G2Cdb::Gene report

Gene id
Gene symbol
Homo sapiens
adaptor-related protein complex 2, mu 1 subunit
G00000670 (Mus musculus)

Databases (7)

ENSG00000161203 (Ensembl human gene)
1173 (Entrez Gene)
1088 (G2Cdb plasticity & disease)
AP2M1 (GeneCards)
601024 (OMIM)
Marker Symbol
Protein Sequence
Q96CW1 (UniProt)

Synonyms (2)

  • AP50
  • mu2

Literature (68)

Pubmed - other

  • Molecular basis for association of PIPKI gamma-p90 with clathrin adaptor AP-2.

    Kahlfeldt N, Vahedi-Faridi A, Koo SJ, Schäfer JG, Krainer G, Keller S, Saenger W, Krauss M and Haucke V

    Institute of Chemistry and Biochemistry, Department of Membrane Biochemistry, Freie Universität Berlin, 14195 Berlin, Germany.

    Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P(2)) is an essential determinant in clathrin-mediated endocytosis (CME). In mammals three type I phosphatidylinositol-4-phosphate 5-kinase (PIPK) enzymes are expressed, with the I gamma-p90 isoform being highly expressed in the brain where it regulates synaptic vesicle (SV) exo-/endocytosis at nerve terminals. How precisely PI(4,5)P(2) metabolism is controlled spatially and temporally is still uncertain, but recent data indicate that direct interactions between type I PIPK and components of the endocytic machinery, in particular the AP-2 adaptor complex, are involved. Here we demonstrated that PIPKI gamma-p90 associates with both the mu and beta2 subunits of AP-2 via multiple sites. Crystallographic data show that a peptide derived from the splice insert of the human PIPKI gamma-p90 tail binds to a cognate recognition site on the sandwich subdomain of the beta2 appendage. Partly overlapping aromatic and hydrophobic residues within the same peptide also can engage the C-terminal sorting signal binding domain of AP-2mu, thereby potentially competing with the sorting of conventional YXXØ motif-containing cargo. Biochemical and structure-based mutagenesis analysis revealed that association of the tail domain of PIPKI gamma-p90 with AP-2 involves both of these sites. Accordingly the ability of overexpressed PIPKI gamma tail to impair endocytosis of SVs in primary neurons largely depends on its association with AP-2 beta and AP-2mu. Our data also suggest that interactions between AP-2 and the tail domain of PIPKI gamma-p90 may serve to regulate complex formation and enzymatic activity. We postulate a model according to which multiple interactions between PIPKI gamma-p90 and AP-2 lead to spatiotemporally controlled PI(4,5)P(2) synthesis during clathrin-mediated SV endocytosis.

    The Journal of biological chemistry 2010;285;4;2734-49

  • Association of genetic variants with chronic kidney disease in individuals with different lipid profiles.

    Yoshida T, Kato K, Yokoi K, Oguri M, Watanabe S, Metoki N, Yoshida H, Satoh K, Aoyagi Y, Nishigaki Y, Nozawa Y and Yamada Y

    Department of Cardiovascular Medicine, Inabe General Hospital, Inabe, Japan.

    The purpose of the present study was to identify genetic variants that confer susceptibility to chronic kidney disease (CKD) in individuals with low or high serum concentrations of triglycerides (TG), high-density lipoprotein (HDL)-cholesterol, or low-density lipoprotein (LDL)-cholesterol, thereby contributing to the personalized prevention of CKD in such individuals. The study population comprised 5944 Japanese individuals, including 1706 subjects with CKD [estimated glomerular filtration rate (eGFR)<60 ml/min/1.73 m2] and 4238 controls (eGFR>or=60 ml/min/1.73 m2). The genotypes for 296 polymorphisms of 202 candidate genes were determined. The Chi-square test, multivariable logistic regression analysis with adjustment for covariates, and a stepwise forward selection procedure revealed that seven different polymorphisms were significantly (P<0.005) associated with the prevalence of CKD in individuals with low or high serum concentrations of TG or HDL- or LDL-cholesterol: the Aright curved arrow G (Glu23Lys) polymorphism of KCNJ11 and the 125592Cright curved arrow A (Thr431Asn) polymorphism of ROCK2 in individuals with low serum TG; the 734Cright curved arrow T (Thr254Ile) polymorphism of ACAT2 and the Cright curved arrow G (Gln27Glu) polymorphism of ADRB2 in individuals with high serum TG; the -1607/1Gright curved arrow 2G polymorphism of MMP1 in individuals with low serum HDL-cholesterol; the Gright curved arrow A (Val158Met) polymorphism of COMT in individuals with low serum LDL-cholesterol; the 584Gright curved arrow A (Gln192Arg) polymorphism of PON1 in individuals with high serum LDL-cholesterol. No polymorphism was associated with CKD in individuals with high serum HDL-cholesterol. These results suggest that polymorphisms associated with CKD may differ among individuals with different lipid profiles. Stratification of subjects according to lipid profiles may thus be important for personalized prevention of CKD based on genetic information.

    International journal of molecular medicine 2009;24;2;233-46

  • The dyslexia-associated protein KIAA0319 interacts with adaptor protein 2 and follows the classical clathrin-mediated endocytosis pathway.

    Levecque C, Velayos-Baeza A, Holloway ZG and Monaco AP

    Wellcome Trust Centre for Human Genetics, Univ. of Oxford, Roosevelt D., Oxford OX3 7BN, UK.

    Recently, genetic studies have implicated KIAA0319 in developmental dyslexia, the most common of the childhood learning disorders. The first functional data indicated that the KIAA0319 protein is expressed on the plasma membrane and may be involved in neuronal migration. Further analysis of the subcellular distribution of the overexpressed protein in mammalian cells indicates that KIAA0319 can colocalize with the early endosomal marker early endosome antigen 1 (EEA1) in large intracellular vesicles, suggesting that it is endocytosed. Antibody internalization assays with full-length KIAA0319 and deletion constructs confirmed that KIAA0319 is internalized and showed the importance of the cytoplasmic juxtamembranal region in this process. The present study has identified the medium subunit (mu2) of adaptor protein 2 (AP-2) as a binding partner of KIAA0319 in a yeast two-hybrid screen. Using Rab5 mutants or depletion of the mu-subunit of AP-2 or clathrin heavy chain by RNA interference, we demonstrate that KIAA0319 follows a clathrin-mediated endocytic pathway. We also identify tyrosine-995 of KIAA0319 as a critical amino acid required for the interaction with AP-2 and subsequent internalization. These results suggest the surface expression of KIAA0319 is regulated by endocytosis, supporting the idea that the internalization and recycling of the protein may be involved in fine tuning its role in neuronal migration.

    Funded by: Wellcome Trust

    American journal of physiology. Cell physiology 2009;297;1;C160-8

  • Different internalization properties of the alpha1a- and alpha1b-adrenergic receptor subtypes: the potential role of receptor interaction with beta-arrestins and AP50.

    Stanasila L, Abuin L, Dey J and Cotecchia S

    Département de Pharmacologie et de Toxicologie, Rue du Bugnon 27, CH-1005 Lausanne, Switzerland.

    The internalization properties of the alpha1a- and alpha1b-adrenergic receptors (ARs) subtypes transiently expressed in human embryonic kidney (HEK) 293 cells were compared using biotinylation experiments and confocal microscopy. Whereas the alpha1b-AR displayed robust agonist-induced endocytosis, the alpha1a-AR did not. Constitutive internalization of the alpha1a-AR was negligible, whereas the alpha1b-AR displayed significant constitutive internalization and recycling. We investigated the interaction of the alpha1-AR subtypes with beta-arrestins 1 and 2 as well as with the AP50 subunit of the clathrin adaptor complex AP2. The results from both coimmunoprecipitation experiments and beta-arrestin translocation assays indicated that the agonistinduced interaction of the alpha1a-AR with beta-arrestins was much weaker than that of the alpha1b-AR. In addition, the alpha1a-AR did not bind AP50. The alpha1b-AR mutant M8, lacking the main phosphorylation sites in the receptor C tail, was unable to undergo endocytosis and was profoundly impaired in binding beta-arrestins despite its binding to AP50. In contrast, the alpha1b-AR mutant DeltaR8, lacking AP50 binding, bound beta-arrestins efficiently, and displayed delayed endocytosis. RNA interference showed that beta-arrestin 2 plays a prominent role in alpha1b-AR endocytosis. The findings of this study demonstrate differences in internalization between the alpha1a- and alpha1b-AR and provide evidence that the lack of significant endocytosis of the alpha1a-AR is linked to its poor interaction with beta-arrestins as well as with AP50. We also provide evidence that the integrity of the phosphorylation sites in the C tail of the alpha1b-AR is important for receptor/beta-arrestin interaction and that this interaction is the main event triggering receptor internalization.

    Molecular pharmacology 2008;74;3;562-73

  • Basal ubiquitin-independent internalization of interferon alpha receptor is prevented by Tyk2-mediated masking of a linear endocytic motif.

    Kumar KG, Varghese B, Banerjee A, Baker DP, Constantinescu SN, Pellegrini S and Fuchs SY

    Department of Animal Biology and Mari Lowe Center for Comparative Oncology Research, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA.

    Linear endocytic motifs of signaling receptors as well as their ubiquitination determine the rate of ligand-induced endocytosis that mediates down-regulation of these receptors and restricts the magnitude and duration of their respective signal transduction pathways. We previously hypothesized that, in the absence of its cognate ligand, type I interferon (IFN), the IFNalpha receptor chain 1 (IFNAR1) receptor chain is protected from basal endocytosis by a hypothetical masking complex that prevents the Tyr-based endocytic motif within IFNAR1 from interacting with components of the adaptin protein complex 2 (AP2). Here we identify a member of the Janus kinase (Jak) family, Tyk2, as a component of such a masking complex. In the absence of ligand or of receptor chain ubiquitination, binding of Janus kinase Tyk2 within the proximity of the Tyr-based linear motif of IFNAR1 is required to prevent IFNAR1 internalization and to maintain its cell surface expression. Furthermore, interaction experiments revealed that Tyk2 physically shields this Tyr-based motif from the recognition by the AP50 subunit of AP2. These data delineate a long-sought ligand- and ubiquitin-independent mechanism by which Tyk2 contributes to both the regulation of total IFNAR1 levels as well as the regulation of the cell surface density of this receptor chain.

    Funded by: NCI NIH HHS: CA92900, R01 CA092900, R01 CA092900-08; NIAID NIH HHS: R21 AI073347, R21 AI073347-02

    The Journal of biological chemistry 2008;283;27;18566-72

  • A diacidic motif in human immunodeficiency virus type 1 Nef is a novel determinant of binding to AP-2.

    Lindwasser OW, Smith WJ, Chaudhuri R, Yang P, Hurley JH and Bonifacino JS

    Cell Biology and Metabolism Branch, National Institute of Child Health and Human Development, Building 18T, Room 101, National Institutes of Health, Bethesda, MD 20892, USA. juan@helix.nih.gov

    A key function of the Nef protein of immunodeficiency viruses is the downregulation of the T-cell and macrophage coreceptor, CD4, from the surfaces of infected cells. CD4 downregulation depends on a conserved (D/E)XXXL(L/I)-type dileucine motif in the C-terminal, flexible loop of Nef, which mediates binding to the clathrin adaptor complexes AP-1, AP-2, and AP-3. We now report the identification of a consensus (D/E)D motif within this loop as a second, conserved determinant of interaction of Nef with AP-2, though not with AP-1 and AP-3. Mutations in this diacidic motif abrogate both AP-2 binding and CD4 downregulation. We also show that a dileucine motif from tyrosinase, both in its native context and in the context of Nef, can bind to AP-2 independently of a diacidic motif. These results thus identify a novel type of AP-2 interaction determinant, support the notion that AP-2 is the key clathrin adaptor for the downregulation of CD4 by Nef, and reveal a previously unrecognized diversity among dileucine sorting signals.

    Funded by: Intramural NIH HHS; NICHD NIH HHS: K22 HD54602

    Journal of virology 2008;82;3;1166-74

  • Ubiquitination of beta-arrestin links seven-transmembrane receptor endocytosis and ERK activation.

    Shenoy SK, Barak LS, Xiao K, Ahn S, Berthouze M, Shukla AK, Luttrell LM and Lefkowitz RJ

    Department of Medicine, Duke University Medical Center, Durham, North Carolina 27710, USA. sudha@receptor-biol.duke.edu

    Beta-arrestin2 and its ubiquitination play crucial roles in both internalization and signaling of seven-transmembrane receptors (7TMRs). To understand the connection between ubiquitination and the endocytic and signaling functions of beta-arrestin, we generated a beta-arrestin2 mutant that is defective in ubiquitination (beta-arrestin2(0K)), by mutating all of the ubiquitin acceptor lysines to arginines and compared its properties with the wild type and a stably ubiquitinated beta-arrestin2-ubiquitin (Ub) chimera. In vitro translated beta-arrestin2 and beta-arrestin2(0K) displayed equivalent binding to recombinant beta(2)-adrenergic receptor (beta(2)AR) reconstituted in vesicles, whereas beta-arrestin2-Ub bound approximately 4-fold more. In cellular coimmunoprecipitation assays, beta-arrestin2(0K) bound nonreceptor partners, such as AP-2 and c-Raf and scaffolded phosphorylated ERK robustly but displayed weak binding to clathrin. Moreover, beta-arrestin2(0K) was recruited only transiently to activated receptors at the membrane, did not enhance receptor internalization, and decreased the amount of phosphorylated ERK assimilated into isolated beta(2)AR complexes. Although the wild type beta-arrestin2 formed ERK signaling complexes with the beta(2)AR at the membrane, a stably ubiquitinated beta-arrestin2-Ub chimera not only stabilized the ERK signalosomes but also led to their endosomal targeting. Interestingly, in cellular fractionation assays, the ubiquitination state of beta-arrestin2 favors its distribution in membrane fractions, suggesting that ubiquitination increases the propensity of beta-arrestin for membrane association. Our findings suggest that although beta-arrestin ubiquitination is dispensable for beta-arrestin cytosol to membrane translocation and its "constitutive" interactions with some cytosolic proteins, it nevertheless is a prerequisite both for the formation of tight complexes with 7TMRs in vivo and for membrane compartment interactions that are crucial for downstream endocytic and signaling processes.

    Funded by: NHLBI NIH HHS: HL080525, HL16037, R01 HL016037, R01 HL080525, R01 HL080525-02; NIDDK NIH HHS: DK55524, R01 DK055524, R56 DK055524

    The Journal of biological chemistry 2007;282;40;29549-62

  • Frequent overexpression of the genes FXR1, CLAPM1 and EIF4G located on amplicon 3q26-27 in squamous cell carcinoma of the lung.

    Comtesse N, Keller A, Diesinger I, Bauer C, Kayser K, Huwer H, Lenhof HP and Meese E

    Department of Human Genetics, Medical School, Saarland University, Homburg, Germany.

    Previously, we reported gene amplification at chromosome 3q26-27 in more than one third of squamous cell carcinomas of the lung. Frequent amplification of eukaryotic translation initiation factor 4G on 3q27.1 indicated a possible role of this amplification in translation initiation. The analysis of 61 squamous cell lung carcinomas shows that the percentage of carcinomas with a 3q27.1 amplification increases in higher malignant tumors. Non-invasive (T1) and minimal-invasive (T2) tumor stages showed similar percentages of amplified and non-amplified tumors, whereas locally-invasive (T3) tumors revealed a statistically significant (p < 0.05) increased percentage of amplified tumors. Microarrays were used to analyze the expression pattern of genes mapping in the amplified domain and its flanking regions (3q25-28) as well as the expression of genes directly or indirectly associated with translation initiation in squamous cell carcinoma, large cell carcinoma, adenocarcinoma and small cell carcinoma. Three genes, namely FXR1, CLAPM1 and EIF4G, are most frequently overexpressed in the center of the amplified domain in squamous cell carcinomas. The eukaryotic translation initiation factors 4A1, 2B and 4B as well as the poly(A)-binding protein PABPC1 where found to be overexpressed in all lung cancer entities. We found, however, no overexpression of eIF4E. Our results contribute to the understanding of the frequent amplification processes in squamous cell carcinomas of the lung and to the understanding of the translation initiation that appears not to require eIF4E in lung carcinogenesis.

    International journal of cancer 2007;120;12;2538-44

  • Proteomics analysis of the interactome of N-myc downstream regulated gene 1 and its interactions with the androgen response program in prostate cancer cells.

    Tu LC, Yan X, Hood L and Lin B

    Institute for Systems Biology, Seattle, Washington 98103, USA.

    NDRG1 is known to play important roles in both androgen-induced cell differentiation and inhibition of prostate cancer metastasis. However, the proteins associated with NDRG1 function are not fully enumerated. Using coimmunoprecipitation and mass spectrometry analysis, we identified 58 proteins that interact with NDRG1 in prostate cancer cells. These proteins include nuclear proteins, adhesion molecules, endoplasmic reticulum (ER) chaperons, proteasome subunits, and signaling proteins. Integration of our data with protein-protein interaction data from the Human Proteome Reference Database allowed us to build a comprehensive interactome map of NDRG1. This interactome map consists of several modules such as a nuclear module and a cell membrane module; these modules explain the reported versatile functions of NDRG1. We also determined that serine 330 and threonine 366 of NDRG1 were phosphorylated and demonstrated that the phosphorylation of NDRG1 was prominently mediated by protein kinase A (PKA). Further, we showed that NDRG1 directly binds to beta-catenin and E-cadherin. However, the phosphorylation of NDRG1 did not interrupt the binding of NDRG1 to E-cadherin and beta-catenin. Finally, we showed that the inhibition of NDRG1 expression by RNA interference decreased the ER inducible chaperon GRP94 expression, directly proving that NDRG1 is involved in the ER stress response. Intriguingly, we observed that many members of the NDRG1 interactome are androgen-regulated and that the NDRG1 interactome links to the androgen response network through common interactions with beta-catenin and heat shock protein 90. Therefore we overlaid the transcriptomic expression changes in the NDRG1 interactome in response to androgen treatment and built a dual dynamic picture of the NDRG1 interactome in response to androgen. This interactome map provides the first road map for understanding the functions of NDRG1 in cells and its roles in human diseases, such as prostate cancer, which can progress from androgen-dependent curable stages to androgen-independent incurable stages.

    Funded by: NCI NIH HHS: 1U54CA119347, 5P01CA085859, 5P50CA097186; NIDA NIH HHS: 1U54DA021519; NIGMS NIH HHS: 1P50GM076547, P50 GM076547

    Molecular & cellular proteomics : MCP 2007;6;4;575-88

  • Molecular determinants for the interaction between AMPA receptors and the clathrin adaptor complex AP-2.

    Kastning K, Kukhtina V, Kittler JT, Chen G, Pechstein A, Enders S, Lee SH, Sheng M, Yan Z and Haucke V

    Department of Membrane Biochemistry, Institute of Chemistry and Biochemistry, Freie Universität Berlin, Takustrasse 6, 14195 Berlin, Germany.

    alpha-Amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA)-type glutamate receptors undergo constitutive and ligand-induced internalization that requires dynamin and the clathrin adaptor complex AP-2. We report here that an atypical basic motif within the cytoplasmic tails of AMPA-type glutamate receptors directly associates with mu2-adaptin by a mechanism similar to the recognition of the presynaptic vesicle protein synaptotagmin 1 by AP-2. A synaptotagmin 1-derived AP-2 binding peptide competes the interaction of the AMPA receptor subunit GluR2 with AP-2mu and increases the number of surface active glutamate receptors in living neurons. Moreover, fusion of the GluR2-derived tail peptide with a synaptotagmin 1 truncation mutant restores clathrin/AP-2-dependent internalization of the chimeric reporter protein. These data suggest that common mechanisms regulate AP-2-dependent internalization of pre- and postsynaptic membrane proteins.

    Funded by: Medical Research Council: G120/972

    Proceedings of the National Academy of Sciences of the United States of America 2007;104;8;2991-6

  • A conserved dileucine motif mediates clathrin and AP-2-dependent endocytosis of the HIV-1 envelope protein.

    Byland R, Vance PJ, Hoxie JA and Marsh M

    Cell Biology Unit, MRC-Laboratory for Molecular Cell Biology and Department of Biochemistry and Molecular Biology, University College London, London WC1E 6BT, United Kingdom.

    During the assembly of enveloped viruses viral and cellular components essential for infectious particles must colocalize at specific membrane locations. For the human and simian immunodeficiency viruses (HIV and SIV), sorting of the viral envelope proteins (Env) to assembly sites is directed by trafficking signals located in the cytoplasmic domain of the transmembrane protein gp41 (TM). A membrane proximal conserved GYxxØ motif mediates endocytosis through interaction with the clathrin adaptor AP-2. However, experiments with SIV(mac239) Env indicate the presence of additional signals. Here we show that a conserved C-terminal dileucine in HIV(HxB2) also mediates endocytosis. Biochemical and morphological assays demonstrate that the C-terminal dileucine motif mediates internalization as efficiently as the GYxxØ motif and that both must be removed to prevent Env internalization. RNAi experiments show that depletion of the clathrin adaptor AP-2 leads to increased plasma membrane expression of HIV Env and that this adaptor is required for efficient internalization mediated by both signals. The redundancy of conserved endocytosis signals and the role of the SIV(mac239) Env GYxxØ motif in SIV pathogenesis, suggest that these motifs have functions in addition to endocytosis, possibly related to Env delivery to the site of viral assembly and/or incorporation into budding virions.

    Funded by: Medical Research Council: MC_U122663296, MC_U122665002, U.1226.00.003.00001.01(65002); NIAID NIH HHS: AI-49784, R01 AI049784

    Molecular biology of the cell 2007;18;2;414-25

  • Internalization of large double-membrane intercellular vesicles by a clathrin-dependent endocytic process.

    Piehl M, Lehmann C, Gumpert A, Denizot JP, Segretain D and Falk MM

    Department of Biological Sciences, Lehigh University, Bethlehem, PA 18015, USA.

    Beyond its well-documented role in vesicle endocytosis, clathrin has also been implicated in the internalization of large particles such as viruses, pathogenic bacteria, and even latex beads. We have discovered an additional clathrin-dependent endocytic process that results in the internalization of large, double-membrane vesicles at lateral membranes of cells that are coupled by gap junctions (GJs). GJ channels bridge apposing cell membranes to mediate the direct transfer of electrical currents and signaling molecules from cell to cell. Here, we report that entire GJ plaques, clusters of GJ channels, can be internalized to form large, double-membrane vesicles previously termed annular gap junctions (AGJs). These internalized AGJ vesicles subdivide into smaller vesicles that are degraded by endo/lysosomal pathways. Mechanistic analyses revealed that clathrin-dependent endocytosis machinery-components, including clathrin itself, the alternative clathrin-adaptor Dab2, dynamin, myosin-VI, and actin are involved in the internalization, inward movement, and degradation of these large, intercellular double-membrane vesicles. These findings contribute to the understanding of clathrin's numerous emerging functions.

    Funded by: NIGMS NIH HHS: GM-55725, R01 GM055725

    Molecular biology of the cell 2007;18;2;337-47

  • Kir6.2 mutations causing neonatal diabetes prevent endocytosis of ATP-sensitive potassium channels.

    Mankouri J, Taneja TK, Smith AJ, Ponnambalam S and Sivaprasadarao A

    Institute of Membrane and Systems Biology, Faculty of Biological Sciences, Leeds University, Leeds, UK.

    ATP-sensitive potassium (KATP) channels couple the metabolic status of a cell to its membrane potential-a property that endows pancreatic beta-cells with the ability to regulate insulin secretion in accordance with changes in blood glucose. The channel comprises four subunits each of Kir6.2 and the sulphonylurea receptor (SUR1). Here, we report that KATP channels undergo rapid internalisation from the plasma membrane by clathrin-mediated endocytosis. We present several lines of evidence to demonstrate that endocytosis is mediated by a tyrosine based signal (330YSKF333) located in the carboxy-terminus of Kir6.2 and that SUR1 has no direct role. We show that genetic mutations, Y330C and F333I, which cause permanent neonatal diabetes mellitus, disrupt this motif and abrogate endocytosis of reconstituted mutant channels. The resultant increase in the surface density of KATP channels would predispose beta-cells to hyperpolarise and may account for reduced insulin secretion in these patients. The data imply that endocytosis of KATP channels plays a crucial role in the (patho)-physiology of insulin secretion.

    Funded by: Biotechnology and Biological Sciences Research Council: BB/D524875/1; Medical Research Council: G0200533

    The EMBO journal 2006;25;17;4142-51

  • Stimulation of phosphatidylinositol kinase type I-mediated phosphatidylinositol (4,5)-bisphosphate synthesis by AP-2mu-cargo complexes.

    Krauss M, Kukhtina V, Pechstein A and Haucke V

    Institute of Chemistry and Biochemistry, Department of Membrane Biochemistry, Freie Universität Berlin, Takustrasse 6, 14195 Berlin, Germany.

    Phosphatidylinositol (4,5)-bisphosphate [PI(4,5)P(2)] is an important factor for a variety of cellular functions ranging from cell signaling to actin cytoskeletal dynamics and endocytic membrane traffic. Here, we have identified the clathrin adaptor complex AP-2 as a regulator of phosphatidylinositol 4-phosphate 5-kinase (PIPK)-mediated PI(4,5)P(2) synthesis. AP-2 directly interacts with the kinase core domain of type I PIPK isozymes via its mu2-subunit in vitro and in native protein extracts. Endocytic cargo protein binding to mu2 leads to a potent stimulation of PIPK activity. These data thus identify a positive feedback loop consisting of endocytic cargo proteins, AP-2mu, and PIPK type I which may provide a specific pool of PI(4,5)P(2) dedicated to clathrin/AP-2-dependent receptor internalization.

    Proceedings of the National Academy of Sciences of the United States of America 2006;103;32;11934-9

  • Phosphorylation of adaptor protein-2 mu2 is essential for Na+,K+-ATPase endocytosis in response to either G protein-coupled receptor or reactive oxygen species.

    Chen Z, Krmar RT, Dada L, Efendiev R, Leibiger IB, Pedemonte CH, Katz AI, Sznajder JI and Bertorello AM

    Department of Medicine, Membrane Signaling Networks, Karolinska Institutet, Karolinska Hospital, Stockholm, Sweden.

    Activation of G protein-coupled receptor by dopamine and hypoxia-generated reactive oxygen species promote Na+,K+-ATPase endocytosis. This effect is clathrin dependent and involves the activation of protein kinase C (PKC)-zeta and phosphorylation of the Na+,K+-ATPase alpha-subunit. Because the incorporation of cargo into clathrin vesicles requires association with adaptor proteins, we studied whether phosphorylation of adaptor protein (AP)-2 plays a role in its binding to the Na+,K+-ATPase alpha-subunit and thereby in its endocytosis. Dopamine induces a time-dependent phosphorylation of the AP-2 mu2 subunit. Using specific inhibitors and dominant-negative mutants, we establish that this effect was mediated by activation of the adaptor associated kinase 1/PKC-zeta isoform. Expression of the AP-2 mu2 bearing a mutation in its phosphorylation site (T156A) prevented Na+,K+-ATPase endocytosis and changes in activity induced by dopamine. Similarly, in lung alveolar epithelial cells, hypoxia-induced endocytosis of Na+,K+-ATPase requires the binding of AP-2 to the tyrosine-based motif (Tyr-537) located in the Na+,K+-ATPase alpha-subunit, and this effect requires phosphorylation of the AP-2 mu2 subunit. We conclude that phosphorylation of AP-2 mu2 subunit is essential for Na+,K+-ATPase endocytosis in response to a variety of signals, such as dopamine or reactive oxygen species.

    Funded by: NHLBI NIH HHS: HL-48129, P01HL-071643; NIDDK NIH HHS: DK62195

    American journal of respiratory cell and molecular biology 2006;35;1;127-32

  • Endocytosis of the glucose transporter GLUT8 is mediated by interaction of a dileucine motif with the beta2-adaptin subunit of the AP-2 adaptor complex.

    Schmidt U, Briese S, Leicht K, Schürmann A, Joost HG and Al-Hasani H

    German Institute of Human Nutrition, Potsdam, Nuthetal, Germany.

    The glucose transporter GLUT8 cycles between intracellular vesicles and the plasma membrane. Like the insulin-responsive glucose transporter GLUT4, GLUT8 is primarily located in intracellular compartments under basal conditions. Whereas translocation of GLUT4 to the plasma membrane is stimulated by insulin, the distribution of GLUT8 is not affected by insulin treatment in adipose cells. However, blocking endocytosis by co-expression of a dominant-negative dynamin GTPase (K44A) or mutation of the N-terminal dileucine (LL(12/13)) motif in GLUT8 leads to accumulation of the glucose transporter at the cell surface in a variety of different cell types. Yeast two-hybrid analyses and GST pulldown assays reveal that the LL signal constitutes a binding site for the beta2-adaptin subunit of the heterotetrameric AP-2 adaptor complex, implicating this motif in targeting of GLUT8 to clathrin-coated vesicles. Moreover, yeast two-hybrid assays provide evidence that the binding site for the LL motif maps to the appendage domain of beta2-adaptin. To analyze the biological significance of the LL/beta2 interaction, we utilized RNA interference to specifically knockdown AP-2. Our results show that RNAi-mediated targeting of the mu2 subunit leads to cellular depletion of AP-2, but not AP-1 adaptor complexes in HeLa cells. As a consequence, GLUT8 accumulates at the plasma membrane at comparable levels to those observed in K44A-transfected cells. Conversely, the intracellular localization of mutant GLUT8-LL/AA is restored by replacing the LL motif in GLUT8 with the transferrin receptor-derived mu2-adaptin binding motif YTRF, indicating that for endocytosis both AP-2 binding motifs can substitute for each other. Thus, our data demonstrate that recruitment of GLUT8 to the endocytic machinery occurs via direct interaction of the dileucine motif with beta2-adaptin, and that endocytosis might be the main site at which GLUT8 is likely to be regulated.

    Journal of cell science 2006;119;Pt 11;2321-31

  • Recruitment of the adaptor protein 2 complex by the human immunodeficiency virus type 2 envelope protein is necessary for high levels of virus release.

    Noble B, Abada P, Nunez-Iglesias J and Cannon PM

    Department of Biochemistry and Molecular Biology, University of Southern California Keck School of Medicine, Los Angeles, USA.

    The envelope (Env) protein of human immunodeficiency virus type 2 (HIV-2) and the HIV-1 Vpu protein stimulate the release of retroviral particles from human cells that restrict virus production, an activity that we call the enhancement of virus release (EVR). We have previously shown that two separate domains in the HIV-2 envelope protein are required for this activity: a glycine-tyrosine-x-x-hydrophobic (GYxxtheta) motif in the cytoplasmic tail and an unmapped region in the ectodomain of the protein. We here report that the cellular partner of the GYxxtheta motif is the adaptor protein complex AP-2. The mutation of this motif or the depletion of AP-2 by RNA interference abrogated EVR activity and changed the cellular distribution of the Env from a predominantly punctate pattern to a more diffuse distribution. Since the L domain of equine infectious anemia virus (EIAV) contains a Yxxtheta motif that interacts with AP-2, we used both wild-type and L domain-defective particles of HIV-1 and EIAV to examine whether the HIV-2 Env EVR function was analogous to L domain activity. We observed that the production of all particles was stimulated by HIV-2 Env or Vpu, suggesting that the L domain and EVR activities play independent roles in the release of retroviruses. Interestingly, we found that the cytoplasmic tail of the murine leukemia virus (MLV) Env could functionally substitute for the HIV-2 Env tail, but it did so in a manner that did not require a Yxxtheta motif or AP-2. The cellular distribution of the chimeric HIV-2/MLV Env was significantly less punctate than the wild-type Env, although confocal analysis revealed an overlap in the steady-state locations of the two proteins. Taken together, these data suggest that the essential GYxxtheta motif in the HIV-2 Env tail recruits AP-2 in order to direct Env to a cellular pathway or location that is necessary for its ability to enhance virus release but that an alternate mechanism provided by the MLV Env tail can functionally substitute.

    Funded by: NCI NIH HHS: CA-59318, P01 CA059318

    Journal of virology 2006;80;6;2924-32

  • Interaction of HIV-1 Gag with the clathrin-associated adaptor AP-2.

    Batonick M, Favre M, Boge M, Spearman P, Höning S and Thali M

    Department of Microbiology and Molecular Genetics, University of Vermont, Burlington, VT 05405, USA.

    The envelope glycoprotein (Env) of HIV-1 interacts with the clathrin-associated adaptor complex AP-2 during the late phase of the viral replication cycle. Upon its synthesis, Env, therefore, is retrieved from the cellular surface unless internalization is inhibited by viral Gag. Here we demonstrate that not only Env, but also HIV-1 Gag, specifically binds to AP-2. Gag-AP-2 association was found to depend on tyrosine residue 132 and valine residue 135 at the matrix-capsid junction in the Gag polyprotein. Results of a morphological analysis of viral egress from cells expressing dominant-negative AP-2 suggest an involvement of AP-2 in confining HIV-1 exit to distinct microdomains. Further, particle release from AP-2-mutant cells was enhanced compared to release from wild-type cells but the infectivity of virus released from these cells was moderately reduced. Together these data attribute a role to the AP-2 complex in the regulation of HIV-1 assembly/release.

    Funded by: NIAID NIH HHS: R01 AI047727, R01AI047727-04

    Virology 2005;342;2;190-200

  • A requirement for membrane cholesterol in the beta-arrestin- and clathrin-dependent endocytosis of LPA1 lysophosphatidic acid receptors.

    Urs NM, Jones KT, Salo PD, Severin JE, Trejo J and Radhakrishna H

    School of Biology, Georgia Institute of Technology, 315 Ferst Drive, Atlanta, GA 30332, USA.

    Lysophosphatidic acid (LPA) stimulates heterotrimeric G protein signaling by activating three closely related receptors, termed LPA(1), LPA(2) and LPA(3). Here we show that in addition to promoting LPA(1) signaling, membrane cholesterol is essential for the association of LPA(1) with beta-arrestin, which leads to signal attenuation and clathrin-dependent endocytosis of LPA(1). Reduction of clathrin heavy chain expression, using small interfering RNAs, inhibited LPA(1) endocytosis. LPA(1) endocytosis was also inhibited in beta-arrestin 1 and 2-null mouse embryo fibroblasts (beta-arrestin 1/2 KO MEFs), but was restored upon re-expression of wild-type beta-arrestin 2. beta-arrestin attenuates LPA signaling as LPA(1)-dependent phosphoinositide hydrolysis was significantly elevated in beta-arrestin 1/2 KO MEFs and was reduced to wild-type levels upon re-expression of wild-type beta-arrestin. Interestingly, extraction of membrane cholesterol with methyl-beta-cyclodextrin inhibited LPA(1) signaling, beta-arrestin membrane recruitment and LPA(1) endocytosis. Cholesterol repletion restored all of these functions. However, neither the stimulation of phosphoinositide hydrolysis by the M(1) acetylcholine receptor nor its endocytosis was affected by cholesterol extraction. LPA treatment increased the detergent resistance of LPA(1) and this was inhibited by cholesterol extraction, suggesting that LPA(1) localizes to detergent-resistant membranes upon ligand stimulation. These data indicate that although LPA(1) is internalized by clathrin- and beta-arrestin dependent endocytosis, membrane cholesterol is critical for LPA(1) signaling, membrane recruitment of beta-arrestins and LPA(1) endocytosis.

    Funded by: NHLBI NIH HHS: HL 67134

    Journal of cell science 2005;118;Pt 22;5291-304

  • Towards a proteome-scale map of the human protein-protein interaction network.

    Rual JF, Venkatesan K, Hao T, Hirozane-Kishikawa T, Dricot A, Li N, Berriz GF, Gibbons FD, Dreze M, Ayivi-Guedehoussou N, Klitgord N, Simon C, Boxem M, Milstein S, Rosenberg J, Goldberg DS, Zhang LV, Wong SL, Franklin G, Li S, Albala JS, Lim J, Fraughton C, Llamosas E, Cevik S, Bex C, Lamesch P, Sikorski RS, Vandenhaute J, Zoghbi HY, Smolyar A, Bosak S, Sequerra R, Doucette-Stamm L, Cusick ME, Hill DE, Roth FP and Vidal M

    Center for Cancer Systems Biology and Department of Cancer Biology, Dana-Farber Cancer Institute, Harvard Medical School, 44 Binney Street, Boston, Massachusetts 02115, USA.

    Systematic mapping of protein-protein interactions, or 'interactome' mapping, was initiated in model organisms, starting with defined biological processes and then expanding to the scale of the proteome. Although far from complete, such maps have revealed global topological and dynamic features of interactome networks that relate to known biological properties, suggesting that a human interactome map will provide insight into development and disease mechanisms at a systems level. Here we describe an initial version of a proteome-scale map of human binary protein-protein interactions. Using a stringent, high-throughput yeast two-hybrid system, we tested pairwise interactions among the products of approximately 8,100 currently available Gateway-cloned open reading frames and detected approximately 2,800 interactions. This data set, called CCSB-HI1, has a verification rate of approximately 78% as revealed by an independent co-affinity purification assay, and correlates significantly with other biological attributes. The CCSB-HI1 data set increases by approximately 70% the set of available binary interactions within the tested space and reveals more than 300 new connections to over 100 disease-associated proteins. This work represents an important step towards a systematic and comprehensive human interactome project.

    Funded by: NCI NIH HHS: R33 CA132073; NHGRI NIH HHS: P50 HG004233, R01 HG001715, RC4 HG006066, U01 HG001715; NHLBI NIH HHS: U01 HL098166

    Nature 2005;437;7062;1173-8

  • HIV Nef-mediated CD4 down-regulation is adaptor protein complex 2 dependent.

    Jin YJ, Cai CY, Zhang X, Zhang HT, Hirst JA and Burakoff SJ

    Skirball Institute of Biomedical Research, New York University School of Medicine, New York, NY 10016, USA. jiny@saturn.med.nyu.edu

    Nef is a crucial viral protein for HIV to replicate at high titers and in the development of AIDS. One Nef function is down-regulating CD4 from the cell surface, which correlates with Nef-enhanced viral pathogenicity. Nef down-regulates CD4 by linking CD4 to clathrin-coated pits. However, the mechanistic connection between the C-terminal dileucine motif of Nef and the component(s) of the clathrin-coated pits has not been pinpointed. In this report we used two AP-2 complex-specific inhibitors: a dominant negative mutant of Eps15 (Eps15DIII) that binds to the alpha subunit of AP-2 complex and a small interference RNA that is specific for the mu2 subunit of AP-2 complex. We show that both HIV Nef- and SIV Nef-mediated CD4 down-regulations were profoundly blocked by the synergistic effect of Eps15DIII and RNA interference of AP-2 expression. The results demonstrate that HIV/SIV Nef-mediated CD4 down-regulation is AP-2 dependent. We also show that the PMA-induced CD4 down-regulation was blocked by these two inhibitors. Therefore, PMA-induced CD4 down-regulation is also AP-2 dependent. The results demonstrate that, like the tyrosine sorting motif-dependent endocytosis (for which the transferrin receptor and the epidermal growth factor receptor are the two prototypes), dileucine sorting motif-dependent endocytosis of Nef and CD4 are also AP-2 dependent.

    Funded by: NIAID NIH HHS: 1R01AI17258

    Journal of immunology (Baltimore, Md. : 1950) 2005;175;5;3157-64

  • Human immunodeficiency virus Nef induces rapid internalization of the T-cell coreceptor CD8alphabeta.

    Stove V, Van de Walle I, Naessens E, Coene E, Stove C, Plum J and Verhasselt B

    Department of Clinical Chemistry, Microbiology and Immunology, Ghent University Hospita, Belgium.

    Human immunodeficiency virus (HIV) Nef is a membrane-associated protein decreasing surface expression of CD4, CD28, and major histocompatibility complex class I on infected cells. We report that Nef strongly down-modulates surface expression of the beta-chain of the CD8alphabeta receptor by accelerated endocytosis, while CD8 alpha-chain expression is less affected. By mutational analysis of the cytoplasmic tail of the CD8 beta-chain, an FMK amino acid motif was shown to be critical for Nef-induced endocytosis. Although independent of CD4, endocytosis of the CD8 beta-chain was abrogated by the same mutations in Nef that affect CD4 down-regulation, suggesting common molecular interactions. The ability to down-regulate the human CD8 beta-chain was conserved in HIV-1, HIV-2, and simian immunodeficiency virus SIVmac239 Nef and required an intact AP-2 complex. The Nef-mediated internalization of receptors, such as CD4, major histocompatibility complex class I, CD28, and CD8alphabeta, may contribute to the subversion of the host immune system and progression towards AIDS.

    Journal of virology 2005;79;17;11422-33

  • PICK1 interacts with ABP/GRIP to regulate AMPA receptor trafficking.

    Lu W and Ziff EB

    Program in Neuroscience and Physiology, New York University School of Medicine, New York, New York 10016, USA.

    PICK1 and ABP/GRIP bind to the AMPA receptor (AMPAR) GluR2 subunit C terminus. Transfer of the receptor from ABP/GRIP to PICK1, facilitated by GluR2 S880 phosphorylation, may initiate receptor trafficking. Here we report protein interactions that regulate these steps. The PICK1 BAR domain interacts intermolecularly with the ABP/GRIP linker II region and intramolecularly with the PICK1 PDZ domain. Binding of PKCalpha or GluR2 to the PICK1 PDZ domain disrupts the intramolecular interaction and facilitates the PICK1 BAR domain association with ABP/GRIP. Interference with the PICK1-ABP/GRIP interaction impairs S880 phosphorylation of GluR2 by PKC and decreases the constitutive surface expression of GluR2, the NMDA-induced endocytosis of GluR2, and recycling of internalized GluR2. We suggest that the PICK1 interaction with ABP/GRIP is a critical step in controlling GluR2 trafficking.

    Funded by: NIMH NIH HHS: MH067229

    Neuron 2005;47;3;407-21

  • Involvement of clathrin and AP-2 in the trafficking of MHC class II molecules to antigen-processing compartments.

    McCormick PJ, Martina JA and Bonifacino JS

    Cell Biology and Metabolism Branch, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892, USA.

    Major histocompatibility complex class II (MHC-II) molecules are composed of two polymorphic chains, alpha and beta, which assemble with an invariant chain, Ii, in the endoplasmic reticulum. The assembled MHC-II complexes are transported to the Golgi complex and then to late endosomes/lysosomes, where Ii is degraded and alphabeta dimers bind peptides derived from exogenous antigens. Targeting of MHC-II molecules to these compartments is mediated by two dileucine-based signals in the cytoplasmic domain of Ii. These signals bind in vitro to two adaptor protein (AP) complexes, AP-1 and AP-2, which are components of clathrin coats involved in vesicle formation and cargo sorting. The physiological roles of these proteins in MHC-II molecule trafficking, however, remain to be addressed. Here, we report the use of RNA interference to examine the involvement of clathrin and four AP complexes (AP-1, AP-2, AP-3, and AP-4) in MHC-II molecule trafficking in vivo. We found that depletion of clathrin or AP-2 caused >10-fold increases in Ii expression on the cell surface and a concomitant decrease in Ii localization to endosomal/lysosomal vesicles. In addition, depletion of clathrin or AP-2 delayed the degradation of Ii and reduced the surface expression of peptide-loaded alphabeta dimers. In contrast, depletion of AP-1, AP-3, or AP-4 had little or no effect. These findings demonstrate that clathrin and AP-2 participate in MHC-II molecule trafficking in vivo. Because AP-2 is only associated with the plasma membrane, these results also indicate that a significant pool of MHC-II molecules traffic to the endosomal-lysosomal system by means of the cell surface.

    Proceedings of the National Academy of Sciences of the United States of America 2005;102;22;7910-5

  • Phosphatidylinositol-(4,5)-bisphosphate regulates sorting signal recognition by the clathrin-associated adaptor complex AP2.

    Höning S, Ricotta D, Krauss M, Späte K, Spolaore B, Motley A, Robinson M, Robinson C, Haucke V and Owen DJ

    Institute for Biochemistry II, University of Göttingen, Heinrich-Düker-Weg 12, 37073 Göttingen, Germany. shoening@uni-koeln.de

    The alpha,beta2,mu2,sigma2 heterotetrameric AP2 complex is recruited exclusively to the phosphatidylinositol-4,5-bisphosphate (PtdIns4,5P(2))-rich plasma membrane where, amongst other roles, it selects motif-containing cargo proteins for incorporation into clathrin-coated vesicles. Unphosphorylated and mu2Thr156-monophosphorylated AP2 mutated in their alphaPtdIns4,5P(2), mu2PtdIns4,5P(2), and mu2Yxxvarphi binding sites were produced, and their interactions with membranes of different phospholipid and cargo composition were measured by surface plasmon resonance. We demonstrate that recognition of Yxxvarphi and acidic dileucine motifs is dependent on corecognition with PtdIns4,5P(2), explaining the selective recruitment of AP2 to the plasma membrane. The interaction of AP2 with PtdIns4,5P(2)/Yxxvarphi-containing membranes is two step: initial recruitment via the alphaPtdIns4,5P(2) site and then stabilization through the binding of mu2Yxxvarphi and mu2PtdIns4,5P(2) sites to their ligands. The second step is facilitated by a conformational change favored by mu2Thr156 phosphorylation. The binding of AP2 to acidic-dileucine motifs occurs at a different site from Yxxvarphi binding and is not enhanced by mu2Thr156 phosphorylation.

    Funded by: Wellcome Trust

    Molecular cell 2005;18;5;519-31

  • Plasticity and rigidity in adaptor protein-2-mediated internalization of the TCR:CD3 complex.

    Szymczak AL and Vignali DA

    Department of Immunology, St. Jude Children's Research Hospital, Memphis, TN 38105, USA.

    Many cell surface proteins are internalized via dileucine- or tyrosine-based motifs within their cytoplasmic domains by the heterotetrameric adaptor protein complex, AP-2. In this study we have examined how AP-2 mediates internalization of large cell surface receptors, such as the eight-chain TCR:CD3 complex. Although most receptors have a single signal that drives internalization, the TCR complex has two (D/E)xxxL(L/I) motifs and 20 YxxØ motifs. Using 293T cells, we show that AP-2 is completely dependent on both signals to mediate TCR internalization, because deletion of either completely blocks this process. Significant plasticity and redundancy were observed in the use of the YxxØ motifs, with a clear hierarchy in their use (CD3delta > CD3gamma >or= CD3zeta > CD3epsilon). Remarkably, a single, membrane-distal YxxØ motif in CD3delta could mediate approximately 75% of receptor internalization, whereas its removal only reduced internalization by approximately 20%. In contrast, significant rigidity was observed in use of the (D/E)xxxL(L/I) motif in CD3gamma. This was due to an absolute requirement for the position of this signal in the context of the TCR complex and for a highly conserved lysine residue, K128, which is not present in CD3delta. These contrasting requirements suggest a general principle by which AP-2 may mediate the internalization of large, multichain complexes.

    Funded by: NCI NIH HHS: CA21765; NIAID NIH HHS: AI39480, AI52199

    Journal of immunology (Baltimore, Md. : 1950) 2005;174;7;4153-60

  • CD4 down-regulation by HIV-1 and simian immunodeficiency virus (SIV) Nef proteins involves both internalization and intracellular retention mechanisms.

    Rose JJ, Janvier K, Chandrasekhar S, Sekaly RP, Bonifacino JS and Venkatesan S

    Laboratory of Molecular Microbiology, NIAID, National Institutes of Health, Bethesda, Maryland 20892, USA.

    Among the pleiotropic effects of Nef proteins of HIV and simian immunodeficiency virus (SIV), down-modulation of cell surface expression of CD4 is a prominent phenotype. It has been presumed that Nef proteins accelerate endocytosis of CD4 by linking the receptor to the AP-2 clathrin adaptor. However, the related AP-1 and AP-3 adaptors have also been shown to interact with Nef, hinting at role(s) for these complexes in the intracellular retention of CD4. By using genetic inhibitors of endocytosis and small interfering RNA-induced knockdown of AP-2, we show that accelerated CD4 endocytosis is not a dominant mechanism of HIV-1 (NL4-3 strain) Nef in epithelial cells, T lymphocyte cell lines, or peripheral blood lymphocytes. Furthermore, we show that both the CD4 recycling from the plasma membrane and the nascent CD4 in transit to the plasma membrane are susceptible to intracellular retention in HIV-1 Nef-expressing cells. In contrast, AP-2-mediated enhanced endocytosis constitutes the predominant mechanism for SIV (MAC-239 strain) Nef-induced down-regulation of human CD4 in human cells.

    The Journal of biological chemistry 2005;280;9;7413-26

  • L1-mediated branching is regulated by two ezrin-radixin-moesin (ERM)-binding sites, the RSLE region and a novel juxtamembrane ERM-binding region.

    Cheng L, Itoh K and Lemmon V

    Department of Neurosciences, Case Western Reserve University, Cleveland, Ohio 44106, USA.

    We investigated how the neural cell adhesion molecule L1 mediates neurite outgrowth through L1-L1 homophilic interactions. Wild-type L1 and L1 with mutations in the cytoplasmic domain (CD) were introduced into L1 knock-out neurons, and transfected neurons were grown on an L1 substrate. Neurite length and branching were compared between wild-type L1 and L1CD mutations. Surprisingly, the L1CD is not required for L1-mediated neurite outgrowth but plays a critical role in neurite branching, through both the juxtamembrane region and the RSLE region. We demonstrate that both regions serve as ezrin-moesin-radixin-binding sites. A truncation mutant that deletes 110 of 114 amino acids of the L1CD still supports neurite outgrowth on an L1 substrate, suggesting that a coreceptor binds to L1 in cis and mediates neurite outgrowth and that L1-ankyrin interactions are not essential for neurite initiation or outgrowth. These data are consistent with a model in which L1 can influence L1-mediated neurite outgrowth and branching through both the L1CD and a coreceptor.

    Funded by: NEI NIH HHS: EY-05285, EY-11373, P30 EY011373, R01 EY005285, R01 EY005285-23; NICHD NIH HHS: HD39884, R01 HD039884, R01 HD039884-06

    The Journal of neuroscience : the official journal of the Society for Neuroscience 2005;25;2;395-403

  • The status, quality, and expansion of the NIH full-length cDNA project: the Mammalian Gene Collection (MGC).

    Gerhard DS, Wagner L, Feingold EA, Shenmen CM, Grouse LH, Schuler G, Klein SL, Old S, Rasooly R, Good P, Guyer M, Peck AM, Derge JG, Lipman D, Collins FS, Jang W, Sherry S, Feolo M, Misquitta L, Lee E, Rotmistrovsky K, Greenhut SF, Schaefer CF, Buetow K, Bonner TI, Haussler D, Kent J, Kiekhaus M, Furey T, Brent M, Prange C, Schreiber K, Shapiro N, Bhat NK, Hopkins RF, Hsie F, Driscoll T, Soares MB, Casavant TL, Scheetz TE, Brown-stein MJ, Usdin TB, Toshiyuki S, Carninci P, Piao Y, Dudekula DB, Ko MS, Kawakami K, Suzuki Y, Sugano S, Gruber CE, Smith MR, Simmons B, Moore T, Waterman R, Johnson SL, Ruan Y, Wei CL, Mathavan S, Gunaratne PH, Wu J, Garcia AM, Hulyk SW, Fuh E, Yuan Y, Sneed A, Kowis C, Hodgson A, Muzny DM, McPherson J, Gibbs RA, Fahey J, Helton E, Ketteman M, Madan A, Rodrigues S, Sanchez A, Whiting M, Madari A, Young AC, Wetherby KD, Granite SJ, Kwong PN, Brinkley CP, Pearson RL, Bouffard GG, Blakesly RW, Green ED, Dickson MC, Rodriguez AC, Grimwood J, Schmutz J, Myers RM, Butterfield YS, Griffith M, Griffith OL, Krzywinski MI, Liao N, Morin R, Morrin R, Palmquist D, Petrescu AS, Skalska U, Smailus DE, Stott JM, Schnerch A, Schein JE, Jones SJ, Holt RA, Baross A, Marra MA, Clifton S, Makowski KA, Bosak S, Malek J and MGC Project Team

    The National Institutes of Health's Mammalian Gene Collection (MGC) project was designed to generate and sequence a publicly accessible cDNA resource containing a complete open reading frame (ORF) for every human and mouse gene. The project initially used a random strategy to select clones from a large number of cDNA libraries from diverse tissues. Candidate clones were chosen based on 5'-EST sequences, and then fully sequenced to high accuracy and analyzed by algorithms developed for this project. Currently, more than 11,000 human and 10,000 mouse genes are represented in MGC by at least one clone with a full ORF. The random selection approach is now reaching a saturation point, and a transition to protocols targeted at the missing transcripts is now required to complete the mouse and human collections. Comparison of the sequence of the MGC clones to reference genome sequences reveals that most cDNA clones are of very high sequence quality, although it is likely that some cDNAs may carry missense variants as a consequence of experimental artifact, such as PCR, cloning, or reverse transcriptase errors. Recently, a rat cDNA component was added to the project, and ongoing frog (Xenopus) and zebrafish (Danio) cDNA projects were expanded to take advantage of the high-throughput MGC pipeline.

    Funded by: PHS HHS: N01-C0-12400

    Genome research 2004;14;10B;2121-7

  • Subunit-specific regulation of NMDA receptor endocytosis.

    Lavezzari G, McCallum J, Dewey CM and Roche KW

    National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, Maryland 20892, USA.

    At excitatory synapses, both NMDA and AMPA receptors are localized to the postsynaptic density (PSD). However, unlike AMPA receptors, synaptic NMDA receptors are stable components of the PSD. Even so, surface-expressed NMDA receptors undergo endocytosis, which is more robust early in development and declines during synaptic development. We investigated the subunit-specific contributions to NMDA receptor endocytosis, specifically defining the endocytic motifs and endocytic pathways preferred by the NR2A and NR2B subunits. We find that NR2A and NR2B have distinct endocytic motifs encoded in their distal C termini and that these interact with clathrin adaptor complexes with differing affinities. We also find that NR2A and NR2B sort into different intracellular pathways after endocytosis, with NR2B preferentially trafficking through recycling endosomes. In mature cultures, we find that NR2B undergoes more robust endocytosis than NR2A, consistent with previous studies showing that NR2A is more highly expressed at stable synaptic sites. Our findings demonstrate fundamental differences between NR2A and NR2B that help clarify developmental changes in NMDA receptor trafficking and surface expression.

    The Journal of neuroscience : the official journal of the Society for Neuroscience 2004;24;28;6383-91

  • EHD2 interacts with the insulin-responsive glucose transporter (GLUT4) in rat adipocytes and may participate in insulin-induced GLUT4 recruitment.

    Park SY, Ha BG, Choi GH, Ryu J, Kim B, Jung CY and Lee W

    Department of Biochemistry, College of Medicine, Dongguk University, Kyungju 780-714, Korea.

    Insulin-induced GLUT4 recruitment to the plasma membrane involves GLUT4 trafficking through multiple subcellular compartments regulated by multiple proteins, many of which are yet to be identified. Here we describe a 65 kDa protein found in purified GLUT4 vesicles of rat adipocytes as a potential GLUT4 traffic regulatory protein. On the basis of MALDI-TOF MS, RT-PCR, gene cloning, protein sequencing, and immunoreactivity data, we identified this protein as EHD2, a member of the EH domain-containing proteins that have been implicated in vesicle trafficking. EHD2 in rat adipocytes was 85% membrane-associated, including approximately 10% in immunopurified GLUT4 vesicles. This association of EHD2 with GLUT4 vesicles occurred in PM and three distinct endosomal fractions and was not significantly affected by cellular insulin treatment. In co-immunoprecipitation experiments, however, EHD2 physically interacted with GLUT4 in each of these fractions, and cellular insulin treatment selectively enhanced this interaction in an endosomal fraction thought to contain GLUT4 exocytic vesicles. EHD2 also interacted with the clathrin adaptor middle chain subunit micro(1), micro(2), and rCALM in GST pull-down experiments. Significantly, an affinity-purified EHD2 antibody and a peptide corresponding to the EHD2 sequence Glu(428)-Glu(535) drastically (by 75% and 35%, respectively) suppressed the insulin-induced increase in the plasma membrane GLUT4 contents in SLO-permeabilized rat adipocytes without affecting the basal GLUT4 distribution. These findings strongly suggest that EHD2 interacts with GLUT4 in rat adipocytes and may play a key role in insulin-induced GLUT4 recruitment to the plasma membrane.

    Biochemistry 2004;43;23;7552-62

  • HIV-1 Tat enters T cells using coated pits before translocating from acidified endosomes and eliciting biological responses.

    Vendeville A, Rayne F, Bonhoure A, Bettache N, Montcourrier P and Beaumelle B

    UMR 5539 CNRS, Département Biologie-Santé, Case 107, Université Montpellier II, 34095 Montpellier Cedex 5, France.

    The HIV-1 Tat protein is secreted by infected cells. Extracellular Tat can affect bystander uninfected T cells and induce numerous biological responses such as apoptosis and cytokine secretion. Tat is likely involved in several immune disorders during AIDS. Nevertheless, it is not known whether Tat triggers cell responses directly upon binding to signaling receptors at the plasma membrane or after delivery to the cytosol. The pathway that enables Tat to reach the cytosol is also unclear. Here we visualized Tat within T-cell-coated pits and endosomes. Moreover, inhibitors of clathrin/AP-2-mediated uptake such as chlorpromazine, activated RhoA, or dominant-negative mutants of Eps15, intersectin, dynamin, or rab5 impaired Tat delivery to the cytosol by preventing its endocytosis. Molecules neutralizing low endosomal pH or Hsp90 inhibitors abolished Tat entry at a later stage by blocking its endosomal translocation, as directly shown using a cell-free translocation assay. Finally, endosomal pH neutralization prevented Tat from inducing T-cell responses such as NF-kappaB activation, apoptosis, and interleukin secretion, indicating that cytosolic delivery is required for Tat signaling. Hence, Tat enters T cells essentially like diphtheria toxin, using clathrin-mediated endocytosis before low-pH-induced and Hsp90-assisted endosomal translocation. Cell responses are then induced from the cytosol.

    Molecular biology of the cell 2004;15;5;2347-60

  • Clathrin-mediated endocytosis in AP-2-depleted cells.

    Motley A, Bright NA, Seaman MN and Robinson MS

    University of Cambridge, Department of Clinical Biochemistry, Cambridge Institute for Medical Research, Cambridge CB2 2XY, UK.

    We have used RNA interference to knock down the AP-2 mu2 subunit and clathrin heavy chain to undetectable levels in HeLaM cells. Clathrin-coated pits associated with the plasma membrane were still present in the AP-2-depleted cells, but they were 12-fold less abundant than in control cells. No clathrin-coated pits or vesicles could be detected in the clathrin-depleted cells, and post-Golgi membrane compartments were swollen. Receptor-mediated endocytosis of transferrin was severely inhibited in both clathrin- and AP-2-depleted cells. Endocytosis of EGF, and of an LDL receptor chimera, were also inhibited in the clathrin-depleted cells; however, both were internalized as efficiently in the AP-2-depleted cells as in control cells. These results indicate that AP-2 is not essential for clathrin-coated vesicle formation at the plasma membrane, but that it is one of several endocytic adaptors required for the uptake of certain cargo proteins including the transferrin receptor. Uptake of the EGF and LDL receptors may be facilitated by alternative adaptors.

    Funded by: Wellcome Trust

    The Journal of cell biology 2003;162;5;909-18

  • The adaptor complex 2 directly interacts with the alpha 1b-adrenergic receptor and plays a role in receptor endocytosis.

    Diviani D, Lattion AL, Abuin L, Staub O and Cotecchia S

    Institut de Pharmacologie et de Toxicologie, Faculté de Médecine, 1005 Lausanne, Switzerland.

    Using the yeast two-hybrid system, we identified the mu 2 subunit of the clathrin adaptor complex 2 as a protein interacting with the C-tail of the alpha 1b-adrenergic receptor (AR). Direct association between the alpha 1b-AR and mu 2 was demonstrated using a solid phase overlay assay. The alpha 1b-AR/mu 2 interaction occurred inside the cells, as shown by the finding that the transfected alpha 1b-AR and the endogenous mu 2 could be coimmunoprecipitated from HEK-293 cell extracts. Mutational analysis of the alpha 1b-AR revealed that the binding site for mu 2 does not involve canonical YXX Phi or dileucine motifs but a stretch of eight arginines on the receptor C-tail. The binding domain of mu 2 for the receptor C-tail involves both its N terminus and the subdomain B of its C-terminal portion. The alpha 1b-AR specifically interacted with mu 2, but not with the mu 1, mu 3, or mu 4 subunits belonging to other AP complexes. The deletion of the mu 2 binding site in the C-tail markedly decreased agonist-induced receptor internalization as demonstrated by confocal microscopy as well as by the results of a surface receptor biotinylation assay. The direct association of the adaptor complex 2 with a G protein-coupled receptor has not been reported so far and might represent a common mechanism underlying clathrin-mediated receptor endocytosis.

    The Journal of biological chemistry 2003;278;21;19331-40

  • The cell surface expression of SAP-binding receptor CD229 is regulated via its interaction with clathrin-associated adaptor complex 2 (AP-2).

    Del Valle JM, Engel P and Martín M

    Department of Cellular Biology and Pathology, University of Barcelona School of Medicine, Institut d'Investigacions Biomèdiques August Pi i Sunyer, Barcelona E-08036, Spain.

    CD229 (Ly9) is a cell surface receptor selectively expressed on T and B lymphocytes, and it belongs to the CD150 receptor family. Like other receptors of this family, CD229 interacts with SAP/SH2D1a protein, mutation of which is responsible for the fatal X-linked lymphoproliferative disease. Receptors of the CD150 family function as costimulatory molecules, regulating cytokine production and cytotoxicity. Thus, their signaling and regulation in lymphocytes may be critical to an understanding of the pathogenesis of the X-linked lymphoproliferative disease. Here we show that CD229 interacts with the mu(2) chain of the AP-2 adaptor complex that links transmembrane proteins to clathrin-coated pits. CD229 was the only member of the CD150 family associated with AP-2. We also show that the mu(2) chain interacts with the Y(470)EKL motif of CD229. The integrity of this site was necessary for CD229 internalization, but it was not involved in SAP recruitment. Moreover, CD229 binds to the AP-2 complex in T and B cell lines, and it is internalized rapidly from the cell surface on T cells after antibody ligation. In contrast, cross-linking of CD229 receptors with intact antibody inhibited CD229 internalization on B cells. However, when F(ab')(2) antibodies were used, CD229 internalization was similar on T and B cells, suggesting that Fcgamma receptors control CD229 cell surface expression. Furthermore, CD229 was regulated by T cell receptor and B cell receptor signaling because coligation with antibodies against anti-CD3 and anti-IgM increased the rate of CD229 endocytosis. These data suggest that CD229 cell surface expression on lymphocytes surface is strongly and differentially regulated within the CD150 family members.

    The Journal of biological chemistry 2003;278;19;17430-7

  • The B cell coreceptor CD22 associates with AP50, a clathrin-coated pit adapter protein, via tyrosine-dependent interaction.

    John B, Herrin BR, Raman C, Wang YN, Bobbitt KR, Brody BA and Justement LB

    Department of Microbiology and Immunology, University of Texas Medical Branch, Galveston, TX 77555, USA.

    The B cell coreceptor CD22 plays an important role in regulating signal transduction via the B cell Ag receptor. Studies have shown that surface expression of CD22 can be modulated in response to binding of ligand (i.e., mAb). Thus, it is possible that alterations in the level of CD22 expression following binding of natural ligand(s) may affect its ability to modulate the Ag receptor signaling threshold at specific points during B cell development and differentiation. Therefore, it is important to delineate the physiologic mechanism by which CD22 expression is controlled. In the current study, yeast two-hybrid analysis was used to demonstrate that CD22 interacts with AP50, the medium chain subunit of the AP-2 complex, via tyrosine-based internalization motifs in its cytoplasmic domain. This interaction was further characterized using yeast two-hybrid analysis revealing that Tyr(843) and surrounding amino acids in the cytoplasmic tail of CD22 comprise the primary binding site for AP50. Subsequent studies using transfectant Jurkat cell lines expressing wild-type or mutant forms of CD22 demonstrated that either Tyr(843) or Tyr(863) is sufficient for mAb-mediated internalization of CD22 and that these motifs are involved in its interaction with the AP-2 complex, as determined by coprecipitation of alpha-adaptin. Finally, experiments were performed demonstrating that treatment of B cells with either intact anti-Ig Ab or F(ab')(2) blocks ligand-mediated internalization of CD22. In conclusion, these studies demonstrate that internalization of CD22 is dependent on its association with the AP-2 complex via tyrosine-based internalization motifs.

    Funded by: NIAID NIH HHS: AI36401, R01 AI036401

    Journal of immunology (Baltimore, Md. : 1950) 2003;170;7;3534-43

  • Protein-protein interactions between large proteins: two-hybrid screening using a functionally classified library composed of long cDNAs.

    Nakayama M, Kikuno R and Ohara O

    Department of Human Gene Research, Kazusa DNA Research Institute, Kisarazu, Chiba 292-0818, Japan. nmanabu@kazusa.or.jp

    Large proteins have multiple domains that are potentially capable of binding many kinds of partners. It is conceivable, therefore, that such proteins could function as an intricate framework of assembly protein complexes. To comprehensively study protein-protein interactions between large KIAA proteins, we have constructed a library composed of 1087 KIAA cDNA clones based on prior functional classifications done in silico. We were guided by two principles that raise the success rate for detecting interactions per tested combination: we avoided testing low-probability combinations, and reduced the number of potential false negatives that arise from the fact that large proteins cannot reliably be expressed in yeast. The latter was addressed by constructing a cDNA library comprised of random fragments encoding large proteins. Cytoplasmic domains of KIAA transmembrane proteins (>1000 amino acids) were used as bait for yeast two-hybrid screening. Our analyses reveal that several KIAA proteins bearing a transmembrane region have the capability of binding to other KIAA proteins containing domains (e.g., PDZ, SH3, rhoGEF, and spectrin) known to be localized to highly specialized submembranous sites, indicating that they participate in cellular junction formation, receptor or channel clustering, and intracellular signaling events. Our representative library should be a very useful resource for detecting previously unidentified interactions because it complements conventional expression libraries, which seldom contain large cDNAs.

    Genome research 2002;12;11;1773-84

  • Subunit H of the V-ATPase binds to the medium chain of adaptor protein complex 2 and connects Nef to the endocytic machinery.

    Geyer M, Yu H, Mandic R, Linnemann T, Zheng YH, Fackler OT and Peterlin BM

    Department of Medicine, University of California, San Francisco, California 94143-0703, USA.

    Nef is an accessory protein of human and simian immunodeficiency viruses (HIV and SIV) that is required for efficient viral infectivity and pathogenicity. It decreases the expression of CD4 on the surface of infected cells. V1H is the regulatory subunit H of the vacuolar membrane ATPase (V-ATPase). Previously, the interaction between Nef and V1H has been found to facilitate the internalization of CD4, suggesting that V1H could connect Nef to the endocytic machinery. In this study, we demonstrate that V1H binds to the C-terminal flexible loop in Nef from HIV-1 and to the medium chain (mu2) of the adaptor protein complex 2 (AP-2) in vitro and in vivo. The interaction sites of V1H and mu2 were mapped to a central region in V1H from positions 133 to 363, which contains 4 armadillo repeats, and to the N-terminal adaptin-binding domain in mu2 from positions 1 to 145. Fusing Nef to V1H reproduced the appropriate trafficking of Nef. This chimera internalized CD4 even in the absence of the C-terminal flexible loop in Nef. Finally, blocking the expression of V1H decreased the enhancement of virion infectivity by Nef. Thus, V1H can function as an adaptor for interactions between Nef and AP-2.

    Funded by: NIAID NIH HHS: 1R01AI38532-01

    The Journal of biological chemistry 2002;277;32;28521-9

  • CK2 and GAK/auxilin2 are major protein kinases in clathrin-coated vesicles.

    Korolchuk VI and Banting G

    Department of Biochemistry, School of Medical Sciences, University of Bristol, Bristol BS8 1TD, UK.

    Several peripheral membrane proteins associated with clathrin-coated vesicles (CCVs) are reversibly phosphorylated, but it is not clear precisely which protein kinases are involved. In order to address this question directly, we have isolated highly purified CCVs from porcine brain. The peripheral membrane proteins have been removed and assayed for kinase activity using the CCV peripheral membrane proteins as substrate. The major kinase activity identified has a molecular mass of 40 kDa, is inhibited by known specific inhibitors of the protein kinase CK2 and is recognised by an antibody specific to CK2. We show that CK2 is responsible for the phosphorylation of the majority of CCV-associated proteins that are subject to phosphorylation. Intriguingly, CK2 is inactive when associated with CCVs but becomes active once the clathrin coat has been removed. The medium subunit of the AP2 adaptor complex (mu2) is not a substrate for CK2, but is phosphorylated by a second kinase that we show to be cyclin G-associated kinase (GAK/auxilin2). Unlike the situation for the CK2 substrates, mu2 is a substrate for GAK/auxilin2, both in intact CCVs and in solution. In addition, we show that the 'stripped' CCV membranes that remain once the peripheral membrane proteins have been removed from CCVs inhibit CK2 but not GAK/auxilin2 activity.

    Traffic (Copenhagen, Denmark) 2002;3;6;428-39

  • Molecular architecture and functional model of the endocytic AP2 complex.

    Collins BM, McCoy AJ, Kent HM, Evans PR and Owen DJ

    Cambridge Institute for Medical Research, University of Cambridge, Department of Clinical Biochemistry, Wellcome Trust/MRC Building, Hills Road, United Kingdom.

    AP2 is the best-characterized member of the family of heterotetrameric clathrin adaptor complexes that play pivotal roles in many vesicle trafficking pathways within the cell. AP2 functions in clathrin-mediated endocytosis, the process whereby cargo enters the endosomal system from the plasma membrane. We describe the structure of the 200 kDa AP2 "core" (alpha trunk, beta2 trunk, mu2, and sigma2) complexed with the polyphosphatidylinositol headgroup mimic inositolhexakisphosphate at 2.6 A resolution. Two potential polyphosphatidylinositide binding sites are observed, one on alpha and one on mu2. The binding site for Yxxphi endocytic motifs is buried, indicating that a conformational change, probably triggered by phosphorylation in the disordered mu2 linker, is necessary to allow Yxxphi motif binding. A model for AP2 recruitment and activation is proposed.

    Cell 2002;109;4;523-35

  • Polarized trafficking and surface expression of the AQP4 water channel are coordinated by serial and regulated interactions with different clathrin-adaptor complexes.

    Madrid R, Le Maout S, Barrault MB, Janvier K, Benichou S and Mérot J

    Service de Biologie Cellulaire, Département de Biologie Cellulaire et Moléculaire, CEA/Saclay, F-91191 Gif-sur-Yvette, Cedex, France.

    Aquaporin 4 (AQP4) is the predominant water channel in the brain. It is targeted to specific membrane domains of astrocytes and plays a crucial role in cerebral water balance in response to brain edema formation. AQP4 is also specifically expressed in the basolateral membranes of epithelial cells. However, the molecular mechanisms involved in its polarized targeting and membrane trafficking remain largely unknown. Here, we show that two independent C-terminal signals determine AQP4 basolateral membrane targeting in epithelial MDCK cells. One signal involves a tyrosine-based motif; the other is encoded by a di-leucine-like motif. We found that the tyrosine-based basolateral sorting signal also determines AQP4 clathrin-dependent endocytosis through direct interaction with the mu subunit of AP2 adaptor complex. Once endocytosed, a regulated switch in mu subunit interaction changes AP2 adaptor association to AP3. We found that the stress-induced kinase casein kinase (CK)II phosphorylates the Ser276 immediately preceding the tyrosine motif, increasing AQP4-mu 3A interaction and enhancing AQP4-lysosomal targeting and degradation. AQP4 phosphorylation by CKII may thus provide a mechanism that regulates AQP4 cell surface expression.

    The EMBO journal 2001;20;24;7008-21

  • Study of the interaction of the medium chain mu 2 subunit of the clathrin-associated adapter protein complex 2 with cytotoxic T-lymphocyte antigen 4 and CD28.

    Follows ER, McPheat JC, Minshull C, Moore NC, Pauptit RA, Rowsell S, Stacey CL, Stanway JJ, Taylor IW and Abbott WM

    Enabling Science and Technology, Biology Department, AstraZeneca, Mereside, Alderley Park, Macclesfield, Cheshire SK10 4TG, UK.

    The medium chain mu 2 subunit (AP50) of the clathrin-associated adapter protein complex 2 (AP-2) interacts specifically with the tyrosine-based signals of several integral membrane proteins through the consensus sequence YXXPhi, where X can be any residue and Phi is a large hydrophobic residue. Using surface plasmon resonance combined with structural information, we have analysed the interaction of AP50 with peptides derived from the cytoplasmic tail of cytotoxic T-lymphocyte antigen 4 (CTLA-4). The crystal structure of AP50 in complex with a CTLA-4-derived peptide was determined to 3.6 A (1 A=0.1 nm) resolution. The binding domain of AP50 (residues 164-435) was expressed in Escherichia coli and purified. In agreement with previous reports, the AP50 domain bound to residues 152-174 of CTLA-4, but not to the same peptide that was phosphorylated at the single tyrosine residue (position 165). The interaction exhibited fast kinetics with rapid on and off rates and a K(d) of 0.7 microM. In order to further understand why AP50 binds to CTLA-4, but not to the homologous receptor CD28, a comparison of binding of AP50 with five peptides with single changes in and around the YXXPhi motif to the equivalent residues of CD28 was made. T162H greatly reduced binding, whereas T161L had little effect. Mutations G163S, V164D and K167N all exhibited reduced binding. Modelling of the single amino acid changes using structural information, was in broad agreement with the binding data, demonstrating that residues outside of the YXXPhi motif are also important in the interaction of membrane proteins with AP50.

    The Biochemical journal 2001;359;Pt 2;427-34

  • Doublecortin interacts with mu subunits of clathrin adaptor complexes in the developing nervous system.

    Friocourt G, Chafey P, Billuart P, Koulakoff A, Vinet MC, Schaar BT, McConnell SK, Francis F and Chelly J

    Laboratoire de Génétique et Physiopathologie des retards mentaux, ICGM, INSERM, CHU, Cochin, 24, rue du Faubourg Saint Jacques, Paris, 75014, France.

    Doublecortin is a microtubule-associated protein required for normal corticogenesis in the developing brain. We carried out a yeast two-hybrid screen to identify interacting proteins. One of the isolated clones encodes the mu1 subunit of the adaptor complex AP-1 involved in clathrin-dependent protein sorting. We found that Doublecortin also interacts in yeast with mu2 from the AP-2 complex. Mutagenesis and pull-down experiments showed that these interactions were mediated through a tyrosine-based sorting signal (YLPL) in the C-terminal part of Doublecortin. The functional relevance of these interactions was suggested by the coimmunoprecipitation of Doublecortin with AP-1 and AP-2 from mouse brain extracts. This interaction was further supported by RNA in situ hybridization and immunofluorescence studies. Taken together these data indicate that a certain proportion of Doublecortin interacts with AP-1 and/or AP-2 in vivo and are consistent with a potential involvement of Doublecortin in protein sorting or vesicular trafficking.

    Molecular and cellular neurosciences 2001;18;3;307-19

  • Human stoned B interacts with AP-2 and synaptotagmin and facilitates clathrin-coated vesicle uncoating.

    Walther K, Krauss M, Diril MK, Lemke S, Ricotta D, Honing S, Kaiser S and Haucke V

    Zentrum Biochemie und Molekulare Zellbiologie, University of Göttingen, Humboldtallee 23, D-37073 Göttingen, Germany.

    Synaptic vesicle biogenesis involves the recycling of synaptic vesicle components by clathrin-mediated endocytosis from the presynaptic membrane. stoned B, a protein encoded by the stoned locus in Drosophila melanogaster has been shown to regulate vesicle recycling by interacting with synaptotagmin. We report here the identification and characterization of a human homolog of stoned B (hStnB). Human stoned B is a brain-specific protein which co-enriches with other endocytic proteins such as AP-2 in a crude synaptic vesicle fraction and at nerve terminals. A domain with homology to the medium chain of adaptor complexes binds directly to both AP-2 and synaptotagmin and competes with AP-2 for the same binding site within synaptotagmin. Finally we show that the mu 2 homology domain of hStnB stimulates the uncoating of both clathrin and AP-2 adaptors from clathrin-coated vesicles. We hypothesize that hStnB regulates synaptic vesicle recycling by facilitating vesicle uncoating.

    EMBO reports 2001;2;7;634-40

  • Mechanism for down-regulation of CD28 by Nef.

    Swigut T, Shohdy N and Skowronski J

    Cold Spring Harbor Laboratory, Cold Spring Harbor, NY 11724, USA.

    SIV and HIV Nef proteins disrupt T-cell receptor machinery by down-modulating cell surface expression of CD4 and expression or signaling of CD3-TCR. Nef also down-modulates class I major histocompatibility complex (MHC) surface expression. We show that SIV and HIV-1 Nefs down-modulate CD28, a major co-stimulatory receptor that mediates effective T-cell activation, by accelerating CD28 endocytosis. The effects of Nef on CD28, CD4, CD3 and class I MHC expression are all genetically separable, indicating that all are selected independently. In cells expressing a Nef-green fluorescent protein (GFP) fusion, CD28 co-localizes with the AP-2 clathrin adaptor and Nef-GFP. Mutations that disrupt Nef interaction with AP-2 disrupt CD28 down-regulation. Furthermore, HIV and SIV Nefs use overlapping but distinct target sites in the membrane-proximal region of the CD28 cytoplasmic domain. Thus, Nef probably induces CD28 endocytosis via the AP-2 pathway, and this involves a ternary complex containing Nef, AP-2 and CD28. The likely consequence of the concerted down-regulation of CD28, CD4 and/or CD3 by Nef is disruption of antigen-specific signaling machineries in infected T cells following a productive antigen recognition event.

    Funded by: NIAID NIH HHS: AI-42561, R01 AI042561

    The EMBO journal 2001;20;7;1593-604

  • The highly conserved C-terminal dileucine motif in the cytosolic domain of the human immunodeficiency virus type 1 envelope glycoprotein is critical for its association with the AP-1 clathrin adaptor [correction of adapter].

    Wyss S, Berlioz-Torrent C, Boge M, Blot G, Höning S, Benarous R and Thali M

    Institute of Microbiology, University of Lausanne, CH-1011 Lausanne, Switzerland.

    Short amino acid sequences in the cytosolic domains of transmembrane proteins are recognized by specialized adaptor [corrected] proteins which are part of coated vesicles utilized to transport membrane proteins between the trans-Golgi network (TGN) and the plasma membrane (forward and backward). Previously, we and others reported that the membrane-proximal tyrosine residues Y712 (human immunodeficiency virus [HIV]) and Y721 (simian immunodeficiency virus [SIV]) in the envelope glycoprotein (Env) of the primate lentiviruses are crucial for the association of Env with clathrin-associated adaptor [corrected] complex AP-2. The same tyrosine-based endocytosis motifs in the cytosolic domains (EnvCD) of transmembrane gp41 of HIV type 1 (HIV-1) and SIV, respectively, were also shown to modulate the interaction with TGN- and endosome-based clathrin-associated complex AP-1. Our findings suggested that EnvCD binding to AP-1, unlike the association of EnvCD with AP-2, is dependent largely on residues other than Y712 and Y721. Here, we tested if motifs downstream of Y712 affect HIV-1 EnvCD-AP-1 binding and Env trafficking. Mutational analysis revealed that the C-terminal leucine-based motif in Env was crucial for the recruitment of AP-1 in vitro and in Env-expressing cells. In addition to affecting Env-AP-1 association, mutations at the C terminus of Env also altered the subcellular localization of Env, suggesting that proper post-Golgi routing of Env depends on its recruitment of AP-1. Finally, the C-terminal dileucine was shown to assist the membrane-proximal Y712 motif in restricting the cell surface expression of Env.

    Journal of virology 2001;75;6;2982-92

  • Association of Trk neurotrophin receptors with components of the cytoplasmic dynein motor.

    Yano H, Lee FS, Kong H, Chuang J, Arevalo J, Perez P, Sung C and Chao MV

    Molecular Neurobiology Program, Skirball Institute for Biomolecular Medicine, Departments of Cell Biology , New York, New York 10016, USA.

    Nerve growth factor (NGF) initiates its trophic effects by long-range signaling through binding, internalization, and transport of a ligand-receptor complex from the axon terminal to the cell body. However, the mechanism by which retrograde transport of NGF takes place has not been elucidated. Here we describe an interaction between the Trk receptor tyrosine kinase and a 14 kDa light chain of cytoplasmic dynein. After transfection in human embryonic kidney 293 cells, this 14 kDa dynein light chain was found to bind to TrkA, TrkB, and TrkC receptors. Mapping experiments indicated that the 14 kDa dynein light chain binds to the distal region of the TrkA juxtamembrane domain. Coimmunoprecipitation experiments in vivo indicate that Trk receptors are in a complex with the 14 kDa light chain and 74 kDa intermediate chain of dynein. Confirming the physiological relevance of this association, a marked accumulation of Trk with the 14 kDa and the 74 kDa dynein components was observed after ligation of the sciatic nerve. The association of Trk receptors with components of cytoplasmic dynein suggests that transport of neurotrophins during vesicular trafficking may occur through a direct interaction of the Trk receptor with the dynein motor machinery.

    Funded by: NEI NIH HHS: EY11307, R01 EY011307; NICHD NIH HHS: HD233-5; NINDS NIH HHS: NS21072

    The Journal of neuroscience : the official journal of the Society for Neuroscience 2001;21;3;RC125

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

    Morris SM and Cooper JA

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

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

    Funded by: NCI NIH HHS: R37-CA41072

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

  • RLIP76, an effector of the GTPase Ral, interacts with the AP2 complex: involvement of the Ral pathway in receptor endocytosis.

    Jullien-Flores V, Mahé Y, Mirey G, Leprince C, Meunier-Bisceuil B, Sorkin A and Camonis JH

    Institut Curie, INSERM (Institut National de la Santé et de la Recherche Scientifique) U-528, 75248 Paris Cedex 05, France.

    RLIP76 is a modular protein that was identified as a putative effector of Ral, a GTPase activated during Ras signaling. To explore further the contribution of the Ral-RLIP76 pathway to Ras signaling, we have looked for partners of RLIP76. Mu2, the medium chain of the AP2 complex is shown to interact with RLIP76. We show also that in vivo endogenous AP2 and RLIP76 form a complex and that this in vivo interaction is independent of cells being stimulated by a growth factor. Furthermore, RLIP76 differentiates AP2 from AP1 in vivo as RLIP76 differentiates mu2 from mu1 in vitro and in two hybrid assays. We show that activated Ral interferes with both tranferrin receptor endocytosis and epidermal growth factor (EGF) receptor endocytosis in HeLa cells. We propose a model where the Ral-RLIP76 pathway connects signal transduction and endocytosis through interaction on one hand between the Ras-Ral pathway and RLIP, on the other hand between RLIP and proteins belonging to the endocytotic machinery.

    Journal of cell science 2000;113 ( Pt 16);2837-44

  • Identification of the universal cofactor (auxilin 2) in clathrin coat dissociation.

    Umeda A, Meyerholz A and Ungewickell E

    Department of Cell Biology, Centre of Anatomy, Hannover Medical School, Germany.

    Uncoating of clathrin-coated vesicles in neuronal cells requires hsc70 in concert with the cofactor auxilin which contains a J-domain as well as a domain with homology to dual specific phosphatases and tensin, known as PTEN. The question of whether an analogous factor operates in other cell types has until now remained unanswered. Here we show that it is the recently discovered and widely expressed cyclin G-associated protein kinase which fulfils the function of neuronal auxilin in hsc70-mediated clathrin coat dissociation. GAK possesses a J-domain, which stimulates the hsc70 ATPase, it competes with auxilin for clathrin binding and at sufficiently high concentrations acts as a clathrin assembly protein. Moreover, GAK binds to the gamma- and alpha-appendage domains of the adaptor proteins AP-1 and AP-2 in vitro and phosphorylates their medium chains. Cells that transiently overexpress GAK are impaired in respect of receptor-mediated endocytosis. In transfected cells clathrin is dislodged from coated pits/vesicles and co-localizes with GFP-GAK in the form of large aggregates. The cellular distribution of membrane-associated adaptors was unaffected by overexpression of GAK. Our results point to a hsc70/auxilin-based uncoating system as a ubiquitous feature of eukaryotic cells.

    European journal of cell biology 2000;79;5;336-42

  • Recycling of furin from the plasma membrane. Functional importance of the cytoplasmic tail sorting signals and interaction with the AP-2 adaptor medium chain subunit.

    Teuchert M, Berghöfer S, Klenk HD and Garten W

    Institut für Virologie der Philipps-Universität Marburg, Robert-Koch Strasse 17, 35037 Marburg, Germany.

    The predominant intracellular localization of the eukaryotic subtilisin-like endoprotease furin is the trans-Golgi network (TGN), but a small fraction is also found on the cell surface. Furin on the cell surface is internalized and delivered to the TGN. The identification of three endocytosis motifs, a tyrosine (YKGL(765)) motif, a leucine-isoleucine (LI(760)) motif, and a phenylalanine (Phe(790)) signal, in the furin cytoplasmic domain suggested that endocytosis of furin occurs via an AP-2/clathrin-dependent pathway. Since little is known about proteins containing multiple sorting components in their cytoplasmic domain, the combination of diverse internalization signals in the furin tail raised the question of their individual role. Here we present data showing that the furin tail interacts with the medium (micro2) subunit of the AP-2 plasma membrane-specific adaptor complex in vitro and that this interaction primarily depends on recognition of the tyrosine-based sorting signal and to less extent on the leucine-isoleucine motif. We further provide evidence that the three endocytosis signals are of different functional importance for furin internalization and retrieval to the TGN in vivo, with the tyrosine-based motif being the major determinant, followed by the phenylalanine signal, whereas the leucine-isoleucine motif is only a minor component. Finally, we report that phosphorylation of the furin tail by casein kinase II is not only important for efficient interaction with micro2 and internalization from the plasma membrane but also determines fast retrieval of the protein from the plasma membrane to the TGN.

    The Journal of biological chemistry 1999;274;51;36781-9

  • The epsins define a family of proteins that interact with components of the clathrin coat and contain a new protein module.

    Rosenthal JA, Chen H, Slepnev VI, Pellegrini L, Salcini AE, Di Fiore PP and De Camilli P

    Howard Hughes Medical Institute and Department of Cell Biology, Yale University School of Medicine, New Haven, Connecticut 06510, USA.

    Epsin (epsin 1) is an interacting partner for the EH domain-containing region of Eps15 and has been implicated in conjunction with Eps15 in clathrin-mediated endocytosis. We report here the characterization of a similar protein (epsin 2), which we have cloned from human and rat brain libraries. Epsin 1 and 2 are most similar in their NH(2)-terminal region, which represents a module (epsin NH(2) terminal homology domain, ENTH domain) found in a variety of other proteins of the data base. The multiple DPW motifs, typical of the central region of epsin 1, are only partially conserved in epsin 2. Both proteins, however, interact through this central region with the clathrin adaptor AP-2. In addition, we show here that both epsin 1 and 2 interact with clathrin. The three NPF motifs of the COOH-terminal region of epsin 1 are conserved in the corresponding region of epsin 2, consistent with the binding of both proteins to Eps15. Epsin 2, like epsin 1, is enriched in brain, is present in a brain-derived clathrin-coated vesicle fraction, is concentrated in the peri-Golgi region and at the cell periphery of transfected cells, and partially colocalizes with clathrin. High overexpression of green fluorescent protein-epsin 2 mislocalizes components of the clathrin coat and inhibits clathrin-mediated endocytosis. The epsins define a new protein family implicated in membrane dynamics at the cell surface.

    Funded by: NCI NIH HHS: CA46128; NINDS NIH HHS: NS1024-01, NS36251; ...

    The Journal of biological chemistry 1999;274;48;33959-65

  • Interactions of the cytoplasmic domains of human and simian retroviral transmembrane proteins with components of the clathrin adaptor complexes modulate intracellular and cell surface expression of envelope glycoproteins.

    Berlioz-Torrent C, Shacklett BL, Erdtmann L, Delamarre L, Bouchaert I, Sonigo P, Dokhelar MC and Benarous R

    CJF 97/03 INSERM, Interactions Moléculaires, Hôte-Pathogène, Institut Cochin de Génétique Moléculaire, 75014 Paris, France.

    The cytoplasmic domains of the transmembrane (TM) envelope proteins (TM-CDs) of most retroviruses have a Tyr-based motif, YXXO, in their membrane-proximal regions. This signal is involved in the trafficking and endocytosis of membrane receptors via clathrin-associated AP-1 and AP-2 adaptor complexes. We have used CD8-TM-CD chimeras to investigate the role of the Tyr-based motif of human immunodeficiency virus type 1 (HIV-1), simian immunodeficiency virus (SIV), and human T-leukemia virus type 1 (HTLV-1) TM-CDs in the cell surface expression of the envelope glycoprotein. Flow cytometry and confocal microscopy studies showed that this motif is a major determinant of the cell surface expression of the CD8-HTLV chimera. The YXXO motif also plays a key role in subcellular distribution of the envelope of lentiviruses HIV-1 and SIV. However, these viruses, which encode TM proteins with a long cytoplasmic domain, have additional determinants distal to the YXXO motif that participate in regulating cell surface expression. We have also used the yeast two-hybrid system and in vitro binding assays to demonstrate that all three retroviral YXXO motifs interact with the micro1 and micro2 subunits of AP complexes and that the C-terminal regions of HIV-1 and SIV TM proteins interact with the beta2 adaptin subunit. The TM-CDs of HTLV-1, HIV-1, and SIV also interact with the whole AP complexes. These results clearly demonstrate that the cell surface expression of retroviral envelope glycoproteins is governed by interactions with adaptor complexes. The YXXO-based signal is the major determinant of this interaction for the HTLV-1 TM, which contains a short cytoplasmic domain, whereas the lentiviruses HIV-1 and SIV have additional determinants distal to this signal that are also involved.

    Journal of virology 1999;73;2;1350-61

  • A structural explanation for the recognition of tyrosine-based endocytotic signals.

    Owen DJ and Evans PR

    Medical Research Council Laboratory of Molecular Biology, Hills Road, Cambridge CB2 2QH, UK.

    Many cell surface proteins are marked for endocytosis by a cytoplasmic sequence motif, tyrosine-X-X-(hydrophobic residue), that is recognized by the mu2 subunit of AP2 adaptors. Crystal structures of the internalization signal binding domain of mu2 complexed with the internalization signal peptides of epidermal growth factor receptor and the trans-Golgi network protein TGN38 have been determined at 2.7 angstrom resolution. The signal peptides adopted an extended conformation rather than the expected tight turn. Specificity was conferred by hydrophobic pockets that bind the tyrosine and leucine in the peptide. In the crystal, the protein forms dimers that could increase the strength and specificity of binding to dimeric receptors.

    Funded by: Medical Research Council: MC_U105178845

    Science (New York, N.Y.) 1998;282;5392;1327-32

  • Endocytic clathrin-coated pit formation is independent of receptor internalization signal levels.

    Santini F, Marks MS and Keen JH

    Kimmel Cancer Institute and the Departments of Microbiology and Immunology and of Biochemistry and Molecular Pharmacology, Thomas Jefferson University, Philadelphia, Pennsylvania 19107, USA.

    The mechanisms responsible for coated pit formation in cells remain unknown, but indirect evidence has argued both for and against a critical role of receptor cytoplasmic domains in the process. If the endocytic motifs of receptors are responsible for recruiting AP2 to the plasma membrane, thereby driving coated pit formation, then the level of constitutively internalized receptors at the membrane would be expected to govern the steady-state level of coated pits in cells. Here we directly test this hypothesis for broad classes of receptors containing three distinct constitutive internalization signals. Chimeric proteins consisting of an integral membrane reporter protein (Tac) coupled to cytoplasmic domains bearing tyrosine-, di-leucine-, or acidic cluster/casein kinase II-based internalization signals were overexpressed to levels that saturated the internalization pathway. Quantitative confocal immunofluorescence microscopy indicated that the number of plasma membrane clathrin-coated pits and the concentration of their structural components were invariant when comparing cells expressing saturating levels of the chimeric receptors to nonexpressing cells or to cells expressing only the Tac reporter lacking cytoplasmic internalization signals. Biochemical analysis showed that the distribution of coat proteins between assembled coated pits and soluble pools was also not altered by receptor overexpression. Finally, the cellular localizations of AP2 and AP1 were similarly unaffected. These results provide a clear indication that receptor endocytic signals do not determine coated pit levels by directly recruiting AP2 molecules. Rather, the findings support a model in which coated pit formation proceeds through recruitment and activation of AP2, likely through a limited number of regulated docking sites that act independently of endocytic signals.

    Funded by: NCI NIH HHS: 5-T32-CA09678, T32 CA009678; NIGMS NIH HHS: GM-28526, R01 GM028526

    Molecular biology of the cell 1998;9;5;1177-94

  • Interaction of endocytic signals from the HIV-1 envelope glycoprotein complex with members of the adaptor medium chain family.

    Ohno H, Aguilar RC, Fournier MC, Hennecke S, Cosson P and Bonifacino JS

    Cell Biology and Metabolism Branch, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20892, USA.

    The envelope glycoprotein (Env) complex of HIV-1 undergoes rapid internalization from the plasma membrane of human cells by virtue of a tyrosine-based endocytic signal (RQGYSPL, residues 704-710) in the cytosolic tail of the protein (J. F. Rowell et al., J. Immunol. 155, 473-488, 1995). Here we demonstrate that this tyrosine-based signal interacts with the mu 2 (medium) chain of the AP-2 clathrin-associated adaptor, a protein complex involved in endocytosis of cell surface receptors. The same signal is also capable of interacting with two other members of the adaptor medium chain family, mu 1 and mu 3A, which are components of the AP-1 and AP-3 adaptor complexes, respectively. Interactions with mu 1 and mu 3A might be responsible for the targeting of the internalized envelope glycoprotein to lysosomes or to the basolateral plasma membrane of polarized epithelial cells. A second potential tyrosine-based signal (LFSYHRL, residues 760-766) also interacts with mu 1, mu 2, and mu 3A, although it is less important for internalization in vivo probably due to its position within the cytosolic tail. Overexpression of chimeric proteins having the HIV-1 Env cytosolic tail increases expression of the transferrin receptor on the cell surface, probably due to saturation of the cellular pool of mu 2 by the overexpressed proteins. These observations suggest that HIV-1 Env utilizes the protein sorting machinery of the host cells for internalization and sorting at various steps of the endocytic and biosynthetic pathways.

    Virology 1997;238;2;305-15

  • Interaction of CTLA-4 with AP50, a clathrin-coated pit adaptor protein.

    Zhang Y and Allison JP

    Department of Molecular and Cellular Biology and Cancer Research Laboratory, University of California, Berkeley, CA 94720, USA.

    CTLA-4 plays a critical role in regulating the immune response. It is mainly located in cytoplasmic vesicles and is expressed only transiently on the surface after T cell activation. In this study, we demonstrate that CTLA-4 is associated with AP50, the medium chain of the clathrin-associated coated pit adaptor protein complex AP2. In a yeast two-hybrid screen, three individual cDNA clones that encode mouse AP50 were isolated, all of which can interact specifically with the cytoplasmic domain of mouse CTLA-4, but not with the cytoplasmic domain of mouse CD28. We have shown that CTLA-4 can bind specifically to AP50 when CTLA-4 and AP50 are cotransfected into human 293T cells. A Y201 to F201 mutation in the YVKM intracellular localization motif of the CTLA-4 cytoplasmic domain significantly diminished its binding to AP50. We also found that AP50 bound to a CTLA-4 peptide containing unphosphorylated Y201 but not to a peptide containing phosphorylated Y201. Conversely, the p85 subunit of phosphatidylinositol 3-kinase and, to a lesser extent, protein tyrosine phosphatase SYP (SHP-2) and SHP (SHP-1) bind only to the CTLA-4 peptide containing phosphorylated Y201. Therefore, the phosphorylation status of Y201 in the CTLA-4 cytoplasmic domain determines the binding specificity of CTLA-4. These results suggest that AP50 and the coated pit adaptor complex AP2 may play an important role in regulating the intracellular trafficking and function of CTLA-4.

    Funded by: NCI NIH HHS: CA40041, R01 CA040041

    Proceedings of the National Academy of Sciences of the United States of America 1997;94;17;9273-8

  • Interaction of CTLA-4 with the clathrin-associated protein AP50 results in ligand-independent endocytosis that limits cell surface expression.

    Chuang E, Alegre ML, Duckett CS, Noel PJ, Vander Heiden MG and Thompson CB

    Gwen Knapp Center for Lupus and Immunology Research, Department of Molecular Genetics and Cell Biology, The University of Chicago, IL 60637, USA.

    CTLA-4 is a lymphocyte cell surface receptor expressed by activated T cells that functions to down-regulate T cell responses induced by TCR and CD28 stimulation. Since CTLA-4 competes with CD28 for binding to the common ligands B7-1 and B7-2, the level of CTLA-4 surface expression is likely to play an important role in its ability to inhibit CD28-dependent T cell activation. The factors that regulate these levels are poorly understood. Recent studies have revealed that following T cell activation, the majority of CTLA-4 is localized intracellularly rather than on the cell surface, and surface CTLA-4 is rapidly reinternalized. In this study, we investigate the molecular mechanism underlying the rapid clearance of CTLA-4 from the cell surface. The data demonstrate that cell surface CTLA-4 is endocytosed into clathrin-coated vesicles even in the absence of ligand. The targeting of CTLA-4 to clathrin-coated vesicles is mediated by the clathrin-associated adaptor complex AP-2. The cytoplasmic domain of CTLA-4 was found to specifically bind to AP50, the medium chain subunit of AP-2 in both yeast two-hybrid and coimmunoprecipitation assays. The interaction requires the peptide sequence 199-GVYVKM-204 in the cytoplasmic tail of CTLA-4. Mutation of the CTLA-4 amino acid residue Y201 abrogates the interaction with AP50, resulting in the accumulation of CTLA-4 at the cell surface. Together these data suggest that the interaction of CTLA-4 with AP50 plays an important role in regulating the cell surface expression of CTLA-4.

    Funded by: NHLBI NIH HHS: HL07605

    Journal of immunology (Baltimore, Md. : 1950) 1997;159;1;144-51

  • Identification of two new mu-adaptin-related proteins, mu-ARP1 and mu-ARP2.

    Wang X and Kilimann MW

    Institut fur Physiologische Chemie, Medizinische Fakultät, Ruhr-Universität Bochum, Germany.

    We report the cDNA cloning, primary structure and tissue distribution of two new proteins homologous to mu-adaptins, the medium chains of the clathrin coat adaptor complexes. Both predicted proteins share 60% amino acid sequence identity with each other and 27-31%, identity with mu1-adaptin (ap47) and mu2-adaptin (ap50). Lower similarity (23-25% identity) is found with two other mu-adaptin-related proteins, p47A/B, and there is similarity over the N-terminal 150 amino acids with the adaptin small chains and deltaCOP. The mRNAs of both molecules are expressed in all tissues analyzed, but with different profiles of relative abundance. mu-ARP1 is most abundant in brain, ovary and lung, whereas mu-ARP2 is prominently expressed in testis. These proteins suggest the existence of as yet uncharacterized types of clathrin- or non-clathrin-associated protein coats in cellular membrane traffic, of which they are probably prototype subunits, and provide molecular markers and probes for their characterization.

    FEBS letters 1997;402;1;57-61

  • Chromosome localization of human genes for clathrin adaptor polypeptides AP2 beta and AP50 and the clathrin-binding protein, VCP.

    Druck T, Gu Y, Prabhala G, Cannizzaro LA, Park SH, Huebner K and Keen JH

    Jefferson Cancer Institute, Jefferson Medical College, Philadelphia, Pennsylvania 19107, USA.

    Clathrin-coated vesicles, involved in endocytosis and Golgi processing, have a surface lattice containing clathrin triskelia and stoichiometric amounts of additional components termed "assembly proteins," or APs. The AP form at the plasma membrane, AP2, is composed of two large subunits of 100-115 kDa, denoted AP2 alpha and AP2 beta, a medium chain of 50 kDa, designated AP50, and a small chain. We have determined human chromosomal locations of genes for a large AP2 beta (CLAPB1) and a medium (CLAPM1) AP subunit and of a novel clathrin-binding protein, VCP, that binds clathrin simultaneously with APs. Chromosomal in situ hybridization of a human genomic clone demonstrated that the CLAPM1 gene mapped to chromosome region 3q28. The gene for the CLAPB1 large subunit was mapped to 17q11.2-q12 by PCR amplification of an AP2 beta fragment from a panel of rodent-human hybrid DNAs. To map the human VCP sequence, a human-specific probe was made by RT-PCR of human mRNA using oligonucleotide primers from conserved regions of the porcine sequence. The amplified human fragment served as probe on Southern blots of hybrid DNAs to determine that the human VCP locus maps to chromosome region 9pter-q34.

    Funded by: NCI NIH HHS: CA51083; NIGMS NIH HHS: GM-28526, GM49217

    Genomics 1995;30;1;94-7

  • Targeting signals and subunit interactions in coated vesicle adaptor complexes.

    Page LJ and Robinson MS

    Department of Clinical Biochemistry, University of Cambridge, Addenbrooke's Hospital, England.

    There are two clathrin-coated vesicle adaptor complexes in the cell, one associated with the plasma membrane and one associated with the TGN. The subunit composition of the plasma membrane adaptor complex is alpha-adaptin, beta-adaptin, AP50, and AP17; while that of the TGN adaptor complex is gamma-adaptin, beta'-adaptin, AP47, and AP19. To search for adaptor targeting signals, we have constructed chimeras between alpha-adaptin and gamma-adaptin within their NH2-terminal domains. We have identified stretches of sequence in the two proteins between amino acids approximately 130 and 330-350 that are essential for targeting. Immunoprecipitation reveals that this region determines whether a construct coassemblies with AP50 and AP17, or with AP47 and AP19. These observations suggest that these other subunits may play an important role in targeting. In contrast, beta- and beta'-adaptins are clearly not involved in this event. Chimeras between the alpha- and gamma-adaptin COOH-terminal domains reveal the presence of a second targeting signal. We have further investigated the interactions between the adaptor subunits using the yeast two-hybrid system. Interactions can be detected between the beta/beta'-adaptins and the alpha/gamma-adaptins, between the beta/beta'-adaptins and the AP50/AP47 subunits, between alpha-adaptin and AP17, and between gamma-adaptin and AP19. These results indicate that the adaptor subunits act in concert to target the complex to the appropriate membrane.

    Funded by: Wellcome Trust

    The Journal of cell biology 1995;131;3;619-30

  • Interaction of tyrosine-based sorting signals with clathrin-associated proteins.

    Ohno H, Stewart J, Fournier MC, Bosshart H, Rhee I, Miyatake S, Saito T, Gallusser A, Kirchhausen T and Bonifacino JS

    Cell Biology and Metabolism Branch, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892, USA.

    Tyrosine-based signals within the cytoplasmic domain of integral membrane proteins mediate clathrin-dependent protein sorting in the endocytic and secretory pathways. A yeast two-hybrid system was used to identify proteins that bind to tyrosine-based signals. The medium chains (mu 1 and mu 2) of two clathrin-associated protein complexes (AP-1 and AP-2, respectively) specifically interacted with tyrosine-based signals of several integral membrane proteins. The interaction was confirmed by in vitro binding assays. Thus, it is likely that the medium chains serve as signal-binding components of the clathrin-dependent sorting machinery.

    Science (New York, N.Y.) 1995;269;5232;1872-5

  • Prediction of the coding sequences of unidentified human genes. III. The coding sequences of 40 new genes (KIAA0081-KIAA0120) deduced by analysis of cDNA clones from human cell line KG-1.

    Nagase T, Miyajima N, Tanaka A, Sazuka T, Seki N, Sato S, Tabata S, Ishikawa K, Kawarabayasi Y, Kotani H et al.

    Kazusa DNA Research Institute, Chiba, Japan.

    We isolated full-length cDNA clones from size-fractionated cDNA libraries of human immature myeloid cell line KG-1, and the coding sequences of 40 genes were newly predicted. A computer search of the GenBank/EMBL databases indicated that the sequences of 14 genes were unrelated to any reported genes, while the remaining 26 genes carried some sequences with similarities to known genes. Significant transmembrane domains were identified in 17 genes, and protein motifs that matched those in the PROSITE motif database were identified in 11 genes. Northern hybridization analysis with 18 different cells and tissues demonstrated that 10 genes were apparently expressed in a cell-specific or tissue-specific manner. Among the genes predicted, half were isolated from the medium-sized cDNA library and the other half from the small-sized cDNA library, and their average sizes were 4 kb and 1.4 kb, respectively. As judged by Northern hybridization profiles, small-sized cDNAs appeared to be expressed more ubiquitously and abundantly in various tissues, compared with that of medium-sized cDNAs.

    DNA research : an international journal for rapid publication of reports on genes and genomes 1995;2;1;37-43

  • Activity and in vitro reassembly of the coated vesicle (H+)-ATPase requires the 50-kDa subunit of the clathrin assembly complex AP-2.

    Liu Q, Feng Y and Forgac M

    Department of Cellular and Molecular Physiology, Tufts University School of Medicine, Boston, Massachusetts 02111.

    We have previously shown that the 50-kDa subunit of the clathrin assembly complex AP-2 (AP50) stoichiometrically binds to and is immunoprecipitated with the vacuolar (H+)-ATPase (V-ATPase) from clathrin-coated vesicles (Myers, M., and Forgac, M. (1993) J. Biol. Chem. 268, 9184-9186). We now report that treatment of stripped coated vesicles with cystine results in a purified V-ATPase complex lacking the AP50 polypeptide. Removal of AP50 can be reversed upon treatment of the vesicles with dithiothreitol. Removal of AP50 reduces the ATPase activity of the purified V-ATPase by 90% relative to the enzyme containing AP50. This inhibition is not reversed upon treatment of the AP50-depleted enzyme with dithiothreitol in the absence of AP50. The reconstituted V-ATPase depleted of AP50 is devoid of ATP-dependent proton transport activity. We observe further that the peripheral V1 subunits are unable to reassemble onto the integral V0 domain in the absence of AP50. The addition of purified AP-2 containing the AP50 polypeptide restores the ability of the V1 subunits to assemble with the V0 sector to give a V-ATPase complex that is functional in ATP-dependent proton transport. These results indicate that the AP50 polypeptide is necessary for both activity and in vitro reassembly of the V-ATPase complex.

    Funded by: NIGMS NIH HHS: GM 34478, R01 GM034478

    The Journal of biological chemistry 1994;269;50;31592-7

  • The 50 kDa protein subunit of assembly polypeptide (AP) AP-2 adaptor from clathrin-coated vesicles is phosphorylated on threonine-156 by AP-1 and a soluble AP50 kinase which co-purifies with the assembly polypeptides.

    Pauloin A and Thurieau C

    INRA, Laboratoire de Biologie Cellulaire et Moléculaire, Centre de Recherche de Jouy, Jouy-en-Josas, France.

    AP50 is a subunit of the assembly polypeptide (AP) subclass AP-2 from bovine brain coated vesicles. It can be phosphorylated in vivo and in vitro on a threonine residue by means of the AP50 kinase activity associated with AP. We have undertaken an analysis of the amino acid sequence around the AP50 phosphorylation site. After phosphorylation in vitro of AP50 followed by tryptic cleavage, only one radioactive peptide was isolated following Mono-Q ion-exchange f.p.l.c. and reverse-phase h.p.l.c. The amino acid sequence of this peptide: Glu146-Glu-Gln-Ser-Gln-Ile-Thr-Ser-Gln-Val-Thr*-Gly-Gly-Ile-Gly-Tr p-Arg162, displayed two threonine residues. Analysis of the yield and radioactivity of the product from automated Edman degradation indicated that only Thr-156 was phosphorylated, reflecting the presence of a single phosphorylation site in AP50. AP phosphorylated the corresponding synthetic peptide on the same threonyl residue. We demonstrated that AP50 was a phosphorylation substrate unable to autophosphorylate. The enzyme involved in the AP50 phosphorylation was shown to be associated with AP-1 and with a soluble protein complex co-purified with APs but resolved from the latter by hydroxyapatite-column exclusion chromatography. This AP50 kinase activity corresponded to a 280 kDa protein complex according to gel-filtration data.

    The Biochemical journal 1993;296 ( Pt 2);409-15

  • Adaptor self-aggregation, adaptor-receptor recognition and binding of alpha-adaptin subunits to the plasma membrane contribute to recruitment of adaptor (AP2) components of clathrin-coated pits.

    Chang MP, Mallet WG, Mostov KE and Brodsky FM

    Department of Pharmacy, School of Pharmacy, University of California, San Francisco 94143-0446.

    Initiation of receptor-mediated endocytosis by nucleation of clathrin-coated pits involves binding of AP2 adaptor molecules to the plasma membrane. This process was reconstituted in vitro, using plasma membrane fragments, prepared by freeze-thaw lysis of cells, and stripped of their endogenous coat proteins, as targets for binding of purified adaptor molecules and their dissociated subunits. The dissociated alpha-adaptin subunit of AP2 bound to plasma membrane fragments, while the dissociated beta-adaptin subunit did not, suggesting that plasma membrane localization of AP2 adaptors is mediated by alpha-adaptin. Membrane binding of intact AP2 adaptor molecules was enhanced by adaptor self-aggregation, which can be modulated by physiological concentrations of inositol phosphates, and may therefore be sensitive to receptor signaling. Adaptor binding was partially inhibited by soluble peptides representing the cytoplasmic domains of the asialoglycoprotein receptor and the polymeric immunoglobulin receptor. These results indicate that direct binding of adaptors to the cytoplasmic domains of receptors contributes to coated pit nucleation but this appears to be a weak interaction, suggesting that an additional recognition signal could be required for high affinity adaptor binding.

    Funded by: NIAID NIH HHS: AI25144; NIGMS NIH HHS: GM26691, GM38093

    The EMBO journal 1993;12;5;2169-80

  • Serine phosphorylation-independent downregulation of cell-surface CD4 by nef.

    Garcia JV and Miller AD

    Program in molecular Medicine, Fred Hutchinson Cancer Research Center, Seattle, Washington 98104.

    A decline in the T-cell population usually marks the onset of progressive immunological disease in individuals infected with the human immunodeficiency virus (HIV). Because CD4+ cells help to coordinate efficient immune responses, some of the defects in the immune function in advanced cases of AIDS may be explained by the disappearance of these cells. Therefore, an understanding of the mechanisms used by HIV to induce the reduction of CD4+ cells is important. Here we use a Moloney murine leukaemia virus-based retroviral vector in order to express the nef gene of HIV-1 in three lymphocytic cell lines expressing CD4. In all cases we find that cell-surface CD4 expression is inversely related to nef expression. However, nef does not alter steady-state levels of CD4 RNA or CD4 protein. Also, nef can downregulate a CD4 triple mutant (Ser----Ala) that is neither phosphorylated nor down-regulated by phorbol esters, indicating that nef is acting by a different mechanism.

    Nature 1991;350;6318;508-11

Gene lists (6)

Gene List Source Species Name Description Gene count
L00000009 G2C Homo sapiens Human PSD Human orthologues of mouse PSD adapted from Collins et al (2006) 1080
L00000016 G2C Homo sapiens Human PSP Human orthologues of mouse PSP adapted from Collins et al (2006) 1121
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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