the DLG1 open reading frame with promoter activity. We analyzed the activity of a series of 5' deletion constructs of the DLG1 promoter and determined the minimal essential sequences that are required for promoter activity as well as cis-elements that regulate transcription. We found, within the DLG1 promoter sequences, consensus-binding sites for the Snail family of transcription factors that repress the expression of epithelial markers and are up-regulated in a variety of tumours. Snail transcription factors repress the transcriptional activity of the DLG1 promoter and, ectopically expressed Snail proteins bind to the native DLG1 promoter. These data suggest a role for Snail transcription factors in the control of DLG1 expression and provide a basis for understanding the transcriptional regulation of DLG1.

    Gene 2008;424;1-2;87-95

  • Regulation of the hDlg/hScrib/Hugl-1 tumour suppressor complex.

    Massimi P, Narayan N, Thomas M, Gammoh N, Strand S, Strand D and Banks L

    International Centre for Genetic Engineering and Biotechnology, Padriciano 99, I-34012 Trieste, Italy. paola@icgeb.org

    The proper function of the Scribble tumour suppressor complex is dependent upon the correct localisation of its components. Previously we observed dynamic relocalisation of the hDlg component under conditions of osmotic stress. We now show that the other two components of the complex, hScrib and Hugl-1 display similar patterns of expression. We demonstrate, by shRNA ablation of hScrib expression, that hDlg and Hugl-1 are in part dependent upon hScrib for their correct localization. However under conditions of osmotic stress this apparent dependency no longer exists: hDlg and Hugl-1 localise to cell membranes independently of hScrib. We also demonstrate an interaction between the three components of the hScrib complex and the tSNARE syntaxin 4, and show that correct localization of the Scrib complex is in part tSNARE dependent. This is the first detailed analysis of the co-localisation and function of the hScrib complex in mammalian cells and demonstrates a direct link between the control of the hScrib complex and vesicle transport pathways.

    Experimental cell research 2008;314;18;3306-17

  • Functional involvement of human discs large tumor suppressor in cytokinesis.

    Unno K, Hanada T and Chishti AH

    Department of Pharmacology, University of Illinois College of Medicine, Chicago, IL 60612, USA.

    Cytokinesis is the final step of cell division that completes the separation of two daughter cells. We found that the human discs large (hDlg) tumor suppressor homologue is functionally involved in cytokinesis. The guanylate kinase (GUK) domain of hDlg mediates the localization of hDlg to the midbody during cytokinesis, and over-expression of the GUK domain in U2OS and HeLa cells impaired cytokinesis. Mouse embryonic fibroblasts (MEFs) derived from dlg mutant mice contained an increased number of multinucleated cells and showed reduced proliferation in culture. A kinesin-like motor protein, GAKIN, which binds directly to the GUK domain of hDlg, exhibited a similar intracellular distribution pattern with hDlg throughout mitosis and localized to the midbody during cytokinesis. However, the targeting of hDlg and GAKIN to the midbody appeared to be independent of each other. The midbody localization of GAKIN required its functional kinesin-motor domain. Treatment of cells with the siRNA specific for hDlg and GAKIN caused formation of multinucleated cells and delayed cytokinesis. Together, these results suggest that hDlg and GAKIN play functional roles in the maintenance of midbody architecture during cytokinesis.

    Funded by: NCI NIH HHS: CA 94414, R01 CA094414, R01 CA094414-06; NHLBI NIH HHS: HL60755, R01 HL060755, R01 HL060755-04

    Experimental cell research 2008;314;17;3118-29

  • An association analysis of synapse-associated protein 97 (SAP97) gene in schizophrenia.

    Sato J, Shimazu D, Yamamoto N and Nishikawa T

    Section of Psychiatry and Behavioral Sciences, Tokyo Medical and Dental University Graduate School, 1-5-45, Bunkyo-ku, Tokyo 113-8519, Japan.

    SAP97 gene encodes the synaptic scaffolding PDZ proteins that interact with the L: -alpha-amino-3-hydroxyl-5-methylisoxazole-4-propionate (AMPA), kainate and N-methyl-D: -aspartate (NMDA) type glutamate receptors. Because the disturbed glutamate neurotransmission has been implicated in the pathophysiology of schizophrenia, we investigated association between the SAP97 gene and schizophrenia. We genotyped 23 SNPs capturing the known common haplotype variations of the gene in a sample comprising 229 schizophrenic patients and 214 matched controls. In a single marker analysis, ten SNPs displayed nominally significant (P < 0.05) association with schizophrenia, although the P values of these SNPs were non-significant after the Bonferroni correction. We also compared haplotype estimates based on case--control genotypes and observed significant association of eight-two- and three- SNP haplotypes with schizophrenia following permutation-based correction. Further examination of the above series of SNPs or haplotypes in each gender revealed significant associations between some of these SNPs or haplotypes and the disorder only in males. The present findings suggest that the SAP97 gene may be a susceptibility factor in male schizophrenics and that the modification of the glutamate receptors-SAP97 signaling pathway could be involved in the disease pathophysiology.

    Journal of neural transmission (Vienna, Austria : 1996) 2008;115;9;1355-65

  • Knockdown of synapse-associated protein Dlg1 reduces syncytium formation induced by human T-cell leukemia virus type 1.

    Yoshida S, Higuchi M, Shoji T, Yos 1f40 hita M, Ishioka K, Takahashi M, Oie M, Tanaka Y, Uchiyama M and Fujii M

    Division of Virology, Niigata University Graduate School of Medical and Dental Sciences, 1-757 Asahimachi-Dori, Niigata 951-8510, Japan.

    Human T-cell leukemia virus type 1 (HTLV-1) spreads through cell-to-cell contact by forming a virological synapse. Based on the finding that HTLV-1 envelope glycoprotein (Env) binds to a PDZ domain containing scaffold protein Dlg1, whose function has been implicated in the organization of neuronal and immunological synapses, we examined the role of Dlg1 in the cell-cell infection by HTLV-1. The coculture of an HTLV-1-infected T-cell line MT-2 with an uninfected MOLT-4 induced syncytium, a marker of cell-cell HTLV-1 infection, but an RNA interference-mediated knockdown of Dlg1 in both cells cooperatively reduced the syncytium formation. In HTLV-1-uninfected 293T cells, Dlg1 induced the clustering of GLUT1, a cellular receptor for HTLV-1, but such clustering was abrogated by a deletion of the PDZ domain binding motif of GLUT1 (GLUT1DeltaC). GLUT1 expression in MDBK cells induced HTLV-1-mediated syncytium formation, and the activity was much greater than that of GLUT1DeltaC. These results suggest that Dlg1, through the interaction with GLUT1 as well as Env, plays a positive role in the syncytium formation induced by HTLV-1.

    Virus genes 2008;37;1;9-15

  • The anchoring protein SAP97 retains Kv1.5 channels in the plasma membrane of cardiac myocytes.

    Abi-Char J, El-Haou S, Balse E, Neyroud N, Vranckx R, Coulombe A and Hatem SN

    INSERM UMR 621, Faculté de Médecine Pierre-Marie Curie, 91 boulevard de l'Hôpital, 75013 Paris, France.

    Membrane- associated guanylate kinase proteins (MAGUKs) are important determinants of localization and organization of ion channels into specific plasma membrane domains. However, their exact role in channel function and cardiac excitability is not known. We examined the effect of synapse-associated protein 97 (SAP97), a MAGUK abundantly expressed in the heart, on the function and localization of Kv1.5 subunits in cardiac myocytes. Recombinant SAP97 or Kv1.5 subunits tagged with green fluorescent protein (GFP) were overexpressed in rat neonatal cardiac myocytes and in Chinese hamster ovary (CHO) cells from adenoviral or plasmidic vectors. Immunocytochemistry, fluorescence recovery after photobleaching, and patch-clamp techniques were used to study the effects of SAP97 on the localization, mobility, and function of Kv1.5 subunits. Adenovirus-mediated SAP97 overexpression in cardiac myocytes resulted in the clustering of endogenous Kv1.5 subunits at myocyte-myocyte contacts and an increase in both the maintained component of the outward K(+) current, I(Kur) (5.64 +/- 0.57 pA/pF in SAP97 myocytes vs. 3.23 +/- 0.43 pA/pF in controls) and the number of 4-aminopyridine-sensitive potassium channels in cell-attached membrane patches. In live myocytes, GFP-Kv1.5 subunits were mobile and organized in clusters at the basal plasma membrane, whereas SAP97 overexpression reduced their mobility. In CHO cells, Kv1.5 channels were diffusely distributed throughout the cell body and freely mobile. When coexpressed with SAP97, Kv subunits were organized in plaquelike clusters and poorly mobile. In conclusion, SAP97 regulates the K(+) current in cardiac myocytes by retaining and immobilizing Kv1.5 subunits in the plasma membrane. This new regulatory mechanism may contribute to the targeting of Kv channels in cardiac myocytes.

    American journal of physiology. Heart and circulatory physiology 2008;294;4;H1851-61

  • Motor protein-dependent transport of AMPA receptors into spines during long-term potentiation.

    Correia SS, Bassani S, Brown TC, Lisé MF, Backos DS, El-Husseini A, Passafaro M and Esteban JA

    Department of Pharmacology, University of Michigan Medical School, 1150 W. Medical Center Dr., Ann Arbor, Michigan 48109-0632, USA.

    The regulated trafficking of neurotransmitter receptors at synapses is critical for synaptic function and plasticity. However, the molecular machinery that controls active transport of receptors into synapses is largely unknown. We found that, in rat hippocampus, the insertion of AMPA receptors (AMPARs) into spines during synaptic plasticity requires a specific motor protein, which we identified as myosin Va. We found that myosin Va associates with AMPARs through its cargo binding domain. This interaction was enhanced by active, GTP-bound Rab11, which is also transported by the motor protein. Myosin Va mediated the CaMKII-triggered translocation of GluR1 receptors from the dendritic shaft into spines, but it was not required for constitutive GluR2 trafficking. Accordingly, myosin Va was specifically required for long-term potentiation, but not for basal synaptic transmission. In summary, we identified the specific motor protein and organelle acceptor that catalyze the directional transport of AMPARs into spines during activity-dependent synaptic plasticity.

    Funded by: NIMH NIH HHS: F31-MH070205, MH070417; Telethon: TCR07006

    Nature neuroscience 2008;11;4;457-66

  • DLG1 is an anchor for the E3 ligase MARCH2 at sites of cell-cell contact.

    Cao Z, Huett A, Kuballa P, Giallourakis C and Xavier RJ

    Gastrointestinal Unit, Massachusetts General Hospital, Harvard Medical School, Boston, MA 02114, USA.

    PDZ domain containing molecular scaffolds plays a central role in organizing synaptic junctions. Observations in Drosophila and mammalian cells have implicated that ubiquitination and endosomal trafficking, of molecular scaffolds are critical to the development and maintenance of cell-cell junctions and cell polarity. To elucidate if there is a connection between these pathways, we applied an integrative genomic strategy, which combined comparative genomics and proteomics with cell biological assays. Given the importance of ubiquitin in regulating endocytic processes, we first identified the subset of E3 ligases with conserved PDZ binding motifs. Among this subset, the MARCH family ubiquitin ligases account for the largest family and MARCH2 has been previously implicated in endosomal trafficking. Next, we tested in an unbiased fashion, if MARCH2 binds PDZ proteins in vivo using a modified tandem affinity purification strategy followed by mass spectrometry. Of note, DLG1 was co-purified from MARCH2, with subsequent confirmation that MARCH2 interacts with full-length DLG1 in a PDZ domain dependent manner. Furthermore, we demonstrated that MARCH2 co-localized with DLG1 at sites of cell-cell contact. In addition, loss of the MARCH2 PDZ binding motif led to loss of MARCH2 localization at cell-cell contact sites and MARCH2 appeared to localize away from cell-cell junctions. In in vivo ubiquitination assays we show that MARCH2 promotes DLG1 ubiquitination. Overall, these results suggest that PDZ ligands with E3 ligase activity may link PDZ domain containing tumor suppressors to endocytic pathways and cell polarity determination.

    Funded by: NIAID NIH HHS: R01 AI062773, R01 AI062773-02; NIDDK NIH HHS: P30 DK040561, P30 DK040561-12, P30 DK043351

    Cellular signalling 2008;20;1;73-82

  • Solution structure of the hDlg/SAP97 PDZ2 domain and its mechanism of interaction with HPV-18 papillomavirus E6 protein.

    Liu Y, Henry GD, Hegde RS and Baleja JD

    Department of Biochemistry, Tufts University School of Medicine, 136 Harrison Avenue, Boston, Massachusetts 02111, USA.

    The E6 protein from high-risk types of human papillomavirus (HPV) binds PDZ-domain containing proteins and targets them for degradation. We used isothermal titration calorimetry to measure the interaction of a peptide from the C-terminus of HPV-18 E6 to the second PDZ domain (PDZ2) from the human homologue of the Drosophila discs large tumor suppressor protein (hDlg). Isothermal titration calorimetry experiments with a series of peptides showed that HPV-18 E6 bound hDlg PDZ2 about 5-fold stronger than HPV-16 E6, that the contribution of Arg154 to binding was about 1 kcal/mol, and that the binding was disabled by phosphorylation at Thr156. We then used NMR to determine the solution structure of the complex of PDZ2 bound to the HPV-18 E6 peptide. The resultant structures were of high quality and had backbone root-mean-square deviations of less than 0.5 A. The structure shows a novel mode of interaction in which six residues of the HPV-18 E6 peptide are contacted by the PDZ2 domain, in contrast to the typical four residues used by class I PDZ domains. Molecular dynamics simulations supported a model in which the C- and N-terminal ends of the peptide had different mobilities within the complex. Comparison of the NMR complex structure to previously determined X-ray structures of PDZ2 by itself and bound to different peptides allows a description of conformational changes required for PDZ2 to bind to HPV-18 E6.

    Funded by: NEI NIH HHS: R01 EY014648; NIAID NIH HHS: AI38001

    Biochemistry 2007;46;38;10864-74

  • The effector domain of human Dlg tumor suppressor acts as a switch that relieves autoinhibition of kinesin-3 motor GAKIN/KIF13B.

    Yamada KH, Hanada T and Chishti AH

    Department of Pharmacology and UIC Cancer Center, University of Illinois College of Medicine, Chicago, Illinois 60612, USA.

    The activity of motor proteins must be tightly regulated in the cells to prevent unnecessary energy consumption and to maintain proper distribution of cellular components. Loading of the cargo molecule is one likely mechanism to activate an inactive motor. Here, we report that the activity of the kinesin-3 motor protein, GAKIN, is regulated by the direct binding of its protein cargo, human discs large (hDlg) tumor suppressor. Recombinant GAKIN exhibits potent microtubule gliding activity but has little microtubule-stimulated ATPase activity in solution, suggesting that it exists in an autoinhibitory form. In vitro binding measurements revealed that defined segments of GAKIN, particularly the MAGUK binding stalk (MBS) domain and the motor domain, mediate intramolecular interactions to confer globular protein conformation. Direct binding of the SH3-I3-GUK module of hDlg to the MBS domain of GAKIN activates the microtubule-stimulated ATPase activity of GAKIN by approximately 10-fold. We propose that the cargo-mediated regulation of motor activity constitutes a general paradigm for the activation of kinesins.

    Funded by: NCI NIH HHS: CA 94414, R01 CA094414, R01 CA094414-06; NHLBI NIH HHS: HL60755, R01 HL060755, R01 HL060755-04

    Biochemistry 2007;46;35;10039-45

  • Changes in localization of human discs large (hDlg) during keratinocyte differentiation are [corrected] associated with expression of alternatively spliced hDlg variants.

    Roberts S, Calautti E, Vanderweil S, Nguyen HO, Foley A, Baden HP and Viel A

    Cancer Research UK Institute for Cancer Studies, University of Birmingham, Edgbaston, Birmingham, UK. s.roberts@bham.ac.uk

    Alternative spliced variants of the human discs large (hDlg) tumour suppressor are characterized by combinations of insertions. Here, using insertions I2- and I3-specific antibodies, we show that I2 and I3 variants have distinct distributions in epidermal and cervical epithelia. In skin and cervix, I3 variants are found in the cytoplasm. Cytoplasmic localization of I3 variants decreases as cervical keratinocytes differentiate, concomitant with relocalization to the cell periphery. I2 variants are found at the cell periphery of differentiated epidermal and cervical keratinocytes. Nuclear localization of I2 variants was evident in both tissues, with concentration of nuclear I2 variants in basal and parabasal cervical keratinocytes. A prominent nuclear localization of hDlg in cells of hyperproliferative layers of psoriatic lesions, but not in mature differentiated keratinocytes, together with I2 redistribution in differentiating keratinocytes, suggests that 11d8 nuclear hDlg functions may be pertinent to growth of undifferentiated cells. Supporting our findings in squamous tissues, a decrease of nuclear hDlg and an increase of membrane-bound and cytoplasmic hDlg upon calcium-induced keratinocyte differentiation were not concomitant processes. Furthermore, we confirm that the exit of I2 variants from the nucleus is linked to stimulation of epithelial differentiation. The dynamic redistribution of hDlg also correlated with a marked increase in the expression of I3 variants while the level of I2 variants showed only a moderate decrease. Because changes in the intracellular distribution of hDlg splice variants, and in their expression levels, correlate with changes in differentiation state we hypothesize that the different hDlg isoforms play distinct roles at various stages of epithelial differentiation.

    Experimental cell research 2007;313;12;2521-30

  • The MAGUK protein MPP7 binds to the polarity protein hDlg1 and facilitates epithelial tight junction formation.

    Stucke VM, Timmerman E, Vandekerckhove J, Gevaert K and Hall A

    Medical Research Council Laboratory for Molecular Cell Biology, Cancer Research UK Oncogene and Signal Transduction Group, University College London, London WC1E 6BT, England.

    Three groups of evolutionarily conserved proteins have been implicated in the establishment of epithelial cell polarity: the apically-localized proteins of the Par (Par3-Par6-aPKC-Cdc42) and Crumbs groups (Crb3-PALS1-PATJ) and the basolaterally localized proteins of the Dlg group (Dlg1-Scribble-Lgl). During epithelial morphogenesis, these proteins participate in a complex network of interdependent interactions that define the position and functional organization of adherens junctions and tight junctions. However, the biochemical pathways through which they control polarity are poorly understood. In this study, we identify an interaction between endogenous hDlg1 and MPP7, a previously uncharacterized MAGUK-p55 subfamily member. We find that MPP7 targets to the lateral surface of epithelial cells via its L27N domain, through an interaction with hDlg1. Loss of either hDlg1 or MPP7 from epithelial Caco-2 cells results in a significant defect in the assembly and maintenance of functional tight junctions. We conclude that the formation of a complex between hDlg1 and MPP7 promotes epithelial cell polarity and tight junction formation.

    Molecular biology of the cell 2007;18;5;1744-55

  • Discs-large homolog 1 regulates smooth muscle orientation in the mouse ureter.

    Mahoney ZX, Sammut B, Xavier RJ, Cunningham J, Go G, Brim KL, Stappenbeck TS, Miner JH and Swat W

    Department of Internal Medicine, Renal Division, Washington University School of Medicine, St. Louis, MO 63110, USA.

    Discs-large homolog 1 (DLGH1) is a mouse ortholog of the Drosophila discs-large (DLG) tumor suppressor protein, a founding member of the PDZ and MAGUK protein families. DLG proteins play important roles in regulating cell proliferation, epithelial cell polarity, and synapse formation and function. Here, we generated a null allele of Dlgh1 and studied its role in urogenital development. Dlgh1(-/-) mice developed severe urinary tract abnormalities, including congenital hydronephrosis, which is the leading cause of renal failure in infants and children. DLGH1 is expressed in the developing ureter; in its absence, the stromal cells that normally lie between the urothelial and smooth muscle layers were missing. Moreover, in ureteric smooth muscle, the circular smooth muscle cells were misaligned in a longitudinal orientation. These abnormalities in the ureter led to severely impaired ureteric peristalsis. Similar smooth muscle defects are observed frequently in patients with ureteropelvic junction obstruction, a common form of hydronephrosis. Our results suggest that (i) besides its well documented role in regulating epithelial polarity, Dlgh1 also regulates smooth muscle orientation, and (ii) human DLG1 mutations may contribute to hereditary forms of hydronephrosis.

    Funded by: NIAID NIH HHS: R01 AI061077, R01AI061077, R21 AI063024, R21AI063024; NIDDK NIH HHS: P30 DK040561, P30 DK040561-11, R01 DK064687, R01DK064687

    Proceedings of the National Academy of Sciences of the United States of America 2006;103;52;19872-7

  • Crystal structure of the second PDZ domain of SAP97 in complex with a GluR-A C-terminal peptide.

    von Ossowski I, Oksanen E, von Ossowski L, Cai C, Sundberg M, Goldman A and Keinänen K

    Department of Biological and Environmental Sciences (Division of Biochemistry), University of Helsinki, Finland.

    Synaptic targeting of GluR-A subunit-containing glutamate receptors involves an interaction with synapse-associated protein 97 (SAP97). The C-terminus of GluR-A, which contains a class I PDZ ligand motif (-x-Ser/Thr-x-phi-COOH where phi is an aliphatic amino acid) associates preferentially with the second PDZ domain of SAP97 (SAP97(PDZ2)). To understand the structural basis of this interaction, we have determined the crystal structures of wild-type and a SAP97(PDZ2) variant in complex with an 18-mer C-terminal peptide (residues 890-907) of GluR-A and of two variant PDZ2 domains in unliganded state at 1.8-2.44 A resolutions. SAP97(PDZ2) folds to a compact globular domain comprising six beta-strands and two alpha-helices, a typical architecture for PDZ domains. In the structure of the peptide complex, only the last four C-terminal residues of the GluR-A are visible, and align as an antiparallel beta-strand in the binding groove of SAP97(PDZ2). The free carboxylate group and the aliphatic side chain of the C-terminal leucine (Leu907), and the hydroxyl group of Thr905 of the GluR-A peptide are engaged in essential class I PDZ interactions. Comparison between the free and complexed structures reveals conformational changes which take place upon peptide binding. The betaAlpha-betaBeta loop moves away from the C-terminal end of alphaB leading to a slight opening of the binding groove, which may better accommodate the peptide ligand. The two conformational states are stabilized by alternative hydrogen bond and coulombic interactions of Lys324 in betaAlpha-betaBeta loop with Asp396 or Thr394 in betaBeta. Results of in vitro binding and immunoprecipitation experiments using a PDZ motif-destroying L907A mutation as well as the insertion of an extra alanine residue between the C-terminal Leu907 and the stop codon are also consistent with a 'classical' type I PDZ interaction between SAP97 and GluR-A C-terminus.

    The FEBS journal 2006;273;22;5219-29

  • Human discs large and scrib are localized at the same regions in colon mucosa and changes in their expression patterns are correlated with loss of tissue architecture during malignant progression.

    Gardiol D, Zacchi A, Petrera F, Stanta G and Banks L

    International Centre for Genetic Engineering and Biotechnology, Trieste, Italy.

    Loss of cell polarity is one of the hallmarks of malignant carcinomas. Most of the understanding about the link between cell polarity and proliferation control comes from studies on the Drosophila tumor suppressors discs large (Dlg), scribble (Scrib) and lethal giant larvae (lgl). Mammalian homologues of these proteins have been described and are conserved in sequence and function. Human Dlg (hDlg) and Scrib were independently shown to be down-regulated during malignant progression. This, and other lines of evidence, points toward the p 1f40 articipation of both hDlg and hScrib in a common pathway involved in polarity control and tumor suppression. We investigated the correlation between the expression of both proteins in tissues and their relative contributions to the maintenance of tissue architecture during colon cancer development. We analyzed the levels and distribution of hDlg and hScrib by immunohistochemistry, using serial sections of the same sample. We used normal and neoplastic colon mucosa, since it offers a good model for analyzing these features in progressive dysplastic stages. The results demonstrate that both proteins localize at the same regions in polarized colon epithelia, and that in normal samples the proteins' distribution varies as cells differentiate at the surface mucosa. In neoplasia, alterations in the expression pattern of hDlg and of hScrib increase during tumor progression; down-regulation of both proteins being associated with lack of epithelial cell polarity and disorganized tissue architecture. The results, therefore, demonstrate that there is an inverse relationship between the levels of hDlg and hScrib expression and the loss of cell polarity and tissue architecture in the colon.

    International journal of cancer 2006;119;6;1285-90

  • A molecular-properties-based approach to understanding PDZ domain proteins and PDZ ligands.

    Giallourakis C, Cao Z, Green T, Wachtel H, Xie X, Lopez-Illasaca M, Daly M, Rioux J and Xavier R

    Massachusetts General Hospital, Center for Computational and Integrative Biology, Harvard University Medical School, Boston, Massachusetts 02114, USA.

    PDZ domain-containing proteins and their interaction partners are mutated in numerous human diseases and function in complexes regulating epithelial polarity, ion channels, cochlear hair cell development, vesicular sorting, and neuronal synaptic communication. Among several properties of a collection of documented PDZ domain-ligand interactions, we discovered embedded in a large-scale expression data set the existence of a significant level of co-regulation between PDZ domain-encoding genes and these ligands. From this observation, we show how integration of expression data, a comparative genomics catalog of 899 mammalian genes with conserved PDZ-binding motifs, phylogenetic analysis, and literature mining can be utilized to infer PDZ complexes. Using molecular studies we map novel interaction partners for the PDZ proteins DLG1 and CARD11. These results provide insight into the diverse roles of PDZ-ligand complexes in cellular signaling and provide a computational framework for the genome-wide evaluation of PDZ complexes.

    Funded by: NIDDK NIH HHS: P30 DK040561, P30 DK040561-11

    Genome research 2006;16;8;1056-72

  • Uncovering quantitative protein interaction networks for mouse PDZ domains using protein microarrays.

    Stiffler MA, Grantcharova VP, Sevecka M and MacBeath G

    Department of Chemistry and Chemical Biology, Harvard University, 12 Oxford Street, Cambridge, Massachusetts 02138, USA.

    One of the principal challenges in systems biology is to uncover the networks of protein-protein interactions that underlie most biological processes. To date, experimental efforts directed at this problem have largely produced only qualitative networks that are replete with false positives and false negatives. Here, we describe a domain-centered approach--compatible with genome-wide investigations--that enables us to measure the equilibrium dissociation constant (K(D)) of recombinant PDZ domains for fluorescently labeled peptides that represent physiologically relevant binding partners. Using a pilot set of 22 PDZ domains, 4 PDZ domain clusters, and 20 peptides, we define a gold standard dataset by determining the K(D) for all 520 PDZ-peptide combinations using fluorescence polarization. We then show that microarrays of PDZ domains identify interactions of moderate to high affinity (K(D) < or = 10 microM) in a high-throughput format with a false positive rate of 14% and a false negative rate of 14%. By combining the throughput of protein microarrays with the fidelity of fluorescence polarization, our domain/peptide-based strategy yields a quantitative network that faithfully recapitulates 85% of previously reported interactions and uncovers new biophysical interactions, many of which occur between proteins that are co-expressed. From a broader perspective, the selectivity data produced by this effort reveal a strong concordance between protein sequence and protein function, supporting a model in which interaction networks evolve through small steps that do not involve dramatic rewiring of the network.

    Funded by: NIGMS NIH HHS: 1 R01 GM072872-01, 5 T32 GM07598-25, R01 GM072872, R01 GM072872-04, T32 GM007598

    Journal of the American Chemical Society 2006;128;17;5913-22

  • Ggamma13 interacts with PDZ domain-containing proteins.

    Li Z, Benard O and Margolskee RF

    Department of Neuroscience, Mount Sinai School of Medicine, New York, New York 10029, USA.

    The G protein gamma13 subunit (Ggamma13) is expressed in taste and retinal and neuronal tissues and plays a key role in taste transduction. We identified PSD95, Veli-2, and other PDZ domain-containing proteins as binding partners for Ggamma13 by yeast two-hybrid and pull-down assays. In two-hybrid assays, Ggamma13 interacted specifically with the third PDZ domain of PSD95, the sole PDZ domain of Veli-2, and the third PDZ domain of SAP97, a PSD95-related protein. Ggamma13 did not interact with the other PDZ domains of PSD95. Coexpression of Ggamma13 with its Gbeta1 partner did not interfere with these two-hybrid interactions. The physical interaction of Ggamma13 with PSD95 in the cellular milieu was confirmed in pull-down assays following heterologous expression in HEK293 cells. The interaction of Ggamma13 with the PDZ domain of PSD95 was via the C-terminal CAAX tail of Ggamma13 (where AA indicates the aliphatic amino acid); alanine substitution of the CTAL sequence at the C terminus of Ggamma13 abolished its interactions with PSD95 in two-hybrid and pull-down assays. Veli-2 and SAP97 were identified in taste tissue and in Ggamma13-expressing taste cells. Coimmunoprecipitation of Ggamma13 and PSD95 from brain and of Ggamma13 and SAP97 from taste tissue indicates that Ggamma13 interacts with these proteins endogenously. This is the first demonstration that PDZ domain proteins interact with heterotrimeric G proteins via the CAAX tail of Ggamma subunits. The interaction of Ggamma13 with PDZ domain-containing proteins may provide a means to target particular Gbetagamma subunits to specific subcellular locations and/or macromolecular complexes involved in signaling pathways.

    Funded by: NIDCD NIH HHS: DC003055, DC003155

    The Journal of biological chemistry 2006;281;16;11066-73

  • Involvement of a cellular ubiquitin-protein ligase E6AP in the ubiquitin-mediated degradation of extensive substrates of high-risk human papillomavirus E6.

    Matsumoto Y, Nakagawa S, Yano T, Takizawa S, Nagasaka K, Nakagawa K, Minaguchi T, Wada O, Ooishi H, Matsumoto K, Yasugi T, Kanda T, Huibregtse JM and Taketani Y

    Department of Obstetrics and Gynecology, Graduate School of Medicine, The University of Tokyo, Hongo, Bunkyo-ku, Tokyo, Japan.

    Human scribble (hScrib), which was identified as substrate of human papillomavirus (HPV) E6 for ubiquitin-mediated degradation dependent on ubiquitin-protein ligase E6AP, is a human homolog of Drosophila neoplastic tumor suppressor scribble, in which mutation causes loss of polarity and overgrowth of epithelia. Drosophila discs large (Dlg) is one of neoplastic tumor suppressors, which genetically links to scribble. E6 also targets human Dlg (hDlg) for ubiquitin-mediated degradation. Ubiquitin-protein ligase involved in this process has not been identified thus far. Here we investigated mechanism underlying degradation of three target proteins of E6, hScrib, hDlg, and p53 by using eighteen HPV 16 E6 mutants with single amino acid substitution. In vitro degradation ability of each E6 mutant was equivalent for these tumor suppressors. We investigated whether E6AP is involved in ubiquitin-mediated degradation of hDlg. In vitro binding assay revealed that hDlg formed ternary complex with E6-E6AP complex. The ability of E6 mutants to degrade these tumor suppressors was correlated with their ability to interact with E6AP. Furthermore, hDlg was targeted for in vitro ubiquitination in the presence of both E6 and E6AP. These data revealed that E6AP is extensively involved in the ubiquitin-mediated degradation of E6-dependent substrates as a cellular E3 ubiquitin-protein ligase.

    Journal of medical virology 2006;78;4;501-7

  • A specific N-terminal residue in Kv1.5 is required for upregulation of the channel by SAP97.

    Mathur R, Choi WS, Eldstrom J, Wang Z, Kim J, Steele DF and Fedida D

    Department of Cellular and Physiological Sciences, University of British Columbia, 2350 Health Sciences Mall, Vancouver, BC, Canada V6T 1Z3.

    We have previously reported that SAP97 enhancement of hKv1.5 currents requires an intact Kv1.5 N-terminus and is independent of the PDZ-binding motif at the C-terminus of the channel [J. Eldstrom, W.S. Choi, D.F. Steele, D. Fedida, SAP97 increases Kv1.5 currents through an indirect N-terminal mechanism, FEBS Lett. 547 (2003) 205-211]. Here, we report that an interaction between the two proteins can be detected under certain conditions but their interaction is irrelevant to the enhancement of channel expression. Instead, a threonine residue at position 15 in the hKv1.5 N-terminus is critically important. Mutation of this residue, which lies within a consensus site for phosphorylation by protein kinase C, to an alanine, completely abrogated the effect of SAP97 on channel expression. Although we were unable to detect phosphorylation of this residue, specific inhibition of kinase C by Calphostin C eliminated the increase in wild-type hKv1.5 currents associated with SAP97 overexpression suggesting a role for this kinase in the response.

    Biochemical and biophysical research communications 2006;342;1;1-8

  • Proteomic analysis of beta1-adrenergic receptor interactions with PDZ scaffold proteins.

    He J, Bellini M, Inuzuka H, Xu J, Xiong Y, Yang X, Castleberry AM and Hall RA

    Department of Biochemistry and Molecular Biology, Capital University of Medical Sciences, Beijing 100054, China.

    Many G protein-coupled receptors possess carboxyl-terminal motifs ideal for interaction with PDZ scaffold proteins, which can control receptor trafficking and signaling in a cell-specific manner. To gain a panoramic view of beta1-adrenergic receptor (beta AR) interactions with PDZ scaffolds, the beta1AR carboxyl terminus was screened against a newly developed proteomic array of PDZ domains. These screens confirmed beta1AR associations with several previously identified PDZ partners, such as PSD-95, MAGI-2, GIPC, and CAL. Moreover, two novel beta1AR-interacting proteins, SAP97 and MAGI-3, were also identified. The beta1AR carboxyl terminus was found to bind specifically to the first PDZ domain of MAGI-3, with the last four amino acids (E-S-K-V) of beta1AR being the key determinants of the interaction. Full-length beta1AR robustly associated with full-length MAGI-3 in cells, and this association was abolished by mutation of the beta1AR terminal valine residue to alanine (V477A), as determined by co-immunoprecipitation experiments and immunofluorescence co-localization studies. MAGI-3 co-expression with beta1AR profoundly impaired beta1AR-mediated ERK1/2 activation but had no apparent effect on beta1AR-mediated cyclic AMP generation or agonist-promoted beta1AR internalization. These findings revealed that the interaction of MAGI-3 with beta1AR can selectively regulate specific aspects of receptor signaling. Moreover, the screens of the PDZ domain proteomic array provide a comprehensive view of beta1AR interactions with PDZ scaffolds, thereby shedding light on the molecular mechanisms by which beta1 AR signaling and trafficking can be regulated in a cell-specific manner.

    The Journal of biological chemistry 2006;281;5;2820-7

  • Distinct enzyme combinations in AKAP signalling complexes permit functional diversity.

    Hoshi N, Langeberg LK and Scott JD

    Howard Hughes Medical Institute, Vollum Institute, Oregon Health and Science University, 3181 S.W. Sam Jackson Park Road, Portland, OR 97239, USA.

    Specificity in cell signalling can be influenced by the targeting of different enzyme combinations to substrates. The A-kinase anchoring protein AKAP79/150 is a multivalent scaffolding protein that coordinates the subcellular localization of second-messenger-regulated enzymes, such as protein kinase A, protein kinase C and protein phosphatase 2B. We developed a new strategy that combines RNA interference of the endogenous protein with a protocol that selects cells that have been rescued with AKAP79/150 forms that are unable to anchor selected enzymes. Using this approach, we show that AKAP79/150 coordinates different enzyme combinations to modulate the activity of two distinct neuronal ion channels: AMPA-type glutamate receptors and M-type potassium channels. Utilization of distinct enzyme combinations in this manner provides a means to expand the repertoire of cellular events that the same AKAP modulates.

    Funded by: NIGMS NIH HHS: GM48231, R01 GM048231, R37 GM048231

    Nature cell biology 2005;7;11;1066-73

  • p38gamma regulates the localisation of SAP97 in the cytoskeleton by modulating its interaction with GKAP.

    Sabio G, Arthur JS, Kuma Y, Peggie M, Carr J, Murray-Tait V, Centeno F, Goedert M, Morrice NA and Cuenda A

    MRC Protein Phosphorylation Unit, University of Dundee, Dundee, UK.

    Activation of the p38 MAP kinase pathways is crucial for the adaptation of mammalian cells to changes in the osmolarity of the environment. Here we identify SAP97/hDlg, the mammalian homologue of the Drosophila tumour suppressor Dlg, as a physiological substrate for the p38gamma MAP kinase (SAPK3/p38gamma) isoform. SAP97/hDlg is a scaffold protein that forms multiprotein complexes with a variety of proteins and is targeted to the cytoskeleton by its association with the protein guanylate kinase-associated protein (GKAP). The SAPK3/p38gamma-catalysed phosphorylation of SAP97/hDlg triggers its dissociation from GKAP and therefore releases it from the cytoskeleton. This is likely to regulate the integrity of intercellular-junctional complexes, and cell shape and volume in response to osmotic stress.

    The EMBO journal 2005;24;6;1134-45

  • Dlgh1 coordinates actin polymerization, synaptic T cell receptor and lipid raft aggregation, and effector function in T cells.

    Round JL, Tomassian T, Zhang M, Patel V, Schoenberger SP and Miceli MC

    Department of Microbiology, Immunology, and Molecular Genetics, University of California, Los Angeles, Los Angeles, CA 90095, USA.

    Lipid raft membrane compartmentalization and membrane-associated guanylate kinase (MAGUK) family molecular scaffolds function in establishing cell polarity and organizing signal transducers within epithelial cell junctions and neuronal synapses. Here, we elucidate a role for the MAGUK protein, Dlgh1, in polarized T cell synapse assembly and T cell function. We find that Dlgh1 translocates to the immune synapse and lipid rafts in response to T cell receptor (TCR)/CD28 engagement and that LckSH3-mediated interactions with Dlgh1 control its membrane targeting. TCR/CD28 engagement induces the formation of endogenous Lck-Dlgh1-Zap70-Wiskott-Aldrich syndrome protein (WASp) complexes in which Dlgh1 acts to facilitate interactions of Lck with Zap70 and WASp. Using small interfering RNA and overexpression approaches, we show that Dlgh1 promotes antigen-induced actin polymerization, synaptic raft and TCR clustering, nuclear factor of activated T cell activity, and cytokine production. We propose that Dlgh1 coordinates TCR/CD28-induced actin-driven T cell synapse assembly, signal transduction, and effector function. These findings highlight common molecular strategies used to regulate cell polarity, synapse assembly, and transducer organization in diverse cel 1f40 lular systems.

    Funded by: NCI NIH HHS: R01 CA065979, R01CA65979; NIGMS NIH HHS: GM07185, T32 GM007185

    The Journal of experimental medicine 2005;201;3;419-30

  • A novel scaffold protein, TANC, possibly a rat homolog of Drosophila rolling pebbles (rols), forms a multiprotein complex with various postsynaptic density proteins.

    Suzuki T, Li W, Zhang JP, Tian QB, Sakagami H, Usuda N, Usada N, Kondo H, Fujii T and Endo S

    Department of Neuroplasticity, Institute on Ageing and Adaptation, Shinshu University Graduate School of Medicine, 3-1-1 Asahi, Matsumoto 390-8621, Japan. suzukit@sch.md.shinshu-u.ac.jp

    We cloned from the rat brain a novel gene, tanc (GenBank Accession No. AB098072), which encoded a protein containing three tetratricopeptide repeats (TPRs), ten ankyrin repeats and a coiled-coil region, and is possibly a rat homolog of Drosophila rolling pebbles (rols). The tanc gene was expressed widely in the adult rat brain. Subcellular distribution, immunohistochemical study of the brain and immunocytochemical studies of cultured neuronal cells indicated the postsynaptic localization of TANC protein of 200 kDa. Pull-down experiments showed that TANC protein bound PSD-95, SAP97, and Homer via its C-terminal PDZ-binding motif, -ESNV, and fodrin via both its ankyrin repeats and the TPRs together with the coiled-coil domain. TANC also bound the alpha subunit of Ca2+/calmodulin-dependent protein kinase II. An immunoprecipitation study showed TANC association with various postsynaptic proteins, including guanylate kinase-associated protein (GKAP), alpha-internexin, and N-methyl-D-aspartate (NMDA)-type glutamate receptor 2B and AMPA-type glutamate receptor (GluR1) subunits. These results suggest that TANC protein may work as a postsynaptic scaffold component by forming a multiprotein complex with various postsynaptic density proteins.

    The European journal of neuroscience 2005;21;2;339-50

  • The notch ligand Delta1 recruits Dlg1 at cell-cell contacts and regulates cell migration.

    Six EM, Ndiaye D, Sauer G, Laâbi Y, Athman R, Cumano A, Brou C, Israël A and Logeat F

    Unité de Biologie Moléculaire de l'Expression Génique, URA 2582 CNRS, Institut Pasteur, 25 Rue du Dr Roux, 75724 Paris Cedex 15, France.

    Delta1 acts as a membrane-bound ligand that interacts with the Notch receptor and plays a critical role in cell fate specification. By using peptide affinity chromatography followed by mass spectrometry, we have identified Dlg1 as a partner of the Delta1 C-terminal region. Dlg1 is a human homolog of the Drosophila Discs large tumor suppressor, a member of the membrane-associated guanylate kinase family of molecular scaffolds. We confirmed this interaction by co-immunoprecipitation experiments between endogenous 1f40 Dlg1 and transduced Delta1 in a 3T3 cell line stably expressing Delta1. Moreover, we showed that deletion of a canonical C-terminal PDZ-binding motif (ATEV) in Delta1 abrogated this interaction. Delta4 also interacted with Dlg1, whereas Jagged1, another Notch ligand, did not. In HeLa cells, transfected Delta1 triggered the accumulation of endogenous Dlg1 at sites of cell-cell contact. Expression of Delta1 also reduced the motility of 3T3 cells. Finally, deletion of the ATEV motif totally abolished these effects but did not interfere with the ability of Delta1 to induce Notch signaling and T cell differentiation in co-culture experiments. These results point to a new, probably cell-autonomous function of Delta1, which is independent of its activity as a Notch ligand.

    The Journal of biological chemistry 2004;279;53;55818-26

  • Differential expression of the human homologue of drosophila discs large oncosuppressor in histologic samples from human papillomavirus-associated lesions as a marker for progression to malignancy.

    Cavatorta AL, Fumero G, Chouhy D, Aguirre R, Nocito AL, Giri AA, Banks L and Gardiol D

    Departamento de Microbiología, Facultad de Ciencias Bioquímicas, Instituto de Biología Molecular y Celular de Rosario, Rosario, Argentina.

    High-risk HPVs play a causal role in the development of cervical cancer, and their E6 oncoproteins target h-Dlg for ubiquitin-mediated proteolysis. The h-Dlg oncosuppressor is associated with cell-cell interactions, and deregulation of these structures leads to defective cell adhesion, loss of cell polarity and unregulated proliferation. We evaluated the contribution of this E6 activity in the progression to malignancy in HPV infections by analyzing h-Dlg expression in HPV-associated lesions. We analyzed h-Dlg in cervical, laryngeal, vulvar, colon and kidney histologic samples by Dlg immunohistochemistry. HPV association was ascertained by a PCR-colorimetric method. Although Dlg was certainly expressed in intraepithelial cervical, vulvar and laryngeal HPV-associated lesions, its cellular and tissue distribution patterns were altered compared to normal tissue. However, marked reduction in Dlg levels was observed in HPV-positive invasive cervical carcinomas. To elucidate whether the loss of Dlg was significant for carcinogenesis in general, we investigated Dlg expression in tumors not associated with HPV. In colon and kidney carcinomas, Dlg was expressed, albeit with a different pattern of distribution with respect to the normal tissue. The loss of Dlg may be considered a late-stage marker in cervical carcinogenesis, but alterations in its expression and localization take place during the different dysplastic stages. Dlg downregulation and/or alterations in its localization may contribute to transformation and may explain some of the characteristics of the malignant cells, such as loss of polarity and high migration ability.

    International journal of cancer 2004;111;3;373-80

  • Large-scale characterization of HeLa cell nuclear phosphoproteins.

    Beausoleil SA, Jedrychowski M, Schwartz D, Elias JE, Villén J, Li J, Cohn MA, Cantley LC and Gygi SP

    Department of Cell Biology, Harvard Medical School, Boston, MA 02115, USA.

    Determining the site of a regulatory phosphorylation event is often essential for elucidating specific kinase-substrate relationships, providing a handle for understanding essential signaling pathways and ultimately allowing insights into numerous disease pathologies. Despite intense research efforts to elucidate mechanisms of protein phosphorylation regulation, efficient, large-scale identification and characterization of phosphorylation sites remains an unsolved problem. In this report we describe an application of existing technology for the isolation and identification of phosphorylation sites. By using a strategy based on strong cation exchange chromatography, phosphopeptides were enriched from the nuclear fraction of HeLa cell lysate. From 967 proteins, 2,002 phosphorylation sites were determined by tandem MS. This unprecedented large collection of sites permitted a detailed accounting of known and unknown kinase motifs and substrates.

    Funded by: NHGRI NIH HHS: HG00041, K22 HG000041, T32 HG000041; NIGMS NIH HHS: GM67945, GMS6203, R01 GM056203, R01 GM067945

    Proceedings of the National Academy of Sciences of the United States of America 2004;101;33;12130-5

  • Human Dlg protein binds to the envelope glycoproteins of human T-cell leukemia virus type 1 and regulates envelope mediated cell-cell fusion in T lymphocytes.

    Blot V, Delamarre L, Perugi F, Pham D, Bénichou S, Benarous R, Hanada T, Chishti AH, Dokhélar MC and Pique C

    Département Biologie Cellulaire, CNRS UMR 8104 and INSERM U567, Institut Cochin, 22 rue Méchain, 75014 Paris, France. vblot@cochin.inserm.fr

    Human homologue of the Drosophila Dlg tumor suppressor (hDlg) is a widely expressed scaffold protein implicated in the organization of multi-protein complexes at cell adhesion sites such as the neuronal synapse. hDlg contains three PDZ domains that mediate its binding to the consensus motifs present at the C-termini of various cell surface proteins, thus inducing their clustering and/or stabilization at the plasma membrane. Using a yeast two-hybrid screen, we identified hDlg as a cellular binding partner of a viral membrane integral protein, the envelope glycoprotein (Env) of human T-cell leukemia virus type 1 (HTLV-1). HTLV-1 is a human retrovirus that infects CD4+ T lymphocytes and is preferentially transmitted via direct contacts between infected and target cells, through a structure referred to as the virological synapse. Here, we demonstrate that hDlg interacts with a classical PDZ domain-binding motif present at the C-terminus of the cytoplasmic domain of HTLV-1 Env and conserved in the related HTLV-2 virus. We further document that, in HTLV-1 infected primary T cells, hDlg and Env are concentrated in restricted areas of the plasma membrane, enriched in molecules involved in T-cell contacts. The presence of Gag proteins responsible for viral assembly and budding in these areas indicated that they constitute platforms for viral assembly and transmission. Finally, a mutant virus unable to bind hDlg exhibited a decreased ability to trigger Env mediated cell fusion between T lymphocytes. We thus propose that hDlg stabilizes HTLV-1 envelope glycoproteins at the virological synapse formed between infected and target cells, hence assisting the cell-to-cell transmission of the virus.

    Funded by: NCI NIH HHS: CA 94414

    Journal of cell science 2004;117;Pt 17;3983-93

  • Protein trafficking and anchoring complexes revealed by proteomic analysis of inward rectifier potassium channel (Kir2.x)-associated proteins.

    Leonoudakis D, Conti LR, Anderson S, Radeke CM, McGuire LM, Adams ME, Froehner SC, Yates JR and Vandenberg CA

    Department of Molecular, Cellular, University of California, Santa Barbara, California 93106, USA.

    Inward rectifier potassium (Kir) channels play important roles in the maintenance and control of cell excitability. Both intracellular trafficking and modulation of Kir channel activity are regulated by protein-protein interactions. We adopted a proteomics approach to identify proteins associated with Kir2 channels via the channel C-terminal PDZ binding motif. Detergent-solubilized rat brain and heart extracts were subjected to affinity chromatography using a Kir2.2 C-terminal matrix to purify channel-interacting proteins. Proteins were identified with multidimensional high pressure liquid chromatography coupled with electrospray ionization tandem mass spectrometry, N-terminal microsequencing, and immunoblotting with specific antibodies. We identified eight members of the MAGUK family of proteins (SAP97, PSD-95, Chapsyn-110, SAP102, CASK, Dlg2, Dlg3, and Pals2), two isoforms of Veli (Veli-1 and Veli-3), Mint1, and actin-binding LIM protein (abLIM) as Kir2.2-associated brain proteins. From heart extract purifications, SAP97, CASK, Veli-3, and Mint1 also were found to associate with Kir2 channels. Furthermore, we demonstrate for the first time that components of the dystrophin-associated protein complex, including alpha1-, beta1-, and beta2-syntrophin, dystrophin, and dystrobrevin, interact with Kir2 channels, as demonstrated by immunoaffinity purification and affinity chromatography from skeletal and cardiac muscle and brain. Affinity pull-down experiments revealed that Kir2.1, Kir2.2, Kir2.3, and Kir4.1 all bind to scaffolding proteins but with different affinities for the dystrophin-associated protein complex and SAP97, CASK, and Veli. Immunofluorescent localization studies demonstrated that Kir2.2 co-localizes with syntrophin, dystrophin, and dystrobrevin at skeletal muscle neuromuscular junctions. These results suggest that Kir2 channels associate with protein complexes that may be important to target and traffic channels to specific subcellular locations, as well as anchor and stabilize channels in the plasma membrane.

    Funded by: NINDS NIH HHS: NS33145, NS43377

    The Journal of biological chemistry 2004;279;21;22331-46

  • Direct binding of the human homologue of the Drosophila disc large tumor suppressor gene to seven-pass transmembrane proteins, tumor endothelial marker 5 (TEM5), and a novel TEM5-like protein.

    Yamamoto Y, Irie K, Asada M, Mino A, Mandai K and Takai Y

    Department of Molecular Biology and Biochemistry, Osaka University Graduate School of Medicine/Faculty of Medicine, Suita 565-0871, Japan.

    The human homologue of the Drosophila discs large tumor suppressor gene (hDlg) is a member of the membrane-associated guanylate kinase family with three PSD-95/Dlg/ZO-1 (PDZ) domains. hDlg has been shown to bind tumor suppressor proteins, adenomatous polyposis coli (APC) and protein tyrosine phosphatase and tensin homologue (PTEN), and several viral oncoproteins, and has been implicated in the negative regulation of cell proliferation. hDlg has furthermore been shown to localize at the plasma membrane of synapses and to scaffold cell surface receptors and channels. In epithelial cells, hDlg localizes at the basolateral plasma membrane, but its localization mechanism is unknown. We searched here for a transmembrane protein that directly bound to hDlg. hDlg bound tumor endothelial marker 5 (TEM5), a seven-pass transmembrane protein that is homologous to the family B of G-protein-coupled receptors (GPCRs). TEM5 has previously been reported to display elevated expression during tumor angiogenesis and neoangiogenesis. The PDZ domains of hDlg bound the C-terminal PDZ-binding motif of TEM5. The expression of TEM5 was detected in endothelial cells of embryonic liver, where hDlg colocalized with TEM5. hDlg furthermore bound a novel seven-pass transmembrane protein, which was homologous to TEM5, and was named here a TEM5-like protein (TEM5-like). These resul 5a8 ts suggest that hDlg localizes at the plasma membrane through TEM5 and TEM5-like and furthermore scaffolds these GPCRs in endothelial cells during tumor angiogenesis and neoangiogenesis.

    Oncogene 2004;23;22;3889-97

  • A multiprotein trafficking complex composed of SAP97, CASK, Veli, and Mint1 is associated with inward rectifier Kir2 potassium channels.

    Leonoudakis D, Conti LR, Radeke CM, McGuire LM and Vandenberg CA

    Department of Molecular, Cellular, and Developmental Biology, and Neuroscience Research Institute, University of California, Santa Barbara, California 93106, USA.

    Strong inward rectifier potassium (Kir2) channels are important in the control of cell excitability, and their functions are modulated by interactions with intracellular proteins. Here we identified a complex of scaffolding/trafficking proteins in brain that associate with Kir2.1, Kir2.2, and Kir2.3 channels. By using a combination of affinity interaction pulldown assays and co-immunoprecipitations from brain and transfected cells, we demonstrated that a complex composed of SAP97, CASK, Veli, and Mint1 associates with Kir2 channels via the C-terminal PDZ-binding motif. We further demonstrated by using in vitro protein interaction assays that SAP97, Veli-1, or Veli-3 binds directly to the Kir2.2 C terminus and recruits CASK. Co-immunoprecipitations indicated that specific Veli isoforms participate in forming distinct protein complexes in brain, where Veli-1 stably associates with CASK and SAP97, Veli-2 associates with CASK and Mint1, and Veli-3 associates with CASK, SAP97, and Mint1. Additionally, immunocytochemistry of rat cerebellum revealed overlapping expression of Kir2.2, SAP97, CASK, Mint1, with Veli-1 in the granule cell layer and Veli-3 in the molecular layer. We propose a model whereby Kir2.2 associates with distinct SAP97-CASK-Veli-Mint1 complexes. In one complex, SAP97 interacts directly with the Kir2 channels and recruits CASK, Veli, and Mint1. Alternatively, Veli-1 or Veli-3 interacts directly with the Kir2 channels and recruits CASK and SAP97; association of Mint1 with the complex requires Veli-3. Expression of Kir2.2 in polarized epithelial cells resulted in targeting of the channels to the basolateral membrane and co-localization with SAP97 and CASK, whereas a dominant interfering form of CASK caused the channels to mislocalize. Therefore, CASK appears to be a central protein of a macromolecular complex that participates in trafficking and plasma membrane localization of Kir2 channels.

    Funded by: NINDS NIH HHS: NS43377

    The Journal of biological chemistry 2004;279;18;19051-63

  • Human homolog of disc-large is required for adherens junction assembly and differentiation of human intestinal epithelial cells.

    Laprise P, Viel A and Rivard N

    Canadian Institutes of Health Research Group on Functional Development and Physiopathology of the Digestive Tract, Département d'Anatomie et Biologie Cellulaire, Faculté de Médecine, Université de Sherbrooke, Québec J1H 5N4, Canada.

    We and others have shown that phosphatidylinositol 3-kinase (PI3K) is recruited to and activated by E-cadherin engagement. This PI3K activation is essential for adherens junction integrity and intestinal epithelial cell differentiation. Here we provide evidence that hDlg, the homolog of disc-large tumor suppressor, is another key regulator of adherens junction integrity and differentiation in mammalian epithelial cells. We report the following. 1) hDlg co-localizes with E-cadherin, but not with ZO-1, at the sites of cell-cell contact in intestinal epithelial cells. 2) Reduction of hDlg expression levels by RNA(i) in intestinal cells not only severely alters adherens junction integrity but also prevents the recruitment of p85/PI3K to E-cadherin-mediated cell-cell contact and inhibits sucrase-isomaltase gene expression. 3) PI3K and hDlg are associated with E-cadherin in a common macromolecular complex in living differentiating intestinal cells. 4) This interaction requires the association of hDlg with E-cadherin and with Src homology domain 2 domains of the p85/PI3K subunit. 5) Phosphorylation of hDlg on serine and threonine residues prevents its interaction with the p85 Src homology domain 2 in subconfluent cells, whereas phosphorylation of hDlg on tyrosine residues is essential. We conclude that hDlg may be a determinant in E-cadherin-mediated adhesion and signaling in mammalian epithelial cells.

    The Journal of biological chemistry 2004;279;11;10157-66

  • Different isoforms of synapse-associated protein, SAP97, are expressed in the heart and have distinct effects on the voltage-gated K+ channel Kv1.5.

    Godreau D, Vranckx R, Maguy A, Goyenvalle C and Hatem SN

    INSERM 460, Medical Hospital Xavier Bichat-Claude Bernard, 46 Rue Henri Huchard, 75018 Paris, France.

    The SAP97 isoforms differ by alternatively spliced insertion domains that regulate protein localization and oligomerization. We used reverse transcription-PCR to identify SAP97 isoforms of human and rat myocardium. In Chinese hamster ovary cells, cloned protein expression was studied using Western blot, confocal imaging of green fluorescent protein-tagged proteins, and patch clamp technique. The two main cardiac SAP97 isoforms contained both I3 and I1B inserts and differed by the I1A insert. Both isoforms co-precipitated with hKv1.5 channels. Only the isoform lacking I1A increased the current (by 215 +/- 22%), whatever the level of channel expression. To examine the involvement of the proline-rich I1A insert in the effect of SAP97, a W623F mutation in the Src homology 3 domain was created, and that restored the effect of the SAP97 on current. SAP97 isoform containing an I1A and I2 domain instead of the I3 domain stimulated the current, whereas SAP97 after deletion of the Src homology 3 and guanylate kinase-like domains did not. In cells co-expressing I3(+I1A) or I3(-I1A), green fluorescent protein-tagged Kv1.5 channels were organized in plaque-like structures at the plasma membrane level, whereas intracellular aggregates of channels predominated with the I2 isoform. The two cardiac SAP97 isoforms have different effects on the hKv1.5 current, depending on their capacity to form channel clusters.

    The Journal of biological chemistry 2003;278;47

  • CaMKII-dependent phosphorylation regulates SAP97/NR2A interaction.

    Gardoni F, Mauceri D, Fiorentini C, Bellone C, Missale C, Cattabeni F and Di Luca M

    Center of Excellence on Neurodegenerative Diseases and Department of Pharmacological Sciences, University of Milano, via Balzaretti 9, 20133 Milano, Italy. Fabrizio.Gardoni@unimi.it

    Synapse-associated protein 97 (SAP97), a member of membrane-associated guanylate kinase protein family, has been implicated in the processes of targeting ionotropic glutamate receptors at postsynaptic sites. Here we show that SAP97 is enriched at the postsynaptic density where it co-localizes with both ionotropic glutamate receptors and downstream signaling proteins such as Ca2+/calmodulin-dependent protein kinase II (CaMKII). SAP97 and alphaCaMKII display a high co-localization pattern in hippocampal neurons as well as in transfected COS-7 cells. Metabolic labeling of hippocampal cultures reveals that N-methyl-D-aspartic acid (NMDA) receptor activation induces CaMKII-dependent phosphorylation of SAP97; co-incubation with the CaMKII-specific inhibitor KN-93 reduces SAP97 phosphorylation to basal levels. Our results show that SAP97 directly interacts with the NR2A subunit of NMDA receptor both in an in vitro "pull-out" assay and in co-immunoprecipitation experiments from homogenates and synaptosomes purified from hippocampal rat tissue. Interestingly, in the postsynaptic density fraction, SAP97 fails to co-precipitate with NR2A. We show here that SAP97 is directly associated with NR2A through its PDZ1 domain, and CaMKII-dependent phosphorylation of SAP97-Ser-232 disrupts NR2A interaction both in an in vitro pull-out assay and in transfected COS-7 cells. Moreover, expression of SAP97(S232D) mutant has effects similar to those observed upon constitutively activating CaMKII. Our findings suggest that SAP97/NR2A interaction is regulated by CaMKII-dependent phosphorylation and provide a novel mechanism for the regulation of synaptic targeting of NMDA receptor subunits.

    The Journal of biological chemistry 2003;278;45;44745-52

  • Regulation of the discs large tumor suppressor by a phosphorylation-dependent interaction with the beta-TrCP ubiquitin ligase receptor.

    Mantovani F and Banks L

    International Centre for Genetic Engineering and Biotechnology, Padriciano 99, Trieste I-34012, Italy. mantovani@area.trieste.it

    The discs large (hDlg) tumor suppressor is intimately involved in the control of cell contact, polarity, and proliferation by interacting with several components of the epithelial junctional complex and with the APC tumor suppressor protein. In epithelial cells, hDlg protein stability is regulated through the ubiquitin-proteasome pathway: hDlg is actively degraded in isolated cells, whereas it accumulates upon cell-cell contact. During neoplastic transformation of epithelial cells, loss of the differentiated morphology and progression toward a metastatic phenotype correlate with down-regulation of hDlg levels and loss of contact-dependent stabilization. Here we show that upon hyperphosphorylation, hDlg interacts with the beta-TrCP ubiquitin ligase receptor through a DSGLPS motif within its Src homology 3 domain. As a consequence, overexpression of beta-TrCP enhances ubiquitination of Dlg protein and decreases its stability, whereas a dominant negative beta-TrCP mutant inhibits this process. Furthermore, a mutant Dlg protein that is unable to bind beta-TrCP displays a higher protein stability and is insensitive to beta-TrCP. Using RNA interference, we also demonstrate that endogenous beta-TrCP regulates hDlg protein levels in epithelial cells. Finally, we show that beta-TrCP selectively induces the degradation of the membrane-cytoplasmic pool, without affecting the nuclear pool of hDlg.

    The Journal of biological chemistry 2003;278;43;42477-86

  • Protein 4.1-mediated membrane targeting of human discs large in epithelial cells.

    Hanada T, Takeuchi A, Sondarva G and Chishti AH

    Department of Medicine, St. Elizabeth's Medical Center, Tufts University School of Medicine, Boston, Massachusetts 02135, USA.

    Human discs large (hDlg) protein binds to protein 4.1R via a motif encoded by an alternatively spliced exon located between the SH3 and the C-terminal guanylate kinase-like domains. To evaluate the functional significance of protein 4.1R binding for subcellular localization of hDlg in vivo, we expressed full-length recombinant constructs of two naturally occurring isoforms of hDlg termed hDlg-I2 and hDlg-I3. The hDlg-I3 but not the hDlg-I2 isoform binds to the FERM (Four.1-Ezrin-Radixin-Moesin) domain of protein 4.1R in vitro. Upon transient transfection into subconfluent Madine-Darby canine kidney (MDCK) epithelial cells, the hDlg-I3 fused with the green fluorescent protein accumulated predominantly at the plasma membrane of cell-cell contact sites, whereas the hDlg-I2 fusion protein distributed in the cytoplasm. In contrast, in stably transfected confluent MDCK cells, both hDlg-I2 and -I3 isoforms localized efficiently to the lateral membrane, consistent with the previous notion that the N-terminal domain of hDlg mediates its membrane targeting in polarized epithelial cells. We introduced a double mutation (I38A/I40A) into the N-terminal domain of hDlg, which disrupted its interaction with DLG2, a key event in the membrane targeting of hDlg. Interestingly, the hDlg-I2 isoform harboring the I38A/I40A mutation mislocalized from the membrane into cytoplasm. Importantly, the hDlg-I3 isoform with the same mutation localized efficiently to the membrane of confluent MDCK cells. Together, our results demonstrate that in addition to the N-terminal targeting domain, the alternatively spliced I3 insertion plays a critical role in recruiting hDlg to the lateral membrane in epithelial cells via its interaction with protein 4.1R.

    Funded by: NCI NIH HHS: CA94414; NHLBI NIH HHS: HL 60755

    The Journal of biological chemistry 2003;278;36;34445-50

  • SAP97 increases Kv1.5 currents through an indirect N-terminal mechanism.

    Eldstrom J, Choi WS, Steele DF and Fedida D

    Department of Physiology, University of British Columbia, 2146 Health Sciences Mall, V6T 1Z3, Vancouver, BC, Canada.

    The functional interaction of the voltage-gated potassium channel hKv1.5 with the PDZ domain containing protein SAP97 has been investigated. In marked contrast with the known dependence of SAP97-induced Kv1 potassium current down-regulation on the channel C-termini, SAP97 increased hKv1.5 current through an indirect interaction with the Kv1.5 N-terminus. Deletion of the Kv1.5 N-terminus eliminated the SAP97-mediated increase in potassium currents whereas deletion of the channel's C-terminal PDZ binding motif had no effect. In contrast with other Kv1-SAP97 interactions, no physical interaction could be detected in vivo or in vitro between the two proteins. The proteins did not co-localize in cardiac myocytes nor did they co-immunoprecipitate from transfected HEK cells. Yeast two-hybrid experiments also failed to detect any interaction between the two proteins, but in one experiment of six, Kv1.5 co-immunoprecipitated very inefficiently with SAP97 from rat ventricular myocytes. Thus, we conclude that the influence of SAP97 on Kv1.5 potassium current levels is dependent upon a novel regulatory mechanism.

    FEBS letters 2003;547;1-3;205-11

  • Selective interaction of megalin with postsynaptic density-95 (PSD-95)-like membrane-associated guanylate kinase (MAGUK) proteins.

    Larsson M, Hjälm G, Sakwe AM, Engström A, Höglund AS, Larsson E, Robinson RC, Sundberg C and Rask L

    Department of Medical Biochemistry and Microbiology, Uppsala University, PO Box 582, SE-751 23 Uppsala, Sweden. Marten.Larsson@imbim.uu.se

    Megalin is an integral membrane receptor belonging to the low-density lipoprotein receptor family. In addition to its role as an endocytotic receptor, megalin has also been proposed to have signalling functions. Using interaction cloning in yeast, we identified the membrane-associated guanylate kinase family member postsynaptic density-95 (PSD-95) as an interaction partner for megalin. PSD-95 and a truncated version of megalin were co-immunoprecipitated from HEK-293 cell lysates overexpressing the two proteins, which confirmed the interaction. The two proteins were found to be co-localized in these cells by confocal microscopy. Immunocytochemical studies showed that cells in the parathyroid, proximal tubuli of the kidney and placenta express both megalin and PSD-95. We found that the interaction between the two proteins is mediated by the binding of the C-terminus of megalin, which has a type I PSD-95/ Drosophila discs-large/zona occludens 1 (PDZ)-binding motif, to the PDZ2 domain of PSD-95. The PSD-95-like membrane-associated guanylate kinase ('MAGUK') family contains three additional members: PSD-93, synapse-associated protein 97 (SAP97) and SAP102. We detected these proteins, apart from SAP102, in parathyroid chief cells, a cell type having a marked expression of megalin. The PDZ2 domains of PSD-93 and SAP102 were also shown to interact with megalin, whereas no interaction was detected for SAP97. The SAP97 PDZ2 domain differed at four positions from the other members of the PSD-95 subfamily. One of these residues was Thr(389), located in the alphaB-helix and part of the hydrophobic pocket of the PDZ2 domain. Surface plasmon resonance experiments revealed that mutation of SAP97 Thr(389) to alanine, as with the other PSD-95-like membrane-associated guanylate kinases, induced binding to megalin.

    The Biochemical journal 2003;373;Pt 2;381-91

  • Myo6 facilitates the translocation of endocytic vesicles from cell peripheries.

    Aschenbrenner L, Lee T and Hasson T

    Section of Cell and Developmental Biology, Division of Biological Sciences, University of California at San Diego, La Jolla, California 92093, USA.

    Immunolocalization studies in epithelial cells revealed myo6 was associated with peripherally located vesicles that contained the transferrin receptor. Pulse-chase experiments after transferrin uptake showed that these vesicles were newly uncoated endocytic vesicles and that myo6 was recruited to these vesicles immediately after uncoating. GIPC, a putative myo6 tail binding protein, was also present. Myo6 was not present on early endosomes, suggesting that myo6 has a transient association with endocytic vesicles and is released upon early endosome fusion. Green fluorescent protein (GFP) fused to myo6 as well as the cargo-binding tail (M6tail) alone targeted to the nascent endocytic vesicles. Overexpression of GFP-M6tail had no effect on a variety of organelle markers; however, GFP-M6tail displaced the endogenous myo6 from nascent vesicles and resulted in a significant delay in transferrin uptake. Pulse-chase experiments revealed that transferrin accumulated in uncoated vesicles within the peripheries of transfected cells and that Rab5 was recruited to the surface of these vesicles. Given sufficient time, the transferrin did traffic to the perinuclear sorting endosome. These data suggest that myo6 is an accessory protein required for the efficient transportation of nascent endocytic vesicles from the actin-rich peripheries of epithelial cells, allowing for timely fusion of endocytic vesicles with the early endosome.

    Molecular biology of the cell 2003;14;7;2728-43

  • Dlg, Scribble and Lgl in cell polarity, cell proliferation and cancer.

    Humbert P, Russell S and Richardson H

    Cell Cycle and Cancer Genetics Laboratory, Peter MacCallum Cancer Institute, St. Andrew's Place, East Melbourne, Victoria 3002, Australia.

    Dlg (Discs large), Scrib (Scribble) and Lgl (Lethal giant larvae) are evolutionarily conserved components of a common genetic pathway that link the seemingly disparate functions of cell polarity and cell proliferation in epithelial cells. dlg, scrib and lgl have been identified as tumour suppressor genes in Drosophila, mutations of which cause similar phenotypes, involving disruption of cell polarity and neoplastic overgrowth of tissues. The molecular mechanisms by which Dlg, Scrib and Lgl proteins regulate cell proliferation are not clear, but there is some evidence that epithelial polarisation is required for this regulation. Dlg, Scrib and Lgl are highly conserved between human and Drosophila, and we discuss evidence that these proteins also play a role in cancer progression in humans.

    BioEssays : news and reviews in molecular, cellular and developmental biology 2003;25;6;542-53

  • Identification of SAP97 as an intracellular binding partner of TACE.

    Peiretti F, Deprez-Beauclair P, Bonardo B, Aubert H, Juhan-Vague I and Nalbone G

    INSERM EPI 99-36, Laboratoire d'Hématologie, Faculté de Médecine, 27 Bd Jean Moulin, Marseilles 13385 Cedex 5, France. Franck.Peiretti@medecine.univ-mrs.fr

    Tumor necrosis factor alpha converting enzyme (TACE) is the metalloprotease-disintegrin responsible for the ectodomain shedding of several proteins, including tumor necrosis factor alpha. Using the yeast two-hybrid system, we identified the scaffolding protein synapse associated protein 97 (SAP97) as a binding partner of the cytoplasmic domain of TACE. By deletions and site-directed mutagenesis, we demonstrated that this interaction involved the PDZ3 domain of SAP97 and the extreme C-terminal amino-acid sequence of TACE. This interaction as well as the identification of the specific domains involved was confirmed in vitro by affinity purification and in mammalian cells by co-immunoprecipitation and alteration of localization analyzed by immunofluorescence microscopy. In addition, confocal microscopy showed that endogenous TACE and SAP97 colocalized in some intracellular areas of COS-7 cells and CACO-2 cells. Furthermore, overexpression of SAP97, unlike that of a mutant form of SAP97 deleted for its PDZ3 domain, altered the ability of TACE to release its substrates. Altogether, these results demonstrate an interaction between TACE and SAP97, which may have a functional implication for the regulation of TACE shedding activity.

    Journal of cell science 2003;116;Pt 10;1949-57

  • Characterization of PISP, a novel single-PDZ protein that binds to all plasma membrane Ca2+-ATPase b-splice variants.

    Goellner GM, DeMarco SJ and Strehler EE

    Department of Biochemistry and Molecular Biology, Mayo Clinic, Rochester, Minnesota 55905, USA.

    Plasma membrane Ca(2+) ATPases (PMCAs) maintain intracellular Ca(2+) homeostasis and participate in the local regulation of Ca(2+) signaling. Spatially separate demands for Ca(2+) regulation require proper membrane targeting of PMCAs, but the mechanism of PMCA targeting is unknown. Using the PMCA2b carboxyl-terminal tail as yeast two-hybrid bait, we isolated a novel PDZ domain-containing protein from a human brain cDNA library. This protein, named PISP for PMCA-interacting single-PDZ protein, consists of 140 amino acids and contains little else besides a single PDZ domain. Pulldown experiments showed that PISP interacts with all PMCA b-splice forms. PISP was found to be ubiquitously expressed and, in MDCK cells, was present in a punctate pattern throughout the cytosol and at the basolateral membrane. When added to microsomal membranes expressing PMCA4b, PISP was unable to stimulate the PMCA-dependent ATPase activity. Our data suggest that PISP is a transiently interacting partner of the PMCA b-splice forms that may play a role in their sorting to or from the plasma membrane.

    Funded by: NIGMS NIH HHS: GM-58710

    Annals of the New York Academy of Sciences 2003;986;461-71

  • Interaction of the plasma membrane Ca2+ pump 4b/CI with the Ca2+/calmodulin-dependent membrane-associated kinase CASK.

    Schuh K, Uldrijan S, Gambaryan S, Roethlein N and Neyses L

    Department of Medicine, University of Wuerzburg, D-97080 Wuerzburg, Germany.

    Spatial and temporal regulation of intracellular Ca(2+) is a key event in many signaling pathways. Plasma membrane Ca(2+)-ATPases (PMCAs) are major regulators of Ca(2+) homeostasis and bind to PDZ (PSD-95/Dlg/ZO-1) domains via their C termini. Various membrane-associated guanylate kinase family members have been identified as interaction partners of PMCAs. In particular, SAP90/PSD95, PSD93/chapsyn-110, SAP97, and SAP102 all bind to the C-terminal tails of PMCA "b" splice variants. Additionally, it has been demonstrated that PMCA4b interacts with neuronal nitric-oxide synthase and that isoform 2b interacts with Na(+)/H(+) exchanger regulatory factor 2, both via a PDZ domain. CASK (calcium/calmodulin-dependent serine protein kinase) contains a calmodulin-dependent protein kinase-like domain followed by PDZ, SH3, and guanylate kinase-like domains. In adult brain CASK is located at neuronal synapses and interacts with various proteins, e.g. neurexin and Veli/LIN-7. In kidney it is localized to renal epithelia. Surprisingly, interaction with the Tbr-1 transcription factor, nuclear transport, binding to DNA T-elements (in a complex with Tbr-1), and transcriptional competence has been shown. Here we show that the C terminus of PMCA4b binds to CASK and that both proteins co-precipitate from brain and kidney tissue lysates. Immunofluorescence staining revealed co-expression of PMCA, CASK, and calbindin-d-28K in distal tubuli of rat kidney sections. To test if physical interaction of both proteins results in functional consequences we constructed a T-element-dependent reporter vector and investigated luciferase activity in HEK293 lysates, previously co-transfected with PMCA4b expression and control vectors. Expression of wild-type PMCA resulted in an 80% decrease in T-element-dependent transcriptional activity, whereas co-expression of a point-mutated PMCA, with nearly eliminated Ca(2+) pumping activity, had only a small influence on regulation of transcriptional activity. These results provide evidence of a new direct Ca(2+)-dependent link from the plasma membrane to the nucleus.

    The Journal of biological chemistry 2003;278;11;9778-83

  • Binding of the human homolog of the Drosophila discs large tumor suppressor protein to the mitochondrial ribosomal protein MRP-S34.

    Ogawa F, Adachi S, Kohu K, Shige K and Akiyama T

    Laboratory of Molecular and Genetic Information, Institute for Molecular and Cellular Biosciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-0032, Japan.

    The human homolog of the Drosophila discs large tumor suppressor protein (hDLG) functions as a scaffolding protein that facilitates the transmission of diverse downstream signals. Here we show that hDLG interacts through its PDZ domains with the carboxy-terminal S/TXV motif of the mitochondrial ribosomal protein S-34 (MRP-S34). Our results suggest that hDLG interacts with MRP-S34 prior to entry of MRP-S34 into the mitochondria and may be involved in the trafficking of MRP-S34.

    Biochemical and biophysical research communications 2003;300;3;789-92

  • Identification of multiple binding partners for the amino-terminal domain of synapse-associated protein 97.

    Karnak D, Lee S and Margolis B

    Department of Biological Chemistry, University of Michigan, Ann Arbor, Michigan 48109, USA.

    Multiprotein complexes mediate static and dynamic functions to establish and maintain cell polarity in both epithelial cells and neurons. Membrane-associated guanylate kinase (MAGUK) proteins are thought to be scaffolding molecules in these processes and bind multiple proteins via their obligate postsynaptic density (PSD)-95/Disc Large/Zona Occludens-1, Src homology 3, and guanylate kinase-like domains. Subsets of MAGUK proteins have additional protein-protein interaction domains. An additional domain we identified in SAP97 called the MAGUK recruitment (MRE) domain binds the LIN-2,7 amino-terminal (L27N) domain of mLIN-2/CASK, a MAGUK known to bind mLIN-7. Here we show that SAP97 binds two other mLIN-7 binding MAGUK proteins. One of these MAGUK proteins, DLG3, coimmunoprecipitates with SAP97 in lysates from rat brain and transfected Madin-Darby canine kidney cells. This interaction requires the MRE domain of SAP97 and surprisingly, both the L27N and L27 carboxyl-terminal (L27C) domains of DLG3. We also demonstrate that SAP97 can interact with the MAGUK protein, DLG2, but not the highly related protein, PALS2. The ability of SAP97 to interact with multiple MAGUK proteins is likely to be important for the targeting of specific protein complexes in polarized cells.

    Funded by: NIDDK NIH HHS: 2-P50-DK39255; NIGMS NIH HHS: 5-T32-GM07544, GM08353

    The Journal of biological chemistry 2002;277;48;46730-5

  • Changes in expression of the human homologue of the Drosophila discs large tumour suppressor protein in high-grade premalignant cervical neoplasias.

    Watson RA, Rollason TP, Reynolds GM, Murray PG, Banks L and Roberts S

    Cancer Research UK Institute for Cancer Studies, The Medical School, University of Birmingham, UK.

    The Drosophila tumour suppressor discs large (Dlg) is a cell-junction localized protein that is required for the maintenance of epithelial cyto-architecture and the negative control of cell proliferation. The mammalian homologue is likely to have a similar mode of action, and therefore functional perturbation of this protein may be linked to the development of epithelial-derived cancers. The finding that several unrelated viral oncoproteins, including the E6 protein of oncogenic human papillomaviruses, bind to the human homologue of Dlg (hDlg) supports this proposition. Employing immunohistochemistry, we show that in uterine cervical squamous epithelia, prominent localization of hDlg at sites of intercellular contact occurs in cells that have left the proliferating basal cell layers and begun maturation. The presence of hDlg at sites of cell:cell contact diminishes, whilst intracellular cytoplasmic levels increase significantly in high-grade, but not low-grade, cervical neoplasias. In invasive squamous cell carcinomas, total cellular hDlg levels are greatly reduced. Our data suggest that loss of hDlg at sites of intercellular contact may be an important step in the development of epithelial cancers.

    Carcinogenesis 2002;23;11;1791-6

  • Formation of complexes between Ca2+.calmodulin and the synapse-associated protein SAP97 requires the SH3 domain-guanylate kinase domain-connecting HOOK region.

    Paarmann I, Spangenberg O, Lavie A and Konrad M

    Department of Molecular Genetics, Max Planck Institute for Biophysical Chemistry, Göttingen D-37070, Germany.

    Mammalian synapse-associated protein SAP97, a structural and functional homolog of Drosophila Dlg, is a membrane-associated guanylate kinase (MAGUK) that is present at pre- and postsynaptic sites as well as in epithelial cell-cell contact sites. It is a multidomain scaffolding protein that shares with other members of the MAGUK protein family a characteristic modular organization composed of three sequential protein interaction motifs known as PDZ domains, followed by an Src homology 3 (SH3) domain, and an enzymatically inactive guanylate kinase (GK)-like domain. Specific binding partners are known for each domain, and different modes of intramolecular interactions have been proposed that particularly involve the SH3 and GK domains and the so-called HOOK region located between these two domains. We identified the HOOK region as a specific site for calmodulin binding and studied the dynamics of complex formation of recombinant calmodulin and SAP97 by surface plasmon resonance spectroscopy. Binding of various SAP97 deletion constructs to immobilized calmodulin was strictly calcium-dependent. From the rate constants of association and dissociation we determined an equilibrium dissociation constant K(d) of 122 nm for the association of calcium-saturated calmodulin and a SAP97 fragment, which encompassed the entire SH3-HOOK-GK module. Comparative structure-based sequence analysis of calmodulin binding regions from various target proteins predicts variable affinities for the interaction of calmodulin with members of the MAGUK protein family. Our findings suggest that calmodulin could regulate the intramolecular interaction between the SH3, HOOK, and GK domains of SAP97.

    The Journal of biological chemistry 2002;277;43;40832-8

  • Selective binding of synapse-associated protein 97 to GluR-A alpha-amino-5-hydroxy-3-methyl-4-isoxazole propionate receptor subunit is determined by a novel sequence motif.

    Cai C, Coleman SK, Niemi K and Keinänen K

    Department of Biosciences, Division of Biochemistry, University of Helsinki, Helsinki FIN-00014, Finland.

    A family of four closely related PDZ domain-containing membrane-associated guanylate kinase homologues (MAGUKs) is involved in the regulation of the amount and functional state of ionotropic glutamate receptors in excitatory synapses. To understand the mechanisms that determine the specificity of these interactions, we examined the structural basis of the highly selective association between the ionotropic GluR subunit GluR-A and synapse-associated protein 97 (SAP97). The C terminus of GluR-A bound to the PDZ domains of SAP97, but not to those of three related MAGUKs, PSD-93, PSD-95, and SAP102. Experiments with single PDZ domains indicated that the strongest contribution was by the second PDZ domain. Unexpectedly, mutation analysis of the GluR-A C terminus revealed that a tripeptide sequence SSG at position -9 to -11 plays an essential role in this binding, in addition to a C-terminal type I PDZ binding motif (leucine at C terminus and threonine at the -2 position). Analysis of the in vitro MAGUK-binding properties of a GluR-D mutant with a one-residue deletion at the C terminus provides further support for the view that an SSG sequence located N-terminally from a type I PDZ binding motif can mediate selective binding to SAP97 and suggest the existence of a novel variation of the PDZ domain-peptide interaction.

    The Journal of biological chemistry 2002;277;35;31484-90

  • Interaction of SAP97 with minus-end-directed actin motor myosin VI. Implications for AMPA receptor trafficking.

    Wu H, Nash JE, Zamorano P and Garner CC

    Department of Neurobiology, University of Alabama, Birmingham, AL 35294-0021, USA.

    SAP97 is a modular protein composed of three PDZ domains, an SH3 domain, and a guanylate kinase-like domain. It has been implicated functionally in the assembly and structural stability of synaptic junctions as well as in the trafficking, recruitment, and localization of specific ion channels and neurotransmitter receptors. The N terminus of SAP97 (S97N) has been shown to play a key role in the selection of binding partners and the localization of SAP97 at adhesion sites, as well as the clustering of ion channels in heterologous cells. Using the S97N domain as bait in a yeast two-hybrid screen, we identified the minus-end-directed actin-based motor, myosin VI, as an S97N binding partner. Moreover, in light membrane fractions prepared from rat brain, we found that myosin VI and SAP97 form a trimeric complex with the alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptor subunit, GluR1. These data suggest that SAP97 may serve as a molecular link between GluR1 and the actin-dependent motor protein myosin VI during the dynamic translocation of AMPA receptors to and from the postsynaptic plasma membrane.

    Funded by: NIA NIH HHS: AG 06569-09, AG 12978-02; NICHD NIH HHS: P50 HD 32901

    The Journal of biological chemistry 2002;277;34;30928-34

  • Postsynaptic targeting of alternative postsynaptic density-95 isoforms by distinct mechanisms.

    Chetkovich DM, Bunn RC, Kuo SH, Kawasaki Y, Kohwi M and Bredt DS

    Department of Physiology, University of California, San Francisco, San Francisco, California 94143-0444, USA.

    Members of the postsynaptic density-95 (PSD95)/synapse-associated protein-90 (SAP90) family of scaffolding proteins contain a common set of modular protein interaction motifs including PDZ (postsynaptic density-95/Discs large/zona occludens-1), Src homology 3, and guanylate kinase domains, which regulate signaling and plasticity at excitatory synapses. We report that N-terminal alternative splicing of PSD95 generates an isoform, PSD95beta that contains an additional "L27" motif, which is also present in SAP97. Using yeast two hybrid and coimmunoprecipitation assays, we demonstrate that this N-terminal L27 domain of PSD-95beta, binds to an L27 domain in the membrane-associated guanylate kinase calcium/calmodulin-dependent serine kinase, and to Hrs, an endosomal ATPase that regulates vesicular trafficking. By transfecting heterologous cells and hippocampal neurons, we find that interactions with the L27 domain regulate synaptic clustering of PSD95beta. Disrupting Hrs-regulated early endosomal sorting in hippocampal neurons selectively blocks synaptic clustering of PSD95beta but does not interfere with trafficking of the palmitoylated isoform, PSD95alpha. These studies identify molecular and functional heterogeneity in synaptic PSD95 complexes and reveal critical roles for L27 domain interactions and Hrs regulated vesicular trafficking in postsynaptic protein clustering.

    The Journal of neuroscience : the official journal of the Society for Neuroscience 2002;22;15;6415-25

  • Association of the kinesin superfamily motor protein KIF1Balpha with postsynaptic density-95 (PSD-95), synapse-associated protein-97, and synaptic scaffolding molecule PSD-95/discs large/zona occludens-1 proteins.

    Mok H, Shin H, Kim S, Lee JR, Yoon J and Kim E

    Department of Biological Sciences, Korea Advanced Institute of Science and Technology, Daejeon 305-701, Korea.

    Mutation in KIF1B, a kinesin superfamily motor protein, causes a peripheral neuropathy known as Charcot-Marie-Tooth disease type 2A (CMT2A). Little is known, however, about how a defective KIF1B gene leads to CMT2A. Here we report that KIF1Balpha, one of the two splice variants of KIF1B, directly interacts through its C-terminal postsynaptic density-95 (PSD-95)/discs large/zona occludens (PDZ) domain-binding motif with PDZ proteins including PSD-95/synapse-associated protein-90 (SAP90), SAP97, and synaptic scaffolding molecule (S-SCAM)-90 (SAP90). KIF1Balpha selectively interacts with PSD-95, SAP97, and S-SCAM in yeast two-hybrid, pull-down, and in vivo coimmunoprecipitation experiments. KIF1Balpha, SAP97, and S-SCAM are widely distributed to both dendrites and axons of cultured neurons and are enriched in the small membrane fraction of the brain. In the flotation assay, KIF1Balpha cofractionates and coimmunoprecipitates with PSD-95, SAP97, and S-SCAM. These results suggest that the PSD-95 family proteins and S-SCAM have a novel function as KIF1Balpha receptors, linking KIF1Balpha to its specific cargos, and are involved in peripheral neuropathies.

    The Journal of neuroscience : the official journal of the Society for Neuroscience 2002;22;13;5253-8

  • The SH3, HOOK and guanylate kinase-like domains of hDLG are important for its cytoplasmic localization.

    Kohu K, Ogawa F and Akiyama T

    Department of Molecular and Genetic Information, Institute for Molecular and Cellular Biosciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113, Japan.

    Background: hDLG, the human homologue of the Drosophila tumour suppressor dlg, functions as a scaffolding protein that facilitates the transmission of diverse downstream signals. hDLG possesses multiple protein-binding domains, including three PDZ domains, an SH3 domain, a HOOK domain and a guanylate kinase-like (GK) domain.

    Results: We studied the significance of the PDZ, SH3, HOOK and GK domains in the cytoplasmic localization of hDLG. We found that mutation of the SH3 or GK domain, but not the PDZ domain, resulted in a re-localization of hDLG to the nucleus. Furthermore, hDLG was found to possess a potential nuclear localization signal in the HOOK d 13d4 omain.

    Conclusion: These results suggest that the SH3, HOOK and GK domains of hDLG are important for its cytoplasmic localization.

    Genes to cells : devoted to molecular & cellular mechanisms 2002;7;7;707-15

  • Direct interaction of Frizzled-1, -2, -4, and -7 with PDZ domains of PSD-95.

    Hering H and Sheng M

    Center for Learning and Memory, Howard Hughes Medical Institute, RIKEN-MIT Neuroscience Research Center, and Department of Brain and Cognitive Sciences, Massachusetts Institute of Technology, 77 Massachusetts Avenue (E18-215), Cambridge, MA 02139, USA.

    In Drosophila, the frizzled gene plays a critical role in the establishment of tissue polarity, but the function of the Frizzled family of proteins in mammals is largely unknown. Recent evidence suggested that Frizzleds are receptors for the Wnt family of secreted glycoproteins which are involved in cell fate determination. However, it is unclear how Frizzled receptors transduce Wnt signals to intracellular signaling components. Here we show that the mouse Frizzled-1, -2, -4 and -7 can bind to proteins of the PSD-95 family, which are implicated in the assembly and localization of multiprotein signaling complexes in the brain. Moreover, PSD-95 can form a ternary complex with Frizzled-2 and the adenomatous polyposis coli protein, a negative regulator of Wnt signaling, suggesting that members of the PSD-95 family may serve to recruit intracellular signaling molecules of the Wnt/Frizzled pathway into the vicinity of the receptor.

    FEBS letters 2002;521;1-3;185-9

  • Compartmentalized NRG signaling and PDZ domain-containing proteins in synapse structure and function.

    Huang YZ, Wang Q, Won S, Luo ZG, Xiong WC and Mei L

    Department of Neurobiology, Pathology, Physical Medicine and Rehabilitation, University of Alabama at Birmingham, 35294-0021, USA.

    The synapse-specific synthesis of the acetylcholine receptor (AChR) is mediated by multiple mechanisms including compartmentalized signaling induced by neuregulin (NRG). This paper presents evidence that NRG receptors--ErbB receptor tyrosine kinases interact with distinct PDZ domain-containing proteins that are localized at the neuromuscular junction (NMJ). ErbB4 associates with the PSD-95 (also known as SAP90)-family members including PSD-95, SAP97, and SAP102 whereas ErbB2 interacts with Erbin and PICK1. Although, ErbB kinases are concentrated at the NMJ, they are not colocalized with the AChR in cultured muscle cells even in the presence of agrin. Co-expression of PSD-95 causes ErbB4 to form clusters in COS cells. We propose that PDZ domain-containing proteins play a role in anchoring ErbB proteins at the neuromuscular junction, and/or mediating downstream signaling pathways. Such mechanisms could be important for the maintenance and function of the synapse.

    Funded by: NICHD NIH HHS: P30HD38985

    International journal of developmental neuroscience : the official journal of the International Society for Developmental Neuroscience 2002;20;3-5;173-85

  • Inward rectifier K+ channel Kir2.3 is localized at the postsynaptic membrane of excitatory synapses.

    Inanobe A, Fujita A, Ito M, Tomoike H, Inageda K and Kurachi Y

    Department of Pharmacology II, Graduate School of Medicine, Osaka University, Suita, Osaka 565-0871, Japan.

    Classical inwardly rectifying K+ channels (Kir2.0) are responsible for maintaining the resting membrane potential near the K+ equilibrium potential in various cells, including neurons. Although Kir2.3 is known to be expressed abundantly in the forebrain, its precise localization has not been identified. Using an antibody specific to Kir2.3, we examined the subcellular localization of Kir2.3 in mouse brain. Kir2.3 immunoreactivity was detected in a granular pattern in restricted areas of the brain, including the olfactory bulb (OB). Immunoelectron microscopy of the OB revealed that Kir2.3 immunoreactivity was specifically clustered on the postsynaptic membrane of asymmetric synapses between granule cells and mitral/tufted cells. The immunoprecipitants for Kir2.3 obtained from brain contained PSD-95 and chapsyn-110, PDZ domain-containing anchoring proteins. In vitro binding assay further revealed that the COOH-terminal end of Kir2.3 is responsible for the association with these anchoring proteins. Therefore, the Kir channel may be involved in formation of the resting membrane potential of the spines and, thus, would affect the response of N-methyl-D-aspartic acid receptor channels at the excitatory postsynaptic membrane.

    American journal of physiology. Cell physiology 2002;282;6;C1396-403

  • The distribution and function of alternatively spliced insertions in hDlg.

    McLaughlin M, Hale R, Ellston D, Gaudet S, Lue RA and Viel

    Department of Molecular and Cellular Biology, Harvard University, Cambridge, Massachusetts 02138, USA.

    hDlg is the human homolog of the Drosophila Discs-large tumor suppressor. As a member of the MAGUK (membrane-associated guanylate kinase) family of scaffolding proteins, hDlg is composed of three PDZ (PSD-95, Dlg, and ZO-1) repeats, an SH3 (Src homology 3) motif, and a GUK (guanylate kinase-like) domain. Additionally, hDlg contains two regions of alternative splicing. Here we identify a novel insertion, I1B, located N-terminal to the PDZ repeats. We further analyze the tissue-specific combinations of insertions and correlate those results with the distribution of protein isoforms. We also identify the functions of the two alternatively spliced regions. The N-terminal alternatively spliced region is capable of binding several SH3 domains and also moderates the level of protein oligomerization. Insertions in the second region are responsible for determining the localization of hDlg, with insertion I3 targeting the protein to the membrane regions of cell-cell contact and insertion I2 targeting the protein to the nucleus.

    Funded by: NIGMS NIH HHS: GM57314

    The Journal of biological chemistry 2002;277;8;6406-12

  • Pilt, a novel peripheral membrane protein at tight junctions in epithelial cells.

    Kawabe H, Nakanishi H, Asada M, Fukuhara A, Morimoto K, Takeuchi M and Takai Y

    Department of Molecular Biology and Biochemistry, Osaka University Graduate School of Medicine/Faculty of Medicine, Suita 565-0871, Japan.

    Tight junctions (TJs) serve as a barrier that prevents solutes and water from passing through the paracellular pathway, and as a fence between the apical and basolateral plasma membranes in epithelial cells. TJs consist of transmembrane proteins (claudin, occludin, and JAM) and many peripheral membrane proteins, including actin filament (F-actin)-binding scaffold proteins (ZO-1, -2, and -3), non-F-actin-binding scaffold proteins (MAGI-1), and cell polarity molecules (ASIP/PAR-3 and PAR-6). We identified here a novel peripheral membrane protein at TJs from a human cDNA library and named it Pilt (for protein incorporated later into TJs), because it was incorporated into TJs later after the claudin-based junctional strands were formed. Pilt consists of 547 amino acids with a calculated M(r) of 60,704. Pilt has a proline-rich domain. In cadherin-deficient L cells stably expressing claudin or JAM, Pilt was not recruited to claudin-based or JAM-based cell-cell contact sites, suggesting that Pilt does not directly interact with claudin or JAM. The present results indicate that Pilt is a novel component of TJs.

    The Journal of biological chemistry 2001;276;51;48350-5

  • Polarity and developmental regulation of two PDZ proteins in the retinal pigment epithelium.

    Bonilha VL and Rodriguez-Boulan E

    Department of Ophthalmology, Margaret Dyson Vision Research Institute, Weill Medical College of Cornell University, 1300 York Avenue, New York, NY 10021, USA.

    Purpose: Identification of binding partners for ezrin, an actin-binding protein crucial for morphogenesis of apical microvilli and basolateral infoldings in RPE cells.

    Methods: Rat eyes, rat primary RPE, the rat RPE-J cell line, and a clonal line of RPE-J cells transfected with human ezrin cDNA were analyzed by immunofluorescence microscopy and immunoblot. Immunofluorescence localization of two ezrin-binding proteins was performed in cryosections of rat eyes of various ages and in monolayers extracted with the detergent Triton X-100 and fixed in paraformaldehyde. The interaction of both proteins with ezrin and gluthathione-S-transferase (GST)-ezrin fusion proteins was analyzed by SDS-PAGE and immunoblot.

    Results: Immunofluorescence microscopy of adult rat eyes detected a polarized distribution of ERM (ezrin, radixin, and moesin)-binding phosphoprotein of 50 kDa (EBP50) at the apical microvilli and synaptic-associated protein of 97 kDa (SAP97) at the basolateral surface of RPE cells, which overlapped with ezrin. These two PDZ (postsynaptic density protein [PSD-95]/disc large [DLG]-A/ZO-1) domain proteins had a similar polarized distribution and high resistance to detergent extractability, indicative of cytoskeletal association, both in primary cultures of rat RPE and in a clonal RPE-J cell line expressing high levels of transfected ezrin. RPE cell lysates from rat retinas of various postnatal ages revealed increasing levels of EBP50 and SAP97 compared with alphav integrin, a protein expressed at constant adult levels from birth. GST pull-down and immunoprecipitation experiments demonstrated a direct interaction between EBP50 and SAP97 and ezrin.

    Conclusions: The data indicate that EBP50 localizes at the apical microvilli, whereas SAP97 localizes at the basolateral surface of RPE cells, probably through a direct interaction with ezrin.

    Funded by: NEI NIH HHS: EY 08538, R01 EY008538

    Investigative ophthalmology & visual science 2001;42;13;3274-82

  • Guanylyl cyclase/PSD-95 interaction: targeting of the nitric oxide-sensitive alpha2beta1 guanylyl cyclase to synaptic membranes.

    Russwurm M, Wittau N and Koesling D

    Pharmakologie und Toxikologie, Medizinische Fakultät MA N1, Ruhr-Universität Bochum, 44780 Bochum, Germany.

    The signaling molecule nitric oxide (NO) exerts most of its effects by the stimulation of the NO-sensitive guanylyl cyclase. Two isoforms of the NO receptor molecule exist: the ubiquitously occurring alpha(1)beta(1) and the alpha(2)beta(1) with a more limited distribution. As the isoforms are functionally indistinguishable, the physiological relevance of these isoforms remained unclear. The neuronal NO synthase has been reported to be associated with PSD-95. Here, we demonstrate the interaction of the so far unnoticed alpha(2)beta(1) isoform with PSD-95 in rat brain as shown by coprecipitation. The interaction is mediated by the alpha(2) C-terminal peptide and the third PDZ domain of PSD-95. As a consequence of the PSD-95 interaction, the so far considered "soluble" alpha(2)beta(1) isoform is recruited to the membrane fraction of synaptosomes, whereas the alpha(1)beta(1) isoform is found in the cytosol. Our results establish the alpha(1)beta(1) as the cytosolic and the alpha(2)beta(1) as the membrane-associated NO-sensitive guanylyl cyclase and suggest the alpha(2)beta(1) isoform as the sensor for the NO formed by the PSD-95-associated neuronal NO synthase.

    The Journal of biological chemistry 2001;276;48;44647-52

  • Synapse-associated protein 97 selectively associates with a subset of AMPA receptors early in their biosynthetic pathway.

    Sans N, Racca C, Petralia RS, Wang YX, McCallum J and Wenthold RJ

    Laboratory of Neurochemistry, National Institute on Deafness and other Communication Disorders, National Institutes of Health, Bethesda, Maryland 20892-8027, USA. sansn@nidcd.nih.gov

    The regulation of AMPA receptors at the postsynaptic membrane is a fundamental component of synaptic plasticity. In the hippocampus, the induction of long-term potentiation increases the delivery of GluR1, a major AMPA receptor subunit in hippocampal pyramidal neurons, to the synaptic plasma membrane through a mechanism that requires the PDZ binding domain of GluR1. Synapse-associated protein 97 (SAP97), a member of the membrane-associated guanylate kinase family, is believed to associate with AMPA receptors (AMPARs) containing the GluR1 subunit, but the functional significance of these interactions is unclear. We investigated the interaction of GluR1 with SAP97, the only PDZ protein known to interact with GluR1. We find that interactions involving SAP97 and GluR1 occur early in the secretory pathway, while the receptors are in the endoplasmic reticulum or cis-Golgi. In contrast, few synaptic receptors associate with SAP97, suggesting that SAP97 dissociates from the receptor complex at the plasma membrane. We also show that internalization of GluR1, as triggered by NMDAR activation, does not require SAP97. These results implicate GluR1-SAP97 interactions in mechanisms underlying AMPA receptor targeting.

    The Journal of neuroscience : the official journal of the Society for Neuroscience 2001;21;19;7506-16

  • Lano, a novel LAP protein directly connected to MAGUK proteins in epithelial cells.

    Saito H, Santoni MJ, Arsanto JP, Jaulin-Bastard F, Le Bivic A, Marchetto S, Audebert S, Isnardon D, Adélaïde J, Birnbaum D and Borg JP

    U119 INSERM, Molecular Oncology, 27 boulevard Lei Roure, 13009 Marseille, France.

    Protein networks asymetrically distributed to basolateral and apical epithelial membranes maintain cell polarity and homeostasis of epithelial tissues. Genetic studies in non-vertebrates assigned two families of basolateral proteins, MAGUK (membrane-associated and guanylate kinase) and LAP (leucine-rich repeats and PDZ) proteins, to a common pathway crucial for the epithelial architecture and acting as a gatekeeper to malignancy. In mammals, three LAP proteins have been described, Densin-180, Erbin, and hScribble. Here, we identify a protein called Lano (LAP and no PDZ) only present in vertebrates and presenting strong identities with LAP proteins. Despite the lack of PDZ domain, Lano is located at the basolateral side of epithelial cells in a similar manner to Erbin and hScribble. Using in vitro and in vivo experiments, we demonstrate that Lano directly interacts with the PDZ domains of MAGUK proteins, including hDLG (human disc large), in epithelial cells. A second pool of Lano is complexed to Erbin. These LAP-MAGUK protein complexes coexist at the basolateral side of epithelial cells. We provide evidence for a direct interaction between LAP and MAGUK proteins, and we propose that various LAP-MAGUK networks targeted to the basolateral side of epithelial cells participate to homeostasis of epithelial tissues and tumor growth.

    The Journal of biological chemistry 2001;276;34;32051-5

  • Plasma membrane Ca2+-atpase isoforms 2b and 4b interact promiscuously and selectively with members of the membrane-associated guanylate kinase family of PDZ (PSD95/Dlg/ZO-1) domain-containing proteins.

    DeMarco SJ and Strehler EE

    Program in Molecular Neuroscience, Department of Biochemistry, Mayo Graduate School, Mayo Clinic, Rochester, Minnesota 55905, USA.

    Spatial and temporal regulation of intracellular Ca(2+) signaling depends on localized Ca(2+) microdomains containing the requisite molecular components for Ca(2+) influx, efflux, and signal transmission. Plasma membrane Ca(2+)-ATPase (PMCA) isoforms of the "b" splice type contain predicted PDZ (PSD95/Dlg/ZO-1) interaction domains. The COOH-terminal tail of PMCA2b isolated the membrane-associated guanylate kinase (MAGUK) protein SAP97/hDlg as a binding partner in a yeast two-hybrid screen. The related MAGUKs SAP90/PSD95, PSD93/chapsyn-110, SAP97, and SAP102 all bound to the COOH-terminal tail of PMCA4b, whereas only the first three bound to the tail of PMCA2b. Coimmunoprecipitations confirmed the interaction selectivity between PMCA4b and SAP102 as opposed to the promiscuity of PMCA2b and 4b in interacting with other SAPs. Confocal immunofluorescence microscopy revealed the exclusive presence and colocalization of PMCA4b and SAP97 in the basolateral membrane of polarized Madin-Darby canine kidney epithelial cells. In hippocampal neurons, PMCA2b was abundant throughout the somatodendritic compartment and often extended into the neck and head of individual spines where it colocalized with SAP90/PSD95. These data show that PMCA "b" splice forms interact promiscuously but also with specificity with different members of the PSD95 family of SAPs. PMCA-SAP interactions may play a role in the recruitment and maintenance of the PMCA at specific membrane domains involved in local Ca(2+) regulation.

    Funded by: NIGMS NIH HHS: GM-58710

    The Journal of biological chemistry 2001;276;24;21594-600

  • Molecular mechanisms regulating the differential association of kainate receptor subunits with SAP90/PSD-95 and SAP97.

    Mehta S, Wu H, Garner CC and Marshall J

    Department Of Molecular Pharmacology, Physiology, and Biotechnology, Brown University, Providence, Rhode Island 02912, USA.

    Recent studies have demonstrated that kainate receptors are associated with members of the SAP90/PSD-95 family (synapse-associated proteins (SAPs)) in neurons and that SAP90 can cluster and modify the electrophysiological properties of GluR6/KA2 kainate receptors when co-expressed in transfected cells. In vivo, SAP90 tightly binds kainate receptor subunits, while SAP97 is only weakly associated, suggesting that this glutamate receptor differentially associates with SAP90/PSD-95 family members. Here, green fluorescent protein (GFP)-tagged chimeras and deletion mutants of SAP97 and SAP90 were employed to define the molecular mechanism underlying their differential association with kainate receptors. Our results show that a weak interaction between GluR6 and the PDZ1 domain of SAP97 can account for the weak association of GluR6 with the full-length SAP97 observed in vivo. Expression studies in HEK293 cells and in vitro binding studies further show that although the individual Src homology 3 and guanylate kinase domains in SAP97 can interact with the C-terminal tail of KA2 subunit, specific intramolecular interactions in SAP97 (e.g. the SAP97 N terminus (S97N) binding to the Src homology 3 domain) interfere with KA2 binding to the full-length molecule. Because receptor subunits are known to segregate to different parts of the neuron, our results imply that differential association of kainate receptors with SAP family proteins may be one mechanism of subcellular localization.

    Funded by: NIA NIH HHS: AG 06569-09, AG 12978-02, AG12978-02; NICHD NIH HHS: P50 HD32901

    The Journal of biological chemistry 2001;276;19;16092-9

  • Sema4c, a transmembrane semaphorin, interacts with a post-synaptic density protein, PSD-95.

    Inagaki S, Ohoka Y, Sugimoto H, Fujioka S, Amazaki M, Kurinami H, Miyazaki N, Tohyama M and Furuyama T

    Group of Neurobiology, School of Allied Health Sciences, Osaka University Faculty of Medicine, Yamadaoka 1-7, Suita-shi, Osaka, 565-0871, Japan. inagaki@sahs.med.osaka-u.ac.jp

    Semaphorins are known to act as chemorepulsive molecules that guide axons during neural development. Sema4C, a group 4 semaphorin, is a transmembrane semaphorin of unknown function. The cytoplasmic domain of Sema4C contains a proline-rich region that may interact with some signaling proteins. In this study, we demonstrate that Sema4C is enriched in the adult mouse brain and associated with PSD-95 isoforms containing PDZ (PSD-95/DLG/ZO-1) domains, such as PSD-95/SAP90, PSD-93/chapsin110, and SAP97/DLG-1, which are concentrated in the post-synaptic density of the brain. In the neocortex, S4C is enriched in the synaptic vesicle fraction and Triton X-100 insoluble post-synaptic density fraction. Immunostaining for Sema4C overlaps that for PSD-95 in superficial layers I-IV of the neocortex. In neocortical culture, S4C is colocalized with PSD-95 in neurons, with a dot-like pattern along the neurites. Sema4C thus may function in the cortical neurons as a bi-directional transmembrane ligand through interacting with PSD-95.

    The Journal of biological chemistry 2001;276;12;9174-81

  • Inward rectifier potassium channel Kir2.2 is associated with synapse-associated protein SAP97.

    Leonoudakis D, Mailliard W, Wingerd K, Clegg D and Vandenberg C

    Department of Molecular, Cellular and Developmental Biology, and Neuroscience Research Institute, University of California, Santa Barbara, CA 93106, USA.

    The strong inwardly rectifying potassium channels Kir2.x are involved in maintenance and control of cell excitability. Recent studies reveal that the function and localization of ion channels are regulated by interactions with members of the membrane-associated guanylate kinase (MAGUK) protein family. To identify novel interacting MAGUK family members, we constructed GST-fusion proteins with the C termini of Kir2.1, Kir2.2 and Kir2.3. GST affinity-pulldown assays from solubilized rat cerebellum and heart membrane proteins revealed an interaction between all three Kir2.x C-terminal fusion proteins and the MAGUK protein synapse-associated protein 97 (SAP97). A truncated form of the C-terminal GST-Kir2.2 fusion protein indicated that the last three amino acids (S-E-I) are essential for association with SAP97. Affinity interactions using GST-fusion proteins containing the modular domains of SAP97 demonstrate that the second PSD-95/Dlg/ZO-1 (PDZ) domain is sufficient for interaction with Kir2.2. Coimmunoprecipitations demonstrated that endogenous Kir2.2 associates with SAP97 in rat cerebellum and heart. Additionally, phosphorylation of the Kir2.2 C terminus by protein kinase A inhibited the association with SAP97. In rat cardiac ventricular myocytes, Kir2.2 and SAP97 colocalized in striated bands corresponding to T-tubules. In rat cerebellum, Kir2.2 was present in a punctate pattern along SAP97-positive processes of Bergmann glia in the molecular layer, and colocalized with astrocytes and granule cells in the granule cell layer. These results identify a direct association of Kir2.1, Kir2.2 and Kir2.3 with the MAGUK family member SAP97 that may form part of a macromolecular signaling complex in many different tissues.

    Funded by: NHLBI NIH HHS: HL41656, IF32HL10239-01

    Journal of cell science 2001;114;Pt 5;987-98

  • hCASK and hDlg associate in epithelia, and their src homology 3 and guanylate kinase domains participate in both intramolecular and intermolecular interactions.

    Nix SL, Chishti AH, Anderson JM and Walther Z

    Departments of Internal Medicine, Cell Biology, and Pathology, Yale University School of Medicine, New Haven, Connecticut 06520, USA.

    Membrane-associated guanylate kinase (MAGUK) proteins act as molecular scaffolds organizing multiprotein complexes at specialized regions of the plasma membrane. All MAGUKs contain a Src homology 3 (SH3) domain and a region homologous to yeast guanylate kinase (GUK). We showed previously that one MAGUK protein, human CASK (hCASK), is widely expressed and associated with epithelial basolateral plasma membranes. We now report that hCASK binds another MAGUK, human discs large (hDlg). Immunofluorescence microscopy demonstrates that hCASK and hDlg colocalize at basolateral membranes of epithelial cells in small and large intestine. These proteins co-precipitate from lysates of an intestinal cell line, Caco-2. The GUK domain of hCASK binds the SH3 domain of hDlg in both yeast two-hybrid and fusion protein binding assays, and it is required for interaction with hDlg in transfected HEK293 cells. In addition, the SH3 and GUK domains of each protein participate in intramolecular binding that in vitro predominates over intermolecular binding. The SH3 and GUK domains of human p55 display the same interactions in yeast two-hybrid assays as those of hCASK. Not all SH3-GUK interactions among these MAGUKs are permissible, however, implying specificity to SH3-GUK interactions in vivo. These results suggest MAGUK scaffold assembly may be regulated through effects on intramolecular SH3-GUK binding.

    Funded by: NCI NIH HHS: CA66263; NIDDK NIH HHS: DK34989, T32DK07017; ...

    The Journal of biological chemistry 2000;275;52;41192-200

  • Stargazin regulates synaptic targeting of AMPA receptors by two distinct mechanisms.

    Chen L, Chetkovich DM, Petralia RS, Sweeney NT, Kawasaki Y, Wenthold RJ, Bredt DS and Nicoll RA

    Department of Cellular and Molecular Pharmacology, University of California, San Francisco 94143, USA.

    Stargazer, an ataxic and epileptic mutant mouse, lacks functional AMPA (alpha-amino-3-hydroxyl-5-methyl-4-isoxazolepropionate) receptors on cerebellar granule cells. Stargazin, the mutated protein, interacts with both AMPA receptor subunits and synaptic PDZ proteins, such as PSD-95. The interaction of stargazin with AMPA receptor subunits is essential for delivering functional receptors to the surface membrane of granule cells, whereas its binding with PSD-95 and related PDZ proteins through a carboxy-terminal PDZ-binding domain is required for targeting the AMPA receptor to synapses. Expression of a mutant stargazin lacking the PDZ-binding domain in hippocampal pyramidal cells disrupts synaptic AMPA receptors, indicating that stargazin-like mechanisms for targeting AMPA receptors may be widespread in the central nervous system.

    Nature 2000;408;6815;936-43

  • Intramolecular interactions regulate SAP97 binding to GKAP.

    Wu H, Reissner C, Kuhlendahl S, Coblentz B, Reuver S, Kindler S, Gundelfinger ED and Garner CC

    University of Alabama at Birmingham, Department of Neurobiology, 1719 Sixth Avenue South CIRC 589, Birmingham, AL 35294-0021, USA.

    Membrane-associated guanylate kinase homologs (MAGUKs) are multidomain proteins found to be central organizers of cellular junctions. In this study, we examined the molecular mechanisms that regulate the interaction of the MAGUK SAP97 with its GUK domain binding partner GKAP (GUK-associated protein). The GKAP-GUK interaction is regulated by a series of intramolecular interactions. Specifically, the association of the Src homology 3 (SH3) domain and sequences situated between the SH3 and GUK domains with the GUK domain was found to interfere with GKAP binding. In contrast, N-terminal sequences that precede the first PDZ domain in SAP97, facilitated GKAP binding via its association with the SH3 domain. Utilizing crystal structure data available for PDZ, SH3 and GUK domains, molecular models of SAP97 were generated. These models revealed that SAP97 can exist in a compact U-shaped conformation in which the N-terminal domain folds back and interacts with the SH3 and GUK domains. These models support the biochemical data and provide new insights into how intramolecular interactions may regulate the association of SAP97 with its binding partners.

    Funded by: NIA NIH HHS: AG 06569-09, AG 12978-02; NICHD NIH HHS: P50 HD32901

    The EMBO journal 2000;19;21;5740-51

  • GAKIN, a novel kinesin-like protein associates with the human homologue of the Drosophila discs large tumor suppressor in T lymphocytes.

    Hanada T, Lin L, Tibaldi EV, Reinherz EL and Chishti AH

    Section of Hematology-Oncology Research, Departments of Medicine, Anatomy, and Cellular Biology, St. Elizabeth's Medical Center, Tufts University School of Medicine, Boston, Massachusetts 02135, USA.

    Reorganization of the cortical cytoskeleton is a hallmark of T lymphocyte activation. Upon binding to antigen presenting cells, the T cells rapidly undergo cytoskeletal re-organization thus forming a cap at the cell-cell contact site leading to receptor clustering, protein segregation, and cellular polarization. Previously, we reported cloning of the human lymphocyte homologue of the Drosophila Discs Large tumor suppressor protein (hDlg). Here we show that a novel protein termed GAKIN binds to the guanylate kinase-like domain of hDlg. Affinity protein purification, peptide sequencing, and cloning of GAKIN cDNA from Jurkat J77 lymphocytes identified GAKIN as a novel member of the kinesin superfamily of motor proteins. GAKIN mRNA is ubiquitously expressed, and the predicted amino acid sequence shares significant sequence similarity with the Drosophila kinesin-73 motor protein. GAKIN sequence contains a motor domain at the NH(2) terminus, a central stalk domain, and a putative microtubule-interacting sequence called the CAP-Gly domain at the COOH terminus. Among the MAGUK superfamily of proteins examined, GAKIN binds to the guanylate kinase-like domain of PSD-95 but not of p55. The hDlg and GAKIN are localized mainly in the cytoplasm of resting T lymphocytes, however, upon CD2 receptor cross-linking the hDlg can translocate to the lymphocyte cap. We propose that the GAKIN-hDlg interaction lays the foundation for a general paradigm of coupling MAGUKs to the microtubule-based cytoskeleton, and that this interaction may be functionally important for the intracellular trafficking of MAGUKs and associated protein complexes in vivo.

    Funded by: NIAID NIH HHS: AI21226

    The Journal of biological chemistry 2000;275;37;28774-84

  • Targeting of PKA to glutamate receptors through a MAGUK-AKAP complex.

    Colledge M, Dean RA, Scott GK, Langeberg LK, Huganir RL and Scott JD

    Howard Hughes Medical Institute, Vollum Institute, Oregon Health Sciences University, Portland 97201, USA.

    Compartmentalization of glutamate receptors with the signaling enzymes that regulate their activity supports synaptic transmission. Two classes of binding proteins organize these complexes: the MAGUK proteins that cluster glutamate receptors and AKAPs that anchor kinases and phosphatases. In this report, we demonstrate that glutamate receptors and PKA are recruited into a macromolecular signaling complex through direct interaction between the MAGUK proteins, PSD-95 and SAP97, and AKAP79/150. The SH3 and GK regions of the MAGUKs mediate binding to the AKAP. Cell-based studies indicate that phosphorylation of AMPA receptors is enhanced by a SAP97-AKAP79 complex that directs PKA to GluR1 via a PDZ domain interaction. As AMPA receptor phosphorylation is implicated in regulating synaptic plasticity, these data suggest that a MAGUK-AKAP complex may be centrally involved.

    Funded by: NIGMS NIH HHS: GM48231

    Neuron 2000;27;1;107-19

  • Characterization of PDZ-binding kinase, a mitotic kinase.

    Gaudet S, Branton D and Lue RA

    Department of Molecular and Cellular Biology, Harvard University, 16 Divinity Avenue, Cambridge, MA 02138, USA.

    hDlg, the human homologue of the Drosophila Discs-large (Dlg) tumor suppressor protein, is known to interact with the tumor suppressor protein APC and the human papillomavirus E6 transforming protein. In a two-hybrid screen, we identified a 322-aa serine/threonine kinase that binds to the PDZ2 domain of hDlg. The mRNA for this PDZ-binding kinase, or PBK, is most abundant in placenta and absent from adult brain tissue. The protein sequence of PBK has all the characteristic protein kinase subdomains and a C-terminal PDZ-binding T/SXV motif. In vitro, PBK binds specifically to PDZ2 of hDlg through its C-terminal T/SXV motif. PBK and hDlg are phosphorylated at mitosis in HeLa cells, and the mitotic phosphorylation of PBK is required for its kinase activity. In vitro, cdc2/cyclin B phosphorylates PBK. This evidence shows how PBK could link hDlg or other PDZ-containing proteins to signal transduction pathways regulating the cell cycle or cellular proliferation.

    Funded by: NIGMS NIH HHS: GM57314-02

    Proceedings of the National Academy of Sciences of the United States of America 2000;97;10;5167-72

  • The APC-hDLG complex negatively regulates cell cycle progression from the G0/G1 to S phase.

    Ishidate T, Matsumine A, Toyoshima K and Akiyama T

    Department of Oncogene Research, Institute for Microbial Diseases, Osaka University, Suita, Japan.

    The adenomatous polyposis coli (APC) gene is mutated in familial adenomatous polyposis and in many sporadic colorectal tumors. The carboxyl-terminal S/TXV motif of the APC gene product interacts with the PDZ domain of hDLG, the human homolog of the Drosophila lethal (1) discs larige-1 (dlg) tumor suppressor. In the present study, we found that overexpression of hDLG suppresses cell proliferation by blocking cell cycle progression from the G0/G1 to S phase. This inhibition of cell cycle progression was abolished when the PDZ, SH3 or guanylate kinase-like domain of hDLG was mutated. Moreover, overexpression of these mutant hDLGs partially interfered with the cell cycle blocking activity of APC. Consistent with this result, mutant APC lacking the S/TXV motif exhibited weaker cell cycle blocking activity than the intact APC. These results suggest that APC-hDLG complex formation plays an important role in transducing the APC cell cycle blocking signal.

    Oncogene 2000;19;3;365-72

  • Anchoring proteins confer G protein sensitivity to an inward-rectifier K(+) channel through the GK domain.

    Hibino H, Inanobe A, Tanemoto M, Fujita A, Doi K, Kubo T, Hata Y, Takai Y and Kurachi Y

    Departments of Pharmacology II, Graduate School of Medicine, Osaka University, 2-2, Yamada-oka, Suita, Osaka 565-0871, Japan.

    Anchoring proteins cluster receptors and ion channels at postsynaptic membranes in the brain. They also act as scaffolds for intracellular signaling molecules including synGAP and NO synthase. Here we report a new function for intracellular anchoring proteins: the regulation of synaptic ion channel function. A neuronal G protein-gated inwardly rectifying K(+) channel, Kir3.2c, can not be activated either by M(2)-muscarinic receptor stimulation or by G(betagamma) overexpression. When coexpressed with SAP97, a member of the PSD/SAP anchoring protein family, the channel became sensitive to G protein stimulation. Although the C-terminus of Kir3. 2c bound to the second PDZ domain of SAP97, functional analyses revealed that the guanylate kinase (GK) domain of SAP97 is crucial for sensitization of the Kir3.2c channel to G protein stimulation. Furthermore, SAPAP1/GKAP, which binds specifically to the GK domain of membrane-associated guanylate kinases, prevented the SAP97-induced sensitization. The function of a synaptic ion channel can therefore be controlled by a network of various intracellular proteins.

    The EMBO journal 2000;19;1;78-83

  • Threonine phosphorylation of the MMAC1/PTEN PDZ binding domain both inhibits and stimulates PDZ binding.

    Adey NB, Huang L, Ormonde PA, Baumgard ML, Pero R, Byreddy DV and Bartel PL

    Myriad Genetics Inc., Salt Lake City, Utah 84108, USA. nils@myriad.com

    Two-hybrid searches with the tumor suppressor MMAC1/PTEN isolated the proteins hDLG and hMAST205. Further two-hybrid analysis and microtiter plate binding assays localized the sites of interaction to PDZ domains from hDLG and hMAST205 and the PDZ binding domain at the COOH terminus of MMAC1/PTEN. A synthetic peptide derived from the MMAC1/PTEN PDZ binding domain (MMAC1/PTEN-PDZBD) was used to coprecipitate proteins from A431 human cell lysate. The recovered proteins were resolved by SDS-PAGE and immobilized on a nitrocellulose membrane. Treatment of this membrane with an anti-hDLG antibody identified a Mr 140,000 band, consistent with the size of hDLG. Treatment of this membrane with the MMAC1/PTEN-PDZBD peptide identified a single prominent band of slightly larger than Mr 200,000 (Mr 200,000 kDa). Threonine phosphorylation of the MMAC1/ PTEN-PDZBD peptide inhibited both microtiter plate binding to the hDLG and hMAST205 PDZ domains and coprecipitation of the Mr 140,000 and > 200,000 proteins, but promoted coprecipitation of proteins of approximately Mr 90,000 and Mr 120,000 from A431 cell lysate. This result suggests phosphorylation of the MMAC1/PTEN PDZ binding domain can both inhibit and promote PDZ interactions.

    Cancer research 2000;60;1;35-7

  • Cypin: a cytosolic regulator of PSD-95 postsynaptic targeting.

    Firestein BL, Firestein BL, Brenman JE, Aoki C, Sanchez-Perez AM, El-Husseini AE and Bredt DS

    Department of Physiology, University of California, San Francisco 94143, USA.

    Postsynaptic density 95 (PSD-95/SAP-90) is a membrane associated guanylate kinase (GK) PDZ protein that scaffolds glutamate receptors and associated signaling networks at excitatory synapses. Affinity chromatography identifies cypin as a major PSD-95-binding protein in brain extracts. Cypin is homologous to a family of hydrolytic bacterial enzymes and shares some similarity with collapsin response mediator protein (CRMP), a cytoplasmic mediator of semaphorin III signalling. Cypin is discretely expressed in neurons and is polarized to basal membranes in intestinal epithelial cells. Overexpression of cypin in hippocampal neurons specifically perturbs postsynaptic trafficking of PSD-95 and SAP-102, an effect not produced by overexpression of other PDZ ligands. In fact, PSD-95 can induce postsynaptic clustering of an otherwise diffusely localized K+ channel, Kv1.4. By regulating postsynaptic protein sorting, cypin may influence synaptic development and plasticity.

    Funded by: NEI NIH HHS: R01-EY08055; NINDS NIH HHS: R01-NS36017

    Neuron 1999;24;3;659-72

  • Differential interaction of the tSXV motifs of the NR1 and NR2A NMDA receptor subunits with PSD-95 and SAP97.

    Bassand P, Bernard A, Rafiki A, Gayet D and Khrestchatisky M

    Université René Descartes (Paris V), INSERM U-29, Paris, France.

    The NR1 and NR2 subunits of the N-methyl-D-aspartate (NMDA) receptor are encoded by distinct genes. In the rat brain, four C-terminal variants of the NR1 subunit (NR1-1 to NR1-4) are encoded by a single gene, and are generated by alternative splicing of the C1 and C2 exon cassettes, while four different genes encode the NR2 subunits (NR2 A-D). Functional NMDA receptors result from the heteromultimeric assembly of NR1 variants with distinct NR2 subunits. The NR2B subunit interacts with post-synaptic density protein 95 (PSD-95), SAP97 and members of the membrane-associated guanylate-like kinase (MAGUK) family of proteins. This interaction occurs through the binding of the C-terminal tSXV intracellular motif of the NR2B subunit to the N-terminal PDZ (PSD-95, discs-large, ZO-1) domains of the PSD-95 and SAP97 proteins. Both NR1-3 and NR1-4 also display a consensus C-terminal tSXV motif. Using the two-hybrid genetic system in yeast and site-directed mutagenesis, we compared the binding of the NR2A, NR1-3 and NR1-4 tSXV motifs with the PDZ domains of PSD-95 and SAP97. The main conclusions of the present report are that: (i) while NR2A displays a strong interaction with PSD-95 and SAP97, the NR1-3 and NR1-4 NMDA receptor subunits do not display any interaction despite the presence of tSXV motifs; (ii) the C-terminal tSXV motif of the NR2A subunit is mandatory but not sufficient for efficient interaction with the PSD-95 and SAP97 proteins; (iii) as yet unidentified upstream sequences of the receptor subunits determine whether the tSXV motifs will bind to the PSD-95 and SAP97 PDZ domains; (iv) different tSXV motifs elicit interactions of variable strengths; and (v) residues in positions -3 and -4 modulate the binding affinity of the C-terminal tSXV motifs. Using immunohistochemistry, we also compared the distribution of the PSD-95, NR2A and SAP97 proteins in adult rat brain, and we show that in the cortex, hippocampus and cerebellum, there is evidence for colocalization of these proteins.

    The European journal of neuroscience 1999;11;6;2031-43

  • Localization of postsynaptic density-93 to dendritic microtubules and interaction with microtubule-associated protein 1A.

    Brenman JE, Topinka JR, Cooper EC, McGee AW, Rosen J, Milroy T, Ralston HJ and Bredt DS

    Department of Physiology, University of California at San Francisco, San Francisco, California 94143-0444, USA.

    Postsynaptic density-93 (PSD-93)/Chapsyn-110 is a member of the membrane-associated guanylate kinase (MAGUK) family of PDZ domain-containing proteins. MAGUKs are widely expressed in the brain and are critical elements of the cytoskeleton and of certain synapses. In the ultrastructural studies that are described here, PSD-93 localizes to both postsynaptic densities and dendritic microtubules of cerebellar Purkinje neurons. The microtubule localization is paralleled by a high-affinity in vivo interaction of PSD-93 via its guanylate kinase (GK) domain with microtubule-associated protein 1A (MAP1A). GK domain truncations that mimic genetically identified mutations of a Drosophila MAGUK, discs-large, disrupt the GK/MAP-1A interaction. Additional biochemical experiments demonstrate that intact MAGUKs do not bind to MAP1A as effectively as do isolated GK domains. This appears to be attributable to an intramolecular inhibition of the GK domain by the PDZs, because GK binding activity of full-length MAGUKs is partially restored by a variety of PDZ ligands, including the C termini of NMDA receptor 2B, adenomatous polyposis coli (APC), and CRIPT. Beyond demonstrating a novel cytoskeletal link for PSD-93, these experiments support a model in which intramolecular interactions between the multiple domains of MAGUKs regulate intermolecular associations and thereby may play a role in the proper targeting and function of MAGUK proteins.

    Funded by: NINDS NIH HHS: NS23347, NS36017

    The Journal of neuroscience : the official journal of the Society for Neuroscience 1998;18;21;8805-13

  • BEGAIN (brain-enriched guanylate kinase-associated protein), a novel neuronal PSD-95/SAP90-binding protein.

    Deguchi M, Hata Y, Takeuchi M, Ide N, Hirao K, Yao I, Irie M, Toyoda A and Takai Y

    Takai Biotimer Project, ERATO, Japan Science and Technology Corporation, c/o JCR Pharmaceuticals Co. Ltd., 2-2-10 Murotani, Nishi-ku, Kobe 651-2241, Japan.

    PSD-95/SAP90 is a synaptic membrane-associated guanylate kinase with three PDZ, one SH3, and one guanylate kinase (GK) domain. PSD-95/SAP90 binds various proteins through the PDZ domains and organizes synaptic junctions. PSD-95/SAP90 also interacts with the postsynaptic density (PSD) fraction-enriched protein, named SAPAP (also called GKAP and DAP), through the GK domain. SAPAP is Triton X-100-insoluble and recruits PSD-95/SAP90 into the Triton X-100-insoluble fraction in the transfected cells, suggesting that SAPAP may fix PSD-95/SAP90 to the PSD. Here we report a novel protein interacting with the GK domain of PSD-95/SAP90, BEGAIN. BEGAIN is specifically expressed in brain and enriched in the PSD fraction. BEGAIN is Triton X-100-soluble in the transfected cells but is recruited to the Triton X-100-insoluble fraction by SAPAP when coexpressed with PSD-95/SAP90. BEGAIN may be a novel PSD component associated with the core complex of PSD-95/SAP90 and SAPAP.

    The Journal of biological chemistry 1998;273;41;26269-72

  • SAP90 binds and clusters kainate receptors causing incomplete desensitization.

    Garcia EP, Mehta S, Blair LA, Wells DG, Shang J, Fukushima T, Fallon JR, Garner CC and Marshall J

    Department of Molecular Pharmacology, Physiology, and Biotechnology, Brown University, Providence, Rhode Island 02912, USA.

    The mechanism of kainate receptor targeting and clustering is still unresolved. Here, we demonstrate that members of the SAP90/PSD-95 family colocalize and associate with kainate receptors. SAP90 and SAP102 coimmunoprecipitate with both KA2 and GluR6, but only SAP97 coimmunoprecipitates with GluR6. Similar to NMDA receptors, GluR6 clustering is mediated by the interaction of its C-terminal amino acid sequence, ETMA, with the PDZ1 domain of SAP90. In contrast, the KA2 C-terminal region binds to, and is clustered by, the SH3 and GK domains of SAP90. Finally, we show that SAP90 coexpressed with GluR6 or GluR6/KA2 receptors alters receptor function by reducing desensitization. These studies suggest that the organization and electrophysiological properties of synaptic kainate receptors are modified by association with members of the SAP90/PSD-95 family.

    Funded by: NICHD NIH HHS: R01 HD023924, R01 HD052083; NINDS NIH HHS: R29 NS 33914-02

    Neuron 1998;21;4;727-39

  • SAP97 is associated with the alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptor GluR1 subunit.

    Leonard AS, Davare MA, Horne MC, Garner CC and Hell JW

    Department of Pharmacology, University of Wisconsin, Madison, Wisconsin 53706-1532, USA.

    Rapid glutamatergic synaptic transmission is mediated by ionotropic glutamate receptors and depends on their precise localization at postsynaptic membranes opposing the presynaptic neurotransmitter release sites. Postsynaptic localization of N-methyl-D-aspartate-type glutamate receptors may be mediated by the synapse-associated proteins (SAPs) SAP90, SAP102, and chapsyn-110. SAPs contain three PDZ domains that can interact with the C termini of proteins such as N-methyl-D-aspartate receptor subunits that carry a serine or threonine at the -2 position and a valine, isoleucine, or leucine at the very C terminus (position 0). We now show that SAP97, a SAP whose function at the synapse has been unclear, is associated with alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA)-type glutamate receptors. AMPA receptors are probably tetramers and are formed by two or more of the four AMPA receptor subunits GluR1-4. GluR1 possesses a C-terminal consensus sequence for interactions with PDZ domains of SAPs. SAP97 was present in AMPA receptor complexes immunoprecipitated from detergent extracts of rat brain. After treatment of rat brain membrane fractions with the cross-linker dithiobis(succinimidylpropionate) and solubilization with sodium dodecylsulfate, SAP97 was associated with GluR1 but not GluR2 or GluR3. In vitro experiments with recombinant proteins indicate that SAP97 specifically associates with the C terminus of GluR1 but not other AMPA receptor subunits. Our findings suggest that SAP97 may be involved in localizing AMPA receptors at postsynaptic sites through its interaction with the GluR1 subunit.

    Funded by: NIA NIH HHS: AG12978; NINDS NIH HHS: R01-NS35563

    The Journal of biological chemistry 1998;273;31;19518-24

  • E-cadherin mediated cell adhesion recruits SAP97 into the cortical cytoskeleton.

    Reuver SM and Garner CC

    Department of Neurobiology, University of Alabama at Birmingham, Birmingham, AL 35294-0021, USA.

    Members of the SAP family of synapse-associated proteins have recently emerged as central players in the molecular organization of synapses. In this study, we have examined the mechanism that localizes one member, SAP97, to sites of cell-cell contact. Utilizing epithelial CACO-2 cells and fibroblast L-cells as model systems, we demonstrate that SAP97 is associated with the submembranous cortical cytoskeleton at cell-cell adhesion sites. Furthermore, we show that its localization into this structure is triggered by E-cadherin. Although SAP97 can be found in an E-cadherin/catenin adhesion complex, this interaction seems to be mediated by the attachment of SAP97 to the cortical cytoskeleton. Our results are consistent with a model in which SAP97 is recruited to sites of cell-cell contact via an E-cadherin induced assembly of the cortical cytoskeleton.

    Funded by: NIA NIH HHS: AG 12978-02; NICHD NIH HHS: P50 HD32901

    Journal of cell science 1998;111 ( Pt 8);1071-80

  • Human homologue of the Drosophila discs large tumor suppressor binds to p56lck tyrosine kinase and Shaker type Kv1.3 potassium channel in T lymphocytes.

    Hanada T, Lin L, Chandy KG, Oh SS and Chishti AH

    Laboratory of Tumor Cell Biology, St. Elizabeth's Medical Center, Tufts University School of Medicine, Boston, Massachusetts 02135, USA.

    Human homologue of the Drosophila discs large tumor suppressor protein (hDlg) belongs to a newly discovered family of proteins termed MAGUKs that appear to have structural as well as signaling functions. Consistent with the multi-domain organization of MAGUKs, hDlg consists of three copies of the PDZ (PSD-95/Discs large/zO-1) domain, an SH3 motif, and a guanylate kinase-like domain. In addition, the hDlg contains an amino-terminal proline-rich domain that is absent in other MAGUKs. To explore the role of hDlg in cell signaling pathways, we used human T lymphocytes as a model system to investigate interaction of hDlg with known tyrosine kinases. In human T lymphocyte cell lines, binding properties of hDlg were studied by immunoprecipitation, immunoblotting, and immune complex kinase assays. Our results show that protein tyrosine kinase activity is associated with the immunoprecipitates of hDlg. Immunoblotting experiments revealed that the immunoprecipitates of hDlg contain p56lck, a member of the Src family of tyrosine kinases. The specificity of the interaction is demonstrated by the lack of p59fyn tyrosine kinase and phosphotidylinositol 3-kinase in the hDlg immunoprecipitates. Direct interaction between hDlg and p56lck is demonstrated using glutathione S-transferase fusion proteins of hDlg and recombinant p56lck expressed in the baculovirus-infected Sf9 cells. The p56lck binding site was localized within the amino-terminal segment of hDlg containing proline-rich domain. In addition, we show in vivo association of hDlg with Kv1.3 channel, which was expressed in T lymphocytes as an epitope-tagged protein using a vaccinia virus expression system. Taken together, these results provide the first evidence of a direct interaction between hDlg and p56lck tyrosine kinase and suggest a novel function of hDlg in coupling tyrosine kinase and voltage-gated potassium channel in T lymphocytes.

    Funded by: NCI NIH HHS: CA66263; NIAID NIH HHS: AI24783; NIGMS NIH HHS: GM-OD54872-01

    The Journal of biological chemistry 1997;272;43;26899-904

  • Binding of human virus oncoproteins to hDlg/SAP97, a mammalian homolog of the Drosophila discs large tumor suppressor protein.

    Lee SS, Weiss RS and Javier RT

    Division of Molecular Virology, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030, USA.

    The 9ORF1 gene encodes an adenovirus E4 region oncoprotein that requires a C-terminal region for transforming activity. Screening a lambdagt11 cDNA expression library with a 9ORF1 protein probe yielded a novel cellular PDZ domain-containing protein, 9BP-1, which binds to wild-type, but not a transformation-defective, C-terminal, mutant 9ORF1 protein. The fact that PDZ domains complex with specific sequences at the free C-terminal end of some proteins led to the recognition that the 9ORF1 C-terminal region contained such a consensus-binding motif. This discovery prompted investigations into whether the 9ORF1 protein associates with additional cellular proteins having PDZ domains. It was found that the 9ORF1 protein interacts directly, in vitro and in vivo, with the PDZ domain-containing protein hDlg/SAP97 (DLG), which is a mammalian homolog of the Drosophila discs large tumor suppressor protein and which also binds the adenomatous polyposis coli tumor suppressor protein. Of interest, in forming complexes, the 9ORF1 protein preferentially associated with the second PDZ domain of DLG, similar to adenomatous polyposis coli protein. Human T cell leukemia virus type 1 Tax and most oncogenic human papillomavirus E6 oncoproteins also possessed PDZ domain-binding motifs at their C termini and, significantly, human T cell leukemia virus type 1 Tax and human papillomavirus 18 E6 proteins bound DLG in vitro. Considering the requirement of the 9ORF1 C-terminal region in transformation, these findings suggest that interactions with the cellular factor DLG may contribute to the tumorigenic potentials of several different human virus oncoproteins.

    Funded by: NCI NIH HHS: CA58541, R01 CA058541

    Proceedings of the National Academy of Sciences of the United States of America 1997;94;13;6670-5

  • DAP-1, a novel protein that interacts with the guanylate kinase-like domains of hDLG and PSD-95.

    Satoh K, Yanai H, Senda T, Kohu K, Nakamura T, Okumura N, Matsumine A, Kobayashi S, Toyoshima K and Akiyama T

    Department of Oncogene Research, Institute for Microbial Diseases, Osaka University, Suita, Japan.

    Background: The human homologue of the Drosophila discs large tumour suppressor protein (hDLG) and closely related proteins such as postsynaptic density protein 95 kDa (PSD-95) are associated with N-methyl-D-aspartate receptors (NMDA-R) and Shaker-type K+ channels, and are thought to be involved in their clustering.

    Results: We have identified a protein named DAP-1 that binds to the guanylate kinase-like domains of hDLG and PSD-95. DAP-1 was found to associate with hDLG, PSD-95, NMDA-R and adenomatous polyposis coli protein (APC). Furthermore, we found that DAP-1 is specifically expressed in the brain and colocalizes with PSD-95 and APC in mouse cerebellum. We also found that DAP-1 is colocalized with PSD-95 and NMDA-R at the synapses in cultured rat hippocampal neurons.

    Conclusion: Our findings suggest that DAP-1 may play several roles in the molecular organization of synapses and neuronal cell signalling by interacting with hDLG and PSD-95, which in turn are associated with receptors, ion channels and APC.

    Genes to cells : devoted to molecular & cellular mechanisms 1997;2;6;415-24

  • Clustering and enhanced activity of an inwardly rectifying potassium channel, Kir4.1, by an anchoring protein, PSD-95/SAP90.

    Horio Y, Hibino H, Inanobe A, Yamada M, Ishii M, Tada Y, Satoh E, Hata Y, Takai Y and Kurachi Y

    Department of Pharmacology II, Faculty of Medicine, Osaka University, Suita, Osaka 565, Japan.

    An inwardly rectifying potassium channel predominantly expressed in glial cells, Kir4.1/KAB-2, has a sequence of Ser-Asn-Val in its carboxyl-terminal end, suggesting a possible interaction with an anchoring protein of the PSD-95 family. We examined the effects of PSD-95 on the distribution and function of Kir4.1 in a mammalian cell line. When Kir4.1 was expressed alone, the channel immunoreactivity was distributed homogeneously. In contrast, when co-expressed with PSD-95, prominent clustering of Kir4.1 in the cell membrane occurred. Kir4.1 was co-immunoprecipitated with PSD-95 in the co-expressed cells. Glutathione S-transferase-fusion protein of COOH terminus of Kir4.1 bound to PSD-95. These interactions disappeared when the Ser-Asn-Val motif was deleted. The magnitude of whole-cell Kir4.1 current was increased by 2-fold in cells co-expressing Kir4.1 and PSD-95 compared with cells expressing Kir4. 1 alone. SAP97, another member of the PSD-95 family, showed similar effects on Kir4.1. Furthermore, we found that Kir4.1 as well as SAP97 distributed not diffusely but clustered in retinal glial cells. Therefore, PSD-95 family proteins may be a physiological regulator of the distribution and function of Kir4.1 in glial cells.

    The Journal of biological chemistry 1997;272;20;12885-8

  • SAPAPs. A family of PSD-95/SAP90-associated proteins localized at postsynaptic density.

    Takeuchi M, Hata Y, Hirao K, Toyoda A, Irie M and Takai Y

    Takai Biotimer Project, ERATO, Japan Science and Technology Corporation, c/o JCR Pharmaceuticals Co., Ltd., 2-2-10 Murotani, Nishi-ku, Kobe 651-22, Japan.

    PSD-95/SAP90 is a member of membrane-associated guanylate kinases localized at postsynaptic density (PSD) in neuronal cells. Membrane-associated guanylate kinases are a family of signaling molecules expressed at various submembrane domains which have the PDZ (DHR) domains, the SH3 domain, and the guanylate kinase domain. PSD-95/SAP90 interacts with N-methyl-D-aspartate receptors 2A/B, Shaker-type potassium channels, and brain nitric oxide synthase through the PDZ (DHR) domains and clusters these molecules at synaptic junctions. However, neither the function of the SH3 domain or the guanylate kinase domain of PSD-95/SAP90, nor the protein interacting with these domains has been identified. We have isolated here a novel protein family consisting of at least four members which specifically interact with PSD-95/SAP90 and its related proteins through the guanylate kinase domain, and named these proteins SAPAPs (SAP90/PSD-95-Associated Proteins). SAPAPs are specifically expressed in neuronal cells and enriched in the PSD fraction. SAPAPs induce the enrichment of PSD-95/SAP90 to the plasma membrane in transfected cells. Thus, SAPAPs may have a potential activity to maintain the structure of PSD by concentrating its components to the membrane area.

    The Journal of biological chemistry 1997;272;18;11943-51

  • GKAP, a novel synaptic protein that interacts with the guanylate kinase-like domain of the PSD-95/SAP90 family of channel clustering molecules.

    Kim E, Naisbitt S, Hsueh YP, Rao A, Rothschild A, Craig AM and Sheng M

    Howard Hughes Medical Institute, Department of Neurobiology, Massachusetts General Hospital and Harvard Medical School, Boston 02114, USA.

    The molecular mechanisms underlying the organization of ion channels and signaling molecules at the synaptic junction are largely unknown. Recently, members of the PSD-95/SAP90 family of synaptic MAGUK (membrane-associated guanylate kinase) proteins have been shown to interact, via their NH2-terminal PDZ domains, with certain ion channels (NMDA receptors and K+ channels), thereby promoting the clustering of these proteins. Although the function of the NH2-terminal PDZ domains is relatively well characterized, the function of the Src homology 3 (SH3) domain and the guanylate kinase-like (GK) domain in the COOH-terminal half of PSD-95 has remained obscure. We now report the isolation of a novel synaptic protein, termed GKAP for guanylate kinase-associated protein, that binds directly to the GK domain of the four known members of the mammalian PSD-95 family. GKAP shows a unique domain structure and appears to be a major constituent of the postsynaptic density. GKAP colocalizes and coimmunoprecipitates with PSD-95 in vivo, and coclusters with PSD-95 and K+ channels/NMDA receptors in heterologous cells. Given their apparent lack of guanylate kinase enzymatic activity, the fact that the GK domain can act as a site for protein-protein interaction has implications for the function of diverse GK-containing proteins (such as p55, ZO-1, and LIN-2/CASK).

    Funded by: NINDS NIH HHS: NS33184, R01 NS033184

    The Journal of cell biology 1997;136;3;669-78

  • Two independent domains of hDlg are sufficient for subcellular targeting: the PDZ1-2 conformational unit and an alternatively spliced domain.

    Lue RA, Brandin E, Chan EP and Branton D

    Department of Molecular and Cellular Biology, Harvard University, Cambridge, Massachusetts 02138, USA.

    hDlg, a human homologue of the Drosophila Dig tumor suppressor, contains two binding sites for protein 4.1, one within a domain containing three PSD-95/Dlg/ZO-1 (PDZ) repeats and another within the alternatively spliced I3 domain. Here, we further define the PDZ-protein 4.1 interaction in vitro and show the functional role of both 4.1 binding sites in situ. A single protease-resistant structure formed by the entirety of both PDZ repeats 1 and 2 (PDZ1-2) contains the protein 4.1-binding site. Both this PDZ1-2 site and the I3 domain associate with a 30-kD NH2-terminal domain of protein 4.1 that is conserved in ezrin/radixin/moesin (ERM) proteins. We show that both protein 4.1 and the ezrin ERM protein interact with the murine form of hDlg in a coprecipitating immune complex. In permeabilized cells and tissues, either the PDZ1-2 domain or the I3 domain alone are sufficient for proper subcellular targeting of exogenous hDlg. In situ, PDZ1-2-mediated targeting involves interactions with both 4.1/ERM proteins and proteins containing the COOH-terminal T/SXV motif. I3-mediated targeting depends exclusively on interactions with 4.1/ERM proteins. Our data elucidates the multivalent nature of membrane-associated guanylate kinase homologue (MAGUK) targeting, thus beginning to define those protein interactions that are critical in MAGUK function.

    Funded by: NIGMS NIH HHS: GM39686

    The Journal of cell biology 1996;135;4;1125-37

  • Interaction between the C terminus of NMDA receptor subunits and multiple members of the PSD-95 family of membrane-associated guanylate kinases.

    Niethammer M, Kim E and Sheng M

    Department of Neurobiology, Harvard Medical School, Boston, Massachusetts 02114, USA.

    Selective concentration and anchoring of ionotropic receptors at the synapse is essential for neuronal signaling. Little is known about the molecules that mediate receptor clustering in the CNS. With use of the yeast two-hybrid system to screen a rat brain cDNA library and by in vitro binding assays, we have identified an interaction between NMDA receptor subunits 2A and 2B (NR2A and NR2B) and three distinct members of the PSD-95/SAP90 family of membrane-associated putative guanylate kinases. The interaction is mediated by binding of the C terminus of the NMDA receptor subunits to the first two PDZ (also known as GLGF or DHR) domains of PSD-95/SAP90, an abundant synaptic protein associated with the membrane cytoskeleton. PSD-95 is also known to bind and cluster Shaker-type voltage-gated K+ channels. Similarities between the C-termini of NR2 subunits and K+ channels suggest a common C-terminal binding motif for PDZ domains. These data suggest that PDZ domains can function as modules for protein-protein interactions. Members of the PSD-95 family might serve to anchor NMDA receptors to the submembrane cytoskeleton and aid in the assembly of signal transduction complexes at postsynaptic sites.

    The Journal of neuroscience : the official journal of the Society for Neuroscience 1996;16;7;2157-63

  • Differential K+ channel clustering activity of PSD-95 and SAP97, two related membrane-associated putative guanylate kinases.

    Kim E and Sheng M

    Howard Hughes Medical Institute, Department of Neurobiology, Massachusetts General Hospital, Boston 02114, USA.

    The molecular mechanisms underlying the clustering and localization of K+ channels in specific microdomains on the neuronal surface are largely unknown. The Shaker subclass of voltage-gated K+ channel alpha-subunits interact through their cytoplasmic C-terminus with a family of membrane-associated putative guanylate kinases, including PSD-95 and SAP97. We show here that heterologous coexpression of either sap97 or PSD-95 with various Shaker-type subunits results in the coclustering of these proteins with the K+ channels. Mutation of the C-terminal sequence (-ETDV) of the Shaker subunit Kv1.4 abolishes its binding to, and prevents its clustering with, SAP97 and PSD-95. Whereas PSD-95 induces plaque-like clusters of K+ channels at the cell surface; however, SAP97 coexpression results in the formation of large round intracellular aggregates into which both SAP97 and the K+ channel proteins are colocalized. The efficiency of surface clustering by PSD-95 varies with different Shaker subunits: striking Kv1.4 clustering occurs in > 60% of cotransfected cells, whereas Kv1.1 and Kv1.2 form convincing clusters with PSD-95 only in approximately 10% of cells.

    Neuropharmacology 1996;35;7;993-1000

  • DLG1: chromosome location of the closest human homologue of the Drosophila discs large tumor suppressor gene.

    Azim AC, Knoll JH, Marfatia SM, Peel DJ, Bryant PJ and Chishti AH

    Department of Biomedical Research, Tufts University School of Medicine, Boston, Massachusetts 02135, USA.

    The Drosophila discs large tumor suppressor protein, Dlg, is the prototype of a newly discovered family of proteins termed MAGUKs (membrane-associated guanylate kinase homologues). MAGUKs are localized at the membrane-cytoskeleton interface, usually at cell-cell junctions, where they appear to have both structural and signaling roles. They contain several distinct domains, including 5a8 a modified guanylate kinase domain, an SH3 motif, and one or three copies of the DHR (GLGF/PDZ) domain. Recessive lethal mutations in the discs large tumor suppressor gene interfere with the formation of septate junctions (thought to be the arthropod equivalent of tight junctions) between epithelial cells, and they cause neoplastic overgrowth of imaginal discs, suggesting a role for cell junctions in proliferation control. A homologue of the Dlg protein, named Hdlg, has been isolated from human B lymphocytes. It shows 65-79% identity to Dlg in the different domains, and it binds to the cytoskeletal protein 4.1. Here, we report that the gene for lymphocyte Hdlg, named DLG1, is located at chromosome band 3q29. This finding identifies a novel site for a candidate tumor suppressor on chromosome 3.

    Funded by: NHLBI NIH HHS: HL37462; NICHD NIH HHS: HD18568

    Genomics 1995;30;3;613-6

  • Clustering of Shaker-type K+ channels by interaction with a family of membrane-associated guanylate kinases.

    Kim E, Niethammer M, Rothschild A, Jan YN and Sheng M

    Howard Hughes Medical Institute, Massachusetts General Hospital, Department of Neurobiology, Harvard Medical School, Boston 02114, USA.

    ANCHORING of ion channels at specific subcellular sites is critical for neuronal signalling, but the mechanisms underlying channel localization and clustering are largely unknown (reviewed in ref. 1). Voltage-gated K+ channels are concentrated in various neuronal domains, including presynaptic terminals, nodes of Ranvier and dendrites, where they regulate local membrane excitability. Here we present functional and biochemical evidence that cell-surface clustering of Shaker-subfamily K+ channels is mediated by the PSD-95 family of membrane-associated putative guanylate kinases, as a result of direct binding of the carboxy-terminal cytoplasmic tails to the K+ channel subunits to two PDZ (also known as GLGF or DHR) domains in the PSD-95 protein. The ability of PDZ domains to function as independent modules for protein-protein interaction, and their presence in other junction-associated molecules (such as ZO-1 (ref. 3) and syntrophin), suggest that PDZ-domain-containing polypeptides may be widely involved in the organization of proteins at sites of membrane specialization.

    Nature 1995;378;6552;85-8

  • Cloning and characterization of hdlg: the human homologue of the Drosophila discs large tumor suppressor binds to protein 4.1.

    Lue RA, Marfatia SM, Branton D and Chishti AH

    Department of Molecular and Cellular Biology, Harvard University, Cambridge, MA 02138.

    The Drosophila discs large tumor suppressor protein, dlg, has been shown to regulate the growth of imaginal discs during embryogenesis [Woods, D. F. & Bryant, P. J. (1991) Cell 66, 451-464]. We cloned and sequenced the complete cDNA for a human B-lymphocyte 100-kDa protein that shares 60% amino acid identity with dlg. This human homologue of Drosophila discs large (hdlg) contains a C-terminal domain homologous to the known guanylate kinases, a src homology 3 region motif, and three dlg homology repeats. Two nonhomologous domains that can contain in-frame insertions result in at least four alternatively spliced isoforms of hdlg. Several hdlg RNA transcripts are widely distributed in human and murine tissues, and the protein is localized to regions of cell-cell contact. Protein 4.1, the defining member of a family that includes talin and merlin/schwannomin, has the same cellular localization as hdlg, and two sites within hdlg associate in vitro with the 30-kDa N-terminal domain of protein 4.1.

    Funded by: NHLBI NIH HHS: HL17411, HL37462, HL51445

    Proceedings of the National Academy of Sciences of the United States of America 1994;91;21;9818-22

  • Ankle joint instability in poliomyelitis.

    Mukherjee A, Varma SK and Natarajan K

    Indian journal of pediatrics 1972;39;289;37-8

Gene lists (9)

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
L00000015 G2C Homo sapiens Human NRC Human orthologues of mouse NRC adapted from Collins et al (2006) 186
L00000016 G2C Homo sapiens Human PSP Human orthologues of mouse PSP adapted from Collins et al (2006) 1121
L00000032 G2C Homo sapiens Pocklington H1 Human orthologues of cluster 1 (mouse) from Pocklington et al (2006) 21
L00000049 G2C Homo sapiens TAP-PSD-95-CORE TAP-PSD-95 pull-down core list (ortho) 120
L00000059 G2C Homo sapiens BAYES-COLLINS-HUMAN-PSD-CONSENSUS Human cortex PSD consensus 748
L00000061 G2C Homo sapiens BAYES-COLLINS-MOUSE-PSD-CONSENSUS Mouse cortex PSD consensus (ortho) 984
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).

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