G2Cdb::Gene report

Gene id
Gene symbol
Homo sapiens
clathrin, heavy chain (Hc)
G00000656 (Mus musculus)

Databases (7)

ENSG00000141367 (Ensembl human gene)
1213 (Entrez Gene)
42 (G2Cdb plasticity & disease)
CLTC (GeneCards)
118955 (OMIM)
Marker Symbol
HGNC:2092 (HGNC)
Protein Sequence
Q00610 (UniProt)

Synonyms (1)

  • Hc

Diseases (2)

Disease Nervous effect Mutations Found Literature Mutations Type Genetic association?
D00000004: Inflammatory myofibroblastic tumour N Y (11485898) Translocation fusion (with another gene) (TF) ?
D00000003: ALK-positive diffuse large B-cell lymphoma N Y (15492998) Translocation fusion (with another gene) (TF) Y
D00000003: ALK-positive diffuse large B-cell lymphoma N Y (15852431) Translocation fusion (with another gene) (TF) Y


  • ALK-positive diffuse large B-cell lymphoma.

    Bubała H, Małdyk J, Włodarska I, Sońta-Jakimczyk D and Szczepański T

    Department of Pediatric Hematology and Oncology, Silesian Medical Academy, Zabrze, Poland.

    Anaplastic lymphoma kinase (ALK) positive diffuse large B-cell lymphoma (DLBCL) is a rare subtype of non-Hodgkins lymphoma. Five such cases have been described in children. We present a 9-year-old boy, in whom diagnosis of DLBCL has been established in addition to congenital multiple enchondromatosis. Immunohistopathological evaluation of tumor biopsy established the final diagnosis of ALK + DLBCL. The clathrin gene (CLTC)-ALK fusion underlying aberrant expression of ALK in the present case was demonstrated by interphase fluorescence in situ hybridization (FISH) using break-apart rearrangement probes for ALK and CLTC. The disease in this patient was highly resistant to applied chemotherapy regimens and to radiotherapy. Analysis of the disease course in our patient and review of other cases reported previously show that ALK + DLBCL can be an aggressive malignancy that can be cured with conventional chemotherapy protocols only at stage of localized disease.

    Pediatric blood & cancer 2006;46;5;649-53

  • ALK-positive diffuse large B-cell lymphoma of the stomach associated with a clathrin-ALK rearrangement.

    McManus DT, Catherwood MA, Carey PD, Cuthbert RJ and Alexander HD

    Department of Tissue Pathology, Belfast City Hospital, Belfast City Hospital Trust, Northern Ireland, United Kingdom.

    ALK-positive diffuse large B-cell lymphoma is a rare, recently characterized lymphoma subtype that shows granular cytoplasmic ALK expression. This report describes a primary gastric ALK-positive B-lineage lymphoma in which a clathrin (CLTC)-ALK fusion was identified by RT-PCR and direct sequencing of the breakpoint. This confirmed the presence of t(2;17)(p23;q23) involving the CLTC gene and is only the 4th report of such a translocation in this lymphoma subtype and the first to describe this tumor within the stomach. As in previous reports, immunophenotyping showed the malignant cell to be a terminally differentiated B-lineage cell characterized by the absence of B-cell antigens and expression of antigens associated with plasma cell differentiation. This case confirms the existence of such a lymphoma subtype arising in extranodal locations and underscores the importance of detailed immunophenotyping and specialized molecular genetic investigations in confirming the diagnosis.

    Human pathology 2004;35;10;1285-8

  • Fusion of the ALK gene to the clathrin heavy chain gene, CLTC, in inflammatory myofibroblastic tumor.

    Bridge JA, Kanamori M, Ma Z, Pickering D, Hill DA, Lydiatt W, Lui MY, Colleoni GW, Antonescu CR, Ladanyi M and Morris SW

    Department of Pathology, Center for Human Molecular Genetics, 983135 University of Nebraska Medical Center, Omaha, NE 68198, USA. jbridge@unmc.edu

    Inflammatory myofibroblastic tumor (IMT) is a rare, but distinctive mesenchymal neoplasm composed of fascicles of bland myofibroblasts admixed with a prominent inflammatory component. Genetic studies of IMTs have demonstrated chromosomal abnormalities of 2p23 and rearrangement of the anaplastic lymphoma kinase (ALK) gene locus. In a subset of IMTs, the ALK C-terminal kinase domain is fused with a tropomyosin N-terminal coiled-coil domain. In the current study, fusion of ALK with the clathrin heavy chain (CTLC) gene localized to 17q23 was detected in two cases of IMT. One of these cases exhibited a 2;17 translocation in addition to other karyotypic anomalies [46,XX,t(2;17)(p23;q23),add(16)(q24)].

    Funded by: NCI NIH HHS: CA21765, CA69129, P30 CA021765, P30 CA036727, P30 CA36727, R01 CA069129

    The American journal of pathology 2001;159;2;411-5

Literature (90)

Pubmed - human_disease

  • ALK-positive diffuse large B-cell lymphoma.

    Bubała H, Małdyk J, Włodarska I, Sońta-Jakimczyk D and Szczepański T

    Department of Pediatric Hematology and Oncology, Silesian Medical Academy, Zabrze, Poland.

    Anaplastic lymphoma kinase (ALK) positive diffuse large B-cell lymphoma (DLBCL) is a rare subtype of non-Hodgkins lymphoma. Five such cases have been described in children. We present a 9-year-old boy, in whom diagnosis of DLBCL has been established in addition to congenital multiple enchondromatosis. Immunohistopathological evaluation of tumor biopsy established the final diagnosis of ALK + DLBCL. The clathrin gene (CLTC)-ALK fusion underlying aberrant expression of ALK in the present case was demonstrated by interphase fluorescence in situ hybridization (FISH) using break-apart rearrangement probes for ALK and CLTC. The disease in this patient was highly resistant to applied chemotherapy regimens and to radiotherapy. Analysis of the disease course in our patient and review of other cases reported previously show that ALK + DLBCL can be an aggressive malignancy that can be cured with conventional chemotherapy protocols only at stage of localized disease.

    Pediatric blood & cancer 2006;46;5;649-53

  • ALK-positive diffuse large B-cell lymphoma of the stomach associated with a clathrin-ALK rearrangement.

    McManus DT, Catherwood MA, Carey PD, Cuthbert RJ and Alexander HD

    Department of Tissue Pathology, Belfast City Hospital, Belfast City Hospital Trust, Northern Ireland, United Kingdom.

    ALK-positive diffuse large B-cell lymphoma is a rare, recently characterized lymphoma subtype that shows granular cytoplasmic ALK expression. This report describes a primary gastric ALK-positive B-lineage lymphoma in which a clathrin (CLTC)-ALK fusion was identified by RT-PCR and direct sequencing of the breakpoint. This confirmed the presence of t(2;17)(p23;q23) involving the CLTC gene and is only the 4th report of such a translocation in this lymphoma subtype and the first to describe this tumor within the stomach. As in previous reports, immunophenotyping showed the malignant cell to be a terminally differentiated B-lineage cell characterized by the absence of B-cell antigens and expression of antigens associated with plasma cell differentiation. This case confirms the existence of such a lymphoma subtype arising in extranodal locations and underscores the importance of detailed immunophenotyping and specialized molecular genetic investigations in confirming the diagnosis.

    Human pathology 2004;35;10;1285-8

  • Fusion of the ALK gene to the clathrin heavy chain gene, CLTC, in inflammatory myofibroblastic tumor.

    Bridge JA, Kanamori M, Ma Z, Pickering D, Hill DA, Lydiatt W, Lui MY, Colleoni GW, Antonescu CR, Ladanyi M and Morris SW

    Department of Pathology, Center for Human Molecular Genetics, 983135 University of Nebraska Medical Center, Omaha, NE 68198, USA. jbridge@unmc.edu

    Inflammatory myofibroblastic tumor (IMT) is a rare, but distinctive mesenchymal neoplasm composed of fascicles of bland myofibroblasts admixed with a prominent inflammatory component. Genetic studies of IMTs have demonstrated chromosomal abnormalities of 2p23 and rearrangement of the anaplastic lymphoma kinase (ALK) gene locus. In a subset of IMTs, the ALK C-terminal kinase domain is fused with a tropomyosin N-terminal coiled-coil domain. In the current study, fusion of ALK with the clathrin heavy chain (CTLC) gene localized to 17q23 was detected in two cases of IMT. One of these cases exhibited a 2;17 translocation in addition to other karyotypic anomalies [46,XX,t(2;17)(p23;q23),add(16)(q24)].

    Funded by: NCI NIH HHS: CA21765, CA69129, P30 CA021765, P30 CA036727, P30 CA36727, R01 CA069129

    The American journal of pathology 2001;159;2;411-5

Pubmed - other

  • Identification of a function-specific mutation of clathrin heavy chain (CHC) required for p53 transactivation.

    Ohata H, Ota N, Shirouzu M, Yokoyama S, Yokota J, Taya Y and Enari M

    Radiobiology Division, National Cancer Center Research Institute, Chuo-ku, Tokyo, Japan.

    The p53 pathway is activated in response to various cellular stresses to protect cells from malignant transformation. We have previously shown that clathrin heavy chain (CHC), which is a cytosolic protein regulating endocytosis, is present in nuclei and binds to p53 to promote p53-mediated transcription. However, details of the binding interface between p53 and CHC remain unclear. Here, we report on the binding mode between p53 and CHC using mutation analyses and a structural model of the interaction generated by molecular dynamics. Structural modeling analyses predict that an Asn1288 residue in CHC is crucial for binding to p53. In fact, substitution of this Asn to Ala of CHC diminished its ability to interact with p53, leading to reduced activity to transactivate p53. Surprisingly, this mutation had little effect on receptor-mediated endocytosis. Thus, the function-specific mutation of CHC will clarify physiological roles of CHC in the regulation of the p53 pathway.

    Journal of molecular biology 2009;394;3;460-71

  • Clathrin-mediated post-Golgi membrane trafficking in the morphogenesis of hepatitis delta virus.

    Huang C, Chang SC, Yang HC, Chien CL and Chang MF

    Institute of Biochemistry and Molecular Biology, Institute of Microbiology, National Taiwan University College of Medicine, Taipei, Taiwan. mfchang@ntu.edu.tw

    Clathrin is involved in the endocytosis and exocytosis of cellular proteins and the process of virus infection. We have previously demonstrated that large hepatitis delta antigen (HDAg-L) functions as a clathrin adaptor, but the detailed mechanisms of clathrin involvement in the morphogenesis of hepatitis delta virus (HDV) are not clear. In this study, we found that clathrin heavy chain (CHC) is a key determinant in the morphogenesis of HDV. HDAg-L with a single amino acid substitution at the clathrin box retained nuclear export activity but failed to interact with CHC and to assemble into virus-like particles. Downregulation of CHC function by a dominant-negative mutant or by short hairpin RNA reduced the efficiency of HDV assembly, but not the secretion of hepatitis B virus subviral particles. In addition, the coexistence of a cell-permeable peptide derived from the C terminus of HDAg-L significantly interfered with the intracellular transport of HDAg-L. HDAg-L, small HBsAg, and CHC were found to colocalize with the trans-Golgi network and were highly enriched on clathrin-coated vesicles. Furthermore, genotype II HDV, which assembles less efficiently than genotype I HDV does, has a putative clathrin box in its HDAg-L but interacted only weakly with CHC. The assembly efficiency of the various HDV genotypes correlates well with the CHC-binding activity of their HDAg-Ls and coincides with the severity of disease outcome. Thus, the clathrin box and the nuclear export signal at the C terminus of HDAg-L are potential new molecular targets for HDV therapy.

    Journal of virology 2009;83;23;12314-24

  • GAK, a regulator of clathrin-mediated membrane traffic, also controls centrosome integrity and chromosome congression.

    Shimizu H, Nagamori I, Yabuta N and Nojima H

    Department of Molecular Genetics, Research Institute for Microbial Diseases, Osaka University, Suita City, Osaka, Japan.

    Cyclin G-associated kinase (GAK) is an association partner of clathrin heavy chain (CHC) and is essential for clathrin-mediated membrane trafficking. Here, we report two novel functions of GAK: maintenance of proper centrosome maturation and of mitotic chromosome congression. Indeed, GAK knockdown by siRNA caused cell-cycle arrest at metaphase, which indicates that GAK is required for proper mitotic progression. We found that this impaired mitotic progression was due to activation of the spindle-assembly checkpoint, which senses protruded, misaligned or abnormally condensed chromosomes in GAK-siRNA-treated cells. GAK knockdown also caused multi-aster formation, which was due to abnormal fragmentation of pericentriolar material, but not of the centrioles. Moreover, GAK and CHC cooperated in the same pathway and interacted in mitosis to regulate the formation of a functional spindle. Taken together, we conclude that GAK and clathrin function cooperatively not only in endocytosis, but also in mitotic progression.

    Journal of cell science 2009;122;Pt 17;3145-52

  • Functional equivalence of the clathrin heavy chains CHC17 and CHC22 in endocytosis and mitosis.

    Hood FE and Royle SJ

    The Physiological Laboratory, School of Biomedical Sciences, University of Liverpool, Liverpool L69 3BX, UK.

    Clathrin is crucial for endocytosis and plays a recently described role in mitosis. Two clathrin heavy chains (CHCs) are found in humans: the ubiquitous CHC17, and CHC22, a CHC that is enriched in skeletal muscle. Functional differences have been proposed for these clathrins despite high sequence similarity. Here, we compared each paralogue in functional assays of endocytosis and mitosis. We find that CHC17 and CHC22 are functionally equivalent. We also describe how previous work on CHC22 has involved a splice variant that is not usually expressed in cells.

    Funded by: Cancer Research UK: A8722

    Journal of cell science 2009;122;Pt 13;2185-90

  • Characterization of clathrin and Syk interaction upon Shiga toxin binding.

    Wälchli S, Aasheim HC, Skånland SS, Spilsberg B, Torgersen ML, Rosendal KR and Sandvig K

    Department of Biochemistry, Institute for Cancer Research, Faculty Division: The Norwegian Radium Hospital, Montebello, Oslo, Norway. sebastien.walchli@rr-research.no

    Shiga toxin (Stx) is a bacterial toxin that binds to its receptor Gb3 at the plasma membrane. It is taken up by endocytosis and transported retrogradely via the Golgi apparatus to the endoplasmic reticulum. The toxin is then translocated to the cytosol where it exerts its toxic effect. We have previously shown that phosphorylation of clathrin heavy chain (CHC) is an early event following Stx binding to HeLa cells, and that this requires the activity of the tyrosine kinase Syk. Here, we have investigated this event in more detail in the B lymphoid cell line Ramos, which expresses high endogenous levels of both Syk and Gb3. We report that efficient endocytosis of Stx in Ramos cells requires Syk activity and that Syk is recruited to the uptake site of Stx. Furthermore, in response to Stx treatment, CHC and Syk were rapidly phosphorylated in a Src family kinase dependent manner at Y1477 and Y352, respectively. We show that these phosphorylated residues act as binding sites for the direct interaction between Syk and CHC. Interestingly, Syk-CHC complex formation could be induced by both Stx and B cell receptor stimulation.

    Cellular signalling 2009;21;7;1161-8

  • SNX4 in complex with clathrin and dynein: implications for endosome movement.

    Skånland SS, Wälchli S, Brech A and Sandvig K

    Centre for Cancer Biomedicine, Faculty Division Norwegian Radium Hospital, University of Oslo, Oslo, Norway.

    Background: Sorting nexins (SNXs) constitute a family of proteins classified by their phosphatidylinositol (PI) binding Phox homology (PX) domain. Some members regulate intracellular trafficking. We have here investigated mechanisms underlying SNX4 mediated endosome to Golgi transport.

    We show that SNX4 forms complexes with clathrin and dynein. The interactions were inhibited by wortmannin, a PI3-kinase inhibitor, suggesting that they form when SNX4 is associated with PI(3)P on endosomes. We further localized the clathrin interacting site on SNX4 to a clathrin box variant. A short peptide containing this motif was sufficient to pull down both clathrin and dynein. Knockdown studies demonstrated that clathrin is not required for the SNX4/dynein interaction. Moreover, clathrin knockdown led to increased Golgi transport of the toxin ricin, as well as redistribution of endosomes.

    We discuss the possibility of clathrin serving as a regulator of SNX4-dependent transport. Upon clathrin release, dynein may bind SNX4 and mediate retrograde movement.

    PloS one 2009;4;6;e5935

  • Double-membrane gap junction internalization requires the clathrin-mediated endocytic machinery.

    Gumpert AM, Varco JS, Baker SM, Piehl M and Falk MM

    Department of Biological Sciences, Lehigh University, 111 Research Drive, Bethlehem, PA 18015, USA.

    Direct cell-cell communication mediated by plasma membrane-spanning gap junction (GJ) channels is vital to all aspects of cellular life. Obviously, GJ intercellular communication (GJIC) requires precise regulation, and it is known that controlled biosynthesis and degradation, and channel opening and closing (gating) are exploited. We discovered that cells internalize GJs in response to various stimuli. Here, we report that GJ internalization is a clathrin-mediated endocytic process that utilizes the vesicle-coat protein clathrin, the adaptor proteins adaptor protein complex 2 and disabled 2, and the GTPase dynamin. To our knowledge, we are first to report that the endocytic clathrin machinery can internalize double-membrane vesicles into cells.

    Funded by: NIGMS NIH HHS: GM 55725, R01 GM055725, R01 GM055725-06A2, R01 GM055725-07, R01 GM055725-08

    FEBS letters 2008;582;19;2887-92

  • Monomeric but not trimeric clathrin heavy chain regulates p53-mediated transcription.

    Ohmori K, Endo Y, Yoshida Y, Ohata H, Taya Y and Enari M

    Radiobiology Division, National Cancer Center Research Institute, Chuo-ku, Tokyo, Japan.

    Tumor suppressor p53 protein is the transcription factor responsible for various genes including DNA repair, growth arrest, apoptosis and antiangiogenesis. Recently, we showed that clathrin heavy chain (CHC), which was originally identified as a cytosolic protein regulating endocytosis, is present in nuclei and functions as a coactivator for p53. Here, we determined the detailed p53-binding site of CHC and a CHC deletion mutant containing this region (CHC833-1406) behaved as a monomer in cells. Monomeric CHC833-1406 still had a higher ability to transactivate p53 than wild-type CHC although this CHC mutant no longer had endocytic function. Moreover, similar to wild-type CHC, monomeric CHC enhances p53-mediated transcription through the recruitment of histone acetyltransferase p300. Immunofluorescent microscopic analysis exhibited that CHC833-1406 is predominantly localized in nuclei, suggesting that there may be a certain regulatory domain for nuclear export in the C-terminus of CHC. Thus, the trimerization domain of CHC is not necessary for the transactivation of p53 target genes and these data provide further evidence that nuclear CHC plays a role distinct from clathrin-mediated endocytosis.

    Oncogene 2008;27;15;2215-27

  • Regulation of clathrin-mediated endocytosis by p53.

    Endo Y, Sugiyama A, Li SA, Ohmori K, Ohata H, Yoshida Y, Shibuya M, Takei K, Enari M and Taya Y

    Radiobiology Division, National Cancer Center Research Institute, Tokyo, Japan.

    The p53 gene encodes a multi-functional protein to prevent tumorigenesis. Although there have been many reports of the nuclear functions of p53, little is known about the cytosolic functions of p53. Here, we found that p53 is present in cytosol as well as nuclei under unstressed conditions and binds to clathrin heavy chain (CHC). CHC is known to play a role in receptor-mediated endocytosis. Based on our findings, we examined the effect of p53 on clathrin-mediated endocytosis of epidermal growth factor receptor (EGFR). Surprisingly, p53 co-localized with CHC at the plasma membrane in response to EGF stimulation. In cells with ablated p53 expression by RNAi, EGFR internalization was delayed and intracellular signaling from EGFR was altered. Thus, our findings provide evidence that cytosolic p53 may participate in the regulation of clathrin-mediated endocytosis to control the correct signaling from EGFR.

    Genes to cells : devoted to molecular & cellular mechanisms 2008;13;4;375-86

  • Clathrin-dependent trafficking of subtilase cytotoxin, a novel AB5 toxin that targets the endoplasmic reticulum chaperone BiP.

    Chong DC, Paton JC, Thorpe CM and Paton AW

    School of Molecular and Biomedical Science, University of Adelaide, SA 5005, Australia.

    Subtilase cytotoxin (SubAB) is the prototype of a new family of AB5 cytotoxins produced by Shiga toxigenic Escherichia coli. Its cytotoxic activity is due to its capacity to enter cells and specifically cleave the endoplasmic reticulum (ER) chaperone BiP. However, its trafficking within target cells has not been investigated previously. In Vero cells, fluorescence colocalization with subcellular markers established that SubAB is trafficked from the cell surface to the ER via a retrograde pathway similar, but not identical, to those of Shiga toxin (Stx) and cholera toxin (Ctx), with their pathways converging at the Golgi. The clathrin inhibitor phenylarsine oxide prevented SubAB entry and BiP cleavage in SubAB-treated Vero, HeLa and N2A cells, while cholesterol depletion did not, demonstrating that, unlike either Stx or Ctx, SubAB internalization is exclusively clathrin-dependent.

    Funded by: NIAID NIH HHS: R01AI-068715, R01AI-59509; NIDDK NIH HHS: P30DK-34928

    Cellular microbiology 2008;10;3;795-806

  • 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

  • The retromer complex and clathrin define an early endosomal retrograde exit site.

    Popoff V, Mardones GA, Tenza D, Rojas R, Lamaze C, Bonifacino JS, Raposo G and Johannes L

    Laboratoire Trafic et Signalisation, UMR144 Curie/CNRS, Institut Curie, 26 rue d'Ulm, 75248 Paris Cedex 05, France.

    Previous studies have indicated a role for clathrin, the clathrin adaptors AP1 and epsinR, and the retromer complex in retrograde sorting from early/recycling endosomes to the trans Golgi network (TGN). However, it has remained unclear whether these protein machineries function on the same or parallel pathways. We show here that clathrin and the retromer subunit Vps26 colocalize at the ultrastructural level on early/recycling endosomes containing Shiga toxin B-subunit, a well-studied retrograde transport cargo. As previously described for clathrin, we find that interfering with Vps26 expression inhibits retrograde transport of the Shiga toxin B-subunit to the TGN. Under these conditions, endosomal tubules that take the Shiga toxin B-subunit out of transferrin-containing early/recycling endosomes appear to be stabilized. This situation differs from that previously described for low-temperature incubation and clathrin-depletion conditions under which Shiga toxin B-subunit labeling was found to overlap with that of the transferrin receptor. In addition, we find that the Shiga toxin B-subunit and the transferrin receptor accumulate close to multivesicular endosomes in clathrin-depleted cells, suggesting that clathrin initiates retrograde sorting on vacuolar early endosomes, and that retromer is then required to process retrograde tubules. Our findings thus establish a role for the retromer complex in retrograde transport of the B-subunit of Shiga toxin, and strongly suggest that clathrin and retromer function in consecutive retrograde sorting steps on early endosomes.

    Journal of cell science 2007;120;Pt 12;2022-31

  • Synaptotagmin I binds intestinal epithelial NHE3 and mediates cAMP- and Ca2+-induced endocytosis by recruitment of AP2 and clathrin.

    Musch MW, Arvans DL, Walsh-Reitz MM, Uchiyama K, Fukuda M and Chang EB

    Dept. of Medicine, MC 6084, The Univ. of Chicago Hospitals, 5841 S. Maryland Ave., Chicago, IL 60637, USA.

    Apical membrane sodium hydrogen exchanger 3 (NHE3), a major pathway for non-nutrient-dependent intestinal Na(+) absorption, is tightly regulated by second messenger systems that affect its functional activity and membrane trafficking. However, the events and components involved in NHE3 regulation are only partially understood. We report that the adaptor protein synaptotagmin I (Syt I) plays a pivotal role in cAMP- and Ca(2+)-induced cargo recognition of NHE3 and initiation of its endocytosis. Both mouse small intestine (jejunum) and Caco-2BBe Syt I coimmunoprecipitated with NHE3, particularly following increases in cellular cAMP or Ca(2+). Following short interfering RNA (siRNA) suppression of Syt I expression, cAMP- and Ca(2+)-induced inhibition of NHE3 activity were still observed but NHE3 endocytosis was blocked, as assessed by (22)Na influx and apical membrane biotin labeling, respectively. Similar effects on NHE3 inhibition and endocytosis were observed by siRNA suppression of either the mu-subunit of the adaptor protein 2 (AP2) complex or the heavy chain of clathrin. Coimmunoprecipitation analyses of NHE3 with these adaptor proteins revealed that cAMP- and Ca(2+)-induced NHE3-Syt I interaction preceded and was required for recruitment of AP2 and the clathrin complex. Confocal microscopy confirmed both the time sequence and protein associations of these events. We conclude that Syt I plays a pivotal role in mediating cAMP- and Ca(2+)-induced endocytosis of NHE3 (but not in inhibition of activity) through cargo recognition of NHE3 and subsequent recruitment of AP2-clathrin assembly required for membrane endocytosis.

    Funded by: NIDDK NIH HHS: DK-38510, DK-42086, DK-47722

    American journal of physiology. Gastrointestinal and liver physiology 2007;292;6;G1549-58

  • 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

  • Large-scale mapping of human protein-protein interactions by mass spectrometry.

    Ewing RM, Chu P, Elisma F, Li H, Taylor P, Climie S, McBroom-Cerajewski L, Robinson MD, O'Connor L, Li M, Taylor R, Dharsee M, Ho Y, Heilbut A, Moore L, Zhang S, Ornatsky O, Bukhman YV, Ethier M, Sheng Y, Vasilescu J, Abu-Farha M, Lambert JP, Duewel HS, Stewart II, Kuehl B, Hogue K, Colwill K, Gladwish K, Muskat B, Kinach R, Adams SL, Moran MF, Morin GB, Topaloglou T and Figeys D

    Protana, Toronto, Ontario, Canada.

    Mapping protein-protein interactions is an invaluable tool for understanding protein function. Here, we report the first large-scale study of protein-protein interactions in human cells using a mass spectrometry-based approach. The study maps protein interactions for 338 bait proteins that were selected based on known or suspected disease and functional associations. Large-scale immunoprecipitation of Flag-tagged versions of these proteins followed by LC-ESI-MS/MS analysis resulted in the identification of 24,540 potential protein interactions. False positives and redundant hits were filtered out using empirical criteria and a calculated interaction confidence score, producing a data set of 6463 interactions between 2235 distinct proteins. This data set was further cross-validated using previously published and predicted human protein interactions. In-depth mining of the data set shows that it represents a valuable source of novel protein-protein interactions with relevance to human diseases. In addition, via our preliminary analysis, we report many novel protein interactions and pathway associations.

    Molecular systems biology 2007;3;89

  • Proteomic and bioinformatic characterization of the biogenesis and function of melanosomes.

    Chi A, Valencia JC, Hu ZZ, Watabe H, Yamaguchi H, Mangini NJ, Huang H, Canfield VA, Cheng KC, Yang F, Abe R, Yamagishi S, Shabanowitz J, Hearing VJ, Wu C, Appella E and Hunt DF

    Department of Chemistry, University of Virginia, Charlottesville, Virginia 22904, USA.

    Melanin, which is responsible for virtually all visible skin, hair, and eye pigmentation in humans, is synthesized, deposited, and distributed in subcellular organelles termed melanosomes. A comprehensive determination of the protein composition of this organelle has been obstructed by the melanin present. Here, we report a novel method of removing melanin that includes in-solution digestion and immobilized metal affinity chromatography (IMAC). Together with in-gel digestion, this method has allowed us to characterize melanosome proteomes at various developmental stages by tandem mass spectrometry. Comparative profiling and functional characterization of the melanosome proteomes identified approximately 1500 proteins in melanosomes of all stages, with approximately 600 in any given stage. These proteins include 16 homologous to mouse coat color genes and many associated with human pigmentary diseases. Approximately 100 proteins shared by melanosomes from pigmented and nonpigmented melanocytes define the essential melanosome proteome. Proteins validated by confirming their intracellular localization include PEDF (pigment-epithelium derived factor) and SLC24A5 (sodium/potassium/calcium exchanger 5, NCKX5). The sharing of proteins between melanosomes and other lysosome-related organelles suggests a common evolutionary origin. This work represents a model for the study of the biogenesis of lysosome-related organelles.

    Funded by: NCRR NIH HHS: RR01744; NHGRI NIH HHS: U01-HG02712; NICHD NIH HHS: HD40179; NIGMS NIH HHS: GM 37537

    Journal of proteome research 2006;5;11;3135-44

  • Role of the AP2 beta-appendage hub in recruiting partners for clathrin-coated vesicle assembly.

    Schmid EM, Ford MG, Burtey A, Praefcke GJ, Peak-Chew SY, Mills IG, Benmerah A and McMahon HT

    Medical Research Council Laboratory of Molecular Biology, Cambridge, United Kingdom.

    Adaptor protein complex 2 alpha and beta-appendage domains act as hubs for the assembly of accessory protein networks involved in clathrin-coated vesicle formation. We identify a large repertoire of beta-appendage interactors by mass spectrometry. These interact with two distinct ligand interaction sites on the beta-appendage (the "top" and "side" sites) that bind motifs distinct from those previously identified on the alpha-appendage. We solved the structure of the beta-appendage with a peptide from the accessory protein Eps15 bound to the side site and with a peptide from the accessory cargo adaptor beta-arrestin bound to the top site. We show that accessory proteins can bind simultaneously to multiple appendages, allowing these to cooperate in enhancing ligand avidities that appear to be irreversible in vitro. We now propose that clathrin, which interacts with the beta-appendage, achieves ligand displacement in vivo by self-polymerisation as the coated pit matures. This changes the interaction environment from liquid-phase, affinity-driven interactions, to interactions driven by solid-phase stability ("matricity"). Accessory proteins that interact solely with the appendages are thereby displaced to areas of the coated pit where clathrin has not yet polymerised. However, proteins such as beta-arrestin (non-visual arrestin) and autosomal recessive hypercholesterolemia protein, which have direct clathrin interactions, will remain in the coated pits with their interacting receptors.

    Funded by: Medical Research Council: G0100100, MC_U105178795

    PLoS biology 2006;4;9;e262

  • Requirement of clathrin heavy chain for p53-mediated transcription.

    Enari M, Ohmori K, Kitabayashi I and Taya Y

    Radiobiology Division, Chuo-ku, Tokyo 104-0045, Japan.

    The p53 protein is a transcription factor that activates various genes responsible for growth arrest and/or apoptosis in response to DNA damage. Here, we report that clathrin heavy chain (CHC) binds to p53 and contributes to p53-mediated transcription. CHC is known to be a cytosolic protein that functions as a vesicle transporter. We found, however, that CHC exists not only in cytosol but also in nuclei. CHC expression enhances p53-dependent transactivation, whereas the reduction of CHC expression by RNA interference (RNAi) attenuates its transcriptional activity. Moreover, CHC binds to the p53-responsive promoter in vivo and stabilizes p53-p300 interaction to promote p53-mediated transcription. Thus, nuclear CHC is required for the transactivation of p53 target genes and plays a distinct role from clathrin-mediated endocytosis.

    Genes & development 2006;20;9;1087-99

  • Differential control of clathrin subunit dynamics measured with EW-FRAP microscopy.

    Loerke D, Wienisch M, Kochubey O and Klingauf J

    Department of Membrane Biophysics, Max-Planck Institute for Biophysical Chemistry, Am Fassberg 11, D-37077 Goettingen, Germany.

    The clathrin triskelion is composed of three light chain (LC) and three heavy chain (HC) subunits. Cellular control of clathrin function is thought to be aimed at the LC subunit, mainly on the basis of structural information. To test this hypothesis in vivo, we used evanescent-wave photobleaching recovery to study clathrin exchange from single pits using LC (LCa and LCb) and HC enhanced green fluorescent protein fusion constructs. The recovery signal was corrected for cytosolic diffusional background, yielding the pure exchange reaction times. For LCa, we measured an unbinding time constant tau(LEa) = 18.9 +/- 1.0 seconds at room temperature, faster than previously published; for LCb, we found tau(LCb) = 10.6 +/- 1.9 seconds and for HC tau(HC) = 15.9 +/- 1.0 seconds. Sucrose treatment, ATP or Ca(2+) depletion blocked exchange of LCa completely, but only partially of HC, lowering its time constant to tau = 10.0 +/- 0.9 seconds, identical to the one for LCb exchange. The latter was also not blocked by Ca(2+) depletion or sucrose. We conclude that HCs bound both to LCa and to LCb contribute side by side to pit formation in vivo, but the affinity of LCa-free HC in pits is reduced, and the Ca(2+)- and ATP-mediated control of clathrin function is lost.

    Traffic (Copenhagen, Denmark) 2005;6;10;918-29

  • A human protein-protein interaction network: a resource for annotating the proteome.

    Stelzl U, Worm U, Lalowski M, Haenig C, Brembeck FH, Goehler H, Stroedicke M, Zenkner M, Schoenherr A, Koeppen S, Timm J, Mintzlaff S, Abraham C, Bock N, Kietzmann S, Goedde A, Toksöz E, Droege A, Krobitsch S, Korn B, Birchmeier W, Lehrach H and Wanker EE

    Max Delbrueck Center for Molecular Medicine, 13092 Berlin-Buch, Germany.

    Protein-protein interaction maps provide a valuable framework for a better understanding of the functional organization of the proteome. To detect interacting pairs of human proteins systematically, a protein matrix of 4456 baits and 5632 preys was screened by automated yeast two-hybrid (Y2H) interaction mating. We identified 3186 mostly novel interactions among 1705 proteins, resulting in a large, highly connected network. Independent pull-down and co-immunoprecipitation assays validated the overall quality of the Y2H interactions. Using topological and GO criteria, a scoring system was developed to define 911 high-confidence interactions among 401 proteins. Furthermore, the network was searched for interactions linking uncharacterized gene products and human disease proteins to regulatory cellular pathways. Two novel Axin-1 interactions were validated experimentally, characterizing ANP32A and CRMP1 as modulators of Wnt signaling. Systematic human protein interaction screens can lead to a more comprehensive understanding of protein function and cellular processes.

    Cell 2005;122;6;957-68

  • Lowe syndrome protein OCRL1 interacts with clathrin and regulates protein trafficking between endosomes and the trans-Golgi network.

    Choudhury R, Diao A, Zhang F, Eisenberg E, Saint-Pol A, Williams C, Konstantakopoulos A, Lucocq J, Johannes L, Rabouille C, Greene LE and Lowe M

    Faculty of Life Sciences, University of Manchester, Manchester M13 9PT, United Kingdom.

    Oculocerebrorenal syndrome of Lowe is caused by mutation of OCRL1, a phosphatidylinositol 4,5-bisphosphate 5-phosphatase localized at the Golgi apparatus. The cellular role of OCRL1 is unknown, and consequently the mechanism by which loss of OCRL1 function leads to disease is ill defined. Here, we show that OCRL1 is associated with clathrin-coated transport intermediates operating between the trans-Golgi network (TGN) and endosomes. OCRL1 interacts directly with clathrin heavy chain and promotes clathrin assembly in vitro. Interaction with clathrin is not, however, required for membrane association of OCRL1. Overexpression of OCRL1 results in redistribution of clathrin and the cation-independent mannose 6-phosphate receptor (CI-MPR) to enlarged endosomal structures that are defective in retrograde trafficking to the TGN. Depletion of cellular OCRL1 also causes partial redistribution of a CI-MPR reporter to early endosomes. These findings suggest a role for OCRL1 in clathrin-mediated trafficking of proteins from endosomes to the TGN and that defects in this pathway might contribute to the Lowe syndrome phenotype.

    Molecular biology of the cell 2005;16;8;3467-79

  • Huntingtin-interacting protein 1 (Hip1) and Hip1-related protein (Hip1R) bind the conserved sequence of clathrin light chains and thereby influence clathrin assembly in vitro and actin distribution in vivo.

    Chen CY and Brodsky FM

    G. W. Hooper Foundation, Department of Biopharmaceutical Sciences, University of California, San Francisco, CA 94143-0552, USA.

    Clathrin heavy and light chains form triskelia, which assemble into polyhedral coats of membrane vesicles that mediate transport for endocytosis and organelle biogenesis. Light chain subunits regulate clathrin assembly in vitro by suppressing spontaneous self-assembly of the heavy chains. The residues that play this regulatory role are at the N terminus of a conserved 22-amino acid sequence that is shared by all vertebrate light chains. Here we show that these regulatory residues and others in the conserved sequence mediate light chain interaction with Hip1 and Hip1R. These related proteins were previously found to be enriched in clathrin-coated vesicles and to promote clathrin assembly in vitro. We demonstrate Hip1R binding preference for light chains associated with clathrin heavy chain and show that Hip1R stimulation of clathrin assembly in vitro is blocked by mutations in the conserved sequence of light chains that abolish interaction with Hip1 and Hip1R. In vivo overexpression of a fragment of clathrin light chain comprising the Hip1R-binding region affected cellular actin distribution. Together these results suggest that the roles of Hip1 and Hip1R in affecting clathrin assembly and actin distribution are mediated by their interaction with the conserved sequence of clathrin light chains.

    Funded by: NCI NIH HHS: CA09043; NIGMS NIH HHS: GM38093

    The Journal of biological chemistry 2005;280;7;6109-17

  • 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

  • A protein interaction network links GIT1, an enhancer of huntingtin aggregation, to Huntington's disease.

    Goehler H, Lalowski M, Stelzl U, Waelter S, Stroedicke M, Worm U, Droege A, Lindenberg KS, Knoblich M, Haenig C, Herbst M, Suopanki J, Scherzinger E, Abraham C, Bauer B, Hasenbank R, Fritzsche A, Ludewig AH, Büssow K, Buessow K, Coleman SH, Gutekunst CA, Landwehrmeyer BG, Lehrach H and Wanker EE

    Max-Delbrueck-Center for Molecular Medicine, 13125 Berlin-Buch, Germany.

    Analysis of protein-protein interactions (PPIs) is a valuable approach for characterizing proteins of unknown function. Here, we have developed a strategy combining library and matrix yeast two-hybrid screens to generate a highly connected PPI network for Huntington's disease (HD). The network contains 186 PPIs among 35 bait and 51 prey proteins. It revealed 165 new potential interactions, 32 of which were confirmed by independent binding experiments. The network also permitted the functional annotation of 16 uncharacterized proteins and facilitated the discovery of GIT1, a G protein-coupled receptor kinase-interacting protein, which enhances huntingtin aggregation by recruitment of the protein into membranous vesicles. Coimmunoprecipitations and immunofluorescence studies revealed that GIT1 and huntingtin associate in mammalian cells under physiological conditions. Moreover, GIT1 localizes to neuronal inclusions, and is selectively cleaved in HD brains, indicating that its distribution and function is altered during disease pathogenesis.

    Funded by: NINDS NIH HHS: NS31862

    Molecular cell 2004;15;6;853-65

  • Proteomic, functional, and domain-based analysis of in vivo 14-3-3 binding proteins involved in cytoskeletal regulation and cellular organization.

    Jin J, Smith FD, Stark C, Wells CD, Fawcett JP, Kulkarni S, Metalnikov P, O'Donnell P, Taylor P, Taylor L, Zougman A, Woodgett JR, Langeberg LK, Scott JD and Pawson T

    Samuel Lunenfeld Research Institute, Mount Sinai Hospital, 600 University Avenue, Toronto, Ontario M5G 1X5, Canada.

    Background: 14-3-3 proteins are abundant and conserved polypeptides that mediate the cellular effects of basophilic protein kinases through their ability to bind specific peptide motifs phosphorylated on serine or threonine.

    Results: We have used mass spectrometry to analyze proteins that associate with 14-3-3 isoforms in HEK293 cells. This identified 170 unique 14-3-3-associated proteins, which show only modest overlap with previous 14-3-3 binding partners isolated by affinity chromatography. To explore this large set of proteins, we developed a domain-based hierarchical clustering technique that distinguishes structurally and functionally related subsets of 14-3-3 target proteins. This analysis revealed a large group of 14-3-3 binding partners that regulate cytoskeletal architecture. Inhibition of 14-3-3 phosphoprotein recognition in vivo indicates the general importance of such interactions in cellular morphology and membrane dynamics. Using tandem proteomic and biochemical approaches, we identify a phospho-dependent 14-3-3 binding site on the A kinase anchoring protein (AKAP)-Lbc, a guanine nucleotide exchange factor (GEF) for the Rho GTPase. 14-3-3 binding to AKAP-Lbc, induced by PKA, suppresses Rho activation in vivo.

    Conclusion: 14-3-3 proteins can potentially engage around 0.6% of the human proteome. Domain-based clustering has identified specific subsets of 14-3-3 targets, including numerous proteins involved in the dynamic control of cell architecture. This notion has been validated by the broad inhibition of 14-3-3 phosphorylation-dependent binding in vivo and by the specific analysis of AKAP-Lbc, a RhoGEF that is controlled by its interaction with 14-3-3.

    Funded by: NIDDK NIH HHS: DK44239

    Current biology : CB 2004;14;16;1436-50

  • Large-scale characterization of HeLa cell nuclear phosphoproteins.

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

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

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

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

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

  • Clathrin isoform CHC22, a component of neuromuscular and myotendinous junctions, binds sorting nexin 5 and has increased expression during myogenesis and muscle regeneration.

    Towler MC, Gleeson PA, Hoshino S, Rahkila P, Manalo V, Ohkoshi N, Ordahl C, Parton RG and Brodsky FM

    The G.W. Hooper Foundation, Department of Microbiology and Immunology and Department of Biopharmaceutical Sciences, University of California, San Francisco, California 94143-0552, USA.

    The muscle isoform of clathrin heavy chain, CHC22, has 85% sequence identity to the ubiquitously expressed CHC17, yet its expression pattern and function appear to be distinct from those of well-characterized clathrin-coated vesicles. In mature muscle CHC22 is preferentially concentrated at neuromuscular and myotendinous junctions, suggesting a role at sarcolemmal contacts with extracellular matrix. During myoblast differentiation, CHC22 expression is increased, initially localized with desmin and nestin and then preferentially segregated to the poles of fused myoblasts. CHC22 expression is also increased in regenerating muscle fibers with the same time course as embryonic myosin, indicating a role in muscle repair. CHC22 binds to sorting nexin 5 through a coiled-coil domain present in both partners, which is absent in CHC17 and coincides with the region on CHC17 that binds the regulatory light-chain subunit. These differential binding data suggest a mechanism for the distinct functions of CHC22 relative to CHC17 in membrane traffic during muscle development, repair, and at neuromuscular and myotendinous junctions.

    Funded by: NIAMS NIH HHS: R01 AR044483, R01 AR44483; NIGMS NIH HHS: GM038093, GM057657, R01 GM038093

    Molecular biology of the cell 2004;15;7;3181-95

  • Analysis of clathrin-mediated endocytosis of epidermal growth factor receptor by RNA interference.

    Huang F, Khvorova A, Marshall W and Sorkin A

    Department of Pharmacology, University of Colorado Health Sciences Center, Denver, Colorado 80111, USA.

    To identify proteins that participate in clathrin-mediated endocytosis of the epidermal growth factor receptor (EGFR), 13 endocytic proteins were depleted in HeLa cells using highly efficient small interfering RNAs that were designed using a novel selection algorithm. The effects of small interfering RNAs on the ligand-induced endocytosis of EGFR were compared with those effects on the constitutive internalization of the transferrin receptor. The knock-downs of clathrin heavy chain and dynamin produced maximal inhibitory effects on the internalization of both receptors. Depletion of alpha, beta2, or micro2 subunits of AP-2 reduced EGF and transferrin internalization rates by 40-60%. Down-regulation of several accessory proteins individually had no effect on endocytosis but caused significant inhibition of EGF and transferrin endocytosis when the homologous proteins were depleted simultaneously. Surprisingly, knockdown of clathrin-assembly lymphoid myeloid leukemia protein, CALM, did not influence transferrin endocytosis but considerably affected EGFR internalization. Thus, CALM is the second protein besides Grb2 that appears to play a specific role in EGFR endocytosis. This study demonstrates that the efficient gene silencing by rationally designed small interfering RNA can be used as an approach to functionally analyze the entire cellular machineries, such as the clathrin-coated pits and vesicles.

    The Journal of biological chemistry 2004;279;16;16657-61

  • T cell receptor engagement leads to phosphorylation of clathrin heavy chain during receptor internalization.

    Crotzer VL, Mabardy AS, Weiss A and Brodsky FM

    The GW Hooper Foundation, Department of Microbiology, University of California San Francisco, 94143, USA.

    T cell receptor (TCR) internalization by clathrin-coated vesicles after encounter with antigen has been implicated in the regulation of T cell responses. We demonstrate that TCR internalization after receptor engagement and TCR signaling involves inducible phosphorylation of clathrin heavy chain (CHC) in both CD4+ and CD8+ human T cells. Studies with mutant Jurkat T cells implicate the Src family kinase Lck as the responsible enzyme and its activity in this process is influenced by the functional integrity of the downstream signaling molecule ZAP-70. CHC phosphorylation positively correlates with ligand-induced TCR internalization in both CD4+ and CD8+ T cells, and CHC phosphorylation as a result of basal Lck activity is also implicated in constitutive TCR endocytosis by CD4+ T cells. Remarkably, irreversible CHC phosphorylation in the presence of pervanadate reduced both constitutive and ligand-induced TCR internalization in CD4+ T cells, and immunofluorescence studies revealed that this inhibition affected the early stages of TCR endocytosis from the plasma membrane. Thus, we propose that CHC phosphorylation and dephosphorylation are involved in TCR internalization and that this is a regulatory mechanism linking TCR signaling to endocytosis.

    Funded by: NIAID NIH HHS: AI45865; NIGMS NIH HHS: GM38093, R01 GM038093

    The Journal of experimental medicine 2004;199;7;981-91

  • A physical and functional map of the human TNF-alpha/NF-kappa B signal transduction pathway.

    Bouwmeester T, Bauch A, Ruffner H, Angrand PO, Bergamini G, Croughton K, Cruciat C, Eberhard D, Gagneur J, Ghidelli S, Hopf C, Huhse B, Mangano R, Michon AM, Schirle M, Schlegl J, Schwab M, Stein MA, Bauer A, Casari G, Drewes G, Gavin AC, Jackson DB, Joberty G, Neubauer G, Rick J, Kuster B and Superti-Furga G

    Cellzome AG, Meyerhofstrasse 1, 69117 Heidelberg, Germany. tewis.bouwmeester@cellzome.com

    Signal transduction pathways are modular composites of functionally interdependent sets of proteins that act in a coordinated fashion to transform environmental information into a phenotypic response. The pro-inflammatory cytokine tumour necrosis factor (TNF)-alpha triggers a signalling cascade, converging on the activation of the transcription factor NF-kappa B, which forms the basis for numerous physiological and pathological processes. Here we report the mapping of a protein interaction network around 32 known and candidate TNF-alpha/NF-kappa B pathway components by using an integrated approach comprising tandem affinity purification, liquid-chromatography tandem mass spectrometry, network analysis and directed functional perturbation studies using RNA interference. We identified 221 molecular associations and 80 previously unknown interactors, including 10 new functional modulators of the pathway. This systems approach provides significant insight into the logic of the TNF-alpha/NF-kappa B pathway and is generally applicable to other pathways relevant to human disease.

    Nature cell biology 2004;6;2;97-105

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

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

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

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

    Nature genetics 2004;36;1;40-5

  • Tom1, a VHS domain-containing protein, interacts with tollip, ubiquitin, and clathrin.

    Yamakami M, Yoshimori T and Yokosawa H

    Department of Biochemistry, Graduate School of Pharmaceutical Sciences, Hokkaido University, Sapporo 060-0812, Japan.

    The gene for Tom1 was initially identified as a specific target of the oncogene v-myb. The Tom1 protein belongs to the VHS domain-containing protein family, and it has a GAT domain in a central part as well as an N-terminal VHS domain. VHS domain-containing proteins, including Hrs/Vps27, STAM, and GGA proteins, have been implicated in intracellular trafficking and sorting, but the role of Tom1 has not yet been elucidated. In this study, we found that Tom1 binds directly with ubiquitin chains and Tollip, which was initially isolated as a mediator of interleukin-1 signaling and has a capacity to bind ubiquitin chains. Gel filtration and subsequent Western blot analysis showed that endogenous Tom1 associates with Tollip to form a complex. In addition, Tom1 was found to be capable of binding to clathrin heavy chain through a typical clathrin-binding motif. Fluorescence microscopic analysis revealed that green fluorescent protein-Tom1 was localized predominantly in the cytoplasm, whereas its mutant with deletion of the clathrin-binding motif had a diffuse localization throughout the cell. Thus, we propose that a Tom1-Tollip complex functions as a factor that links polyubiquitinated proteins to clathrin.

    The Journal of biological chemistry 2003;278;52;52865-72

  • Sorting nexin 9 participates in clathrin-mediated endocytosis through interactions with the core components.

    Lundmark R and Carlsson SR

    Department of Medical Biochemistry and Biophysics, Umeå University, S-901 87 Umeå, Sweden.

    Sorting nexin 9 (SNX9) belongs to a family of proteins, the sorting nexins, that are characterized by the presence of a subclass of the phosphoinositide-binding phox domain. SNX9 has in its amino terminus a Src homology 3 domain and a region with predicted low complexity followed by a carboxyl-terminal part containing the phox domain. We previously found that SNX9 is one of the major proteins in hematopoietic cells that binds to the alpha and beta2-appendages of adaptor protein complex 2 (AP-2), a protein with a critical role in the formation of clathrin-coated vesicles at the plasma membrane. In the present study we show that clathrin and dynamin-2, two other essential molecules in the endocytic process, also interact with SNX9. We found that both AP-2 and clathrin bind to the low complexity region in SNX9 in a cooperative manner, whereas dynamin-2 binds to the Src homology 3 domain. In the cytosol, SNX9 is present in a 14.5 S complex containing dynamin-2 and an unidentified 41-kDa protein. In HeLa cells, SNX9 co-localized with both AP-2 and dynamin-2 at the plasma membrane or on vesicular structures derived from it but not with the early endosomal marker EEA1 or with AP-1. The results suggest that SNX9 may be recruited together with dynamin-2 and become co-assembled with AP-2 and clathrin at the plasma membrane. Overexpression in both K562 and HeLa cells of truncated forms of SNX9 interfered with the uptake of transferrin, consistent with a role of SNX9 in endocytosis.

    The Journal of biological chemistry 2003;278;47;46772-81

  • Effect of clathrin heavy chain- and alpha-adaptin-specific small inhibitory RNAs on endocytic accessory proteins and receptor trafficking in HeLa cells.

    Hinrichsen L, Harborth J, Andrees L, Weber K and Ungewickell EJ

    Department of Cell Biology in the Center of Anatomy, Hannover Medical School, D-30623 Hannover, Germany.

    To assess the contribution of individual endocytic proteins to the assembly of clathrin coated pits, we depleted the clathrin heavy chain and the alpha-adaptin subunit of AP-2 in HeLa-cells using RNA interference. 48 h after transfection with clathrin heavy chain-specific short interfering RNA both, the heavy and light chains were depleted by more than 80%. Residual clathrin was mainly membrane-associated, and an increase in shallow pits was noted. The membrane-association of adaptors, clathrin assembly lymphoid myeloid leukemia protein (CALM), epsin, dynamin, and Eps15 was only moderately affected by the knockdown and all proteins still displayed a punctate staining distribution. Clathrin depletion inhibited the uptake of transferrin but not that of the epidermal growth factor. However, efficient sorting of the epidermal growth factor into hepatocyte growth factor-regulated tyrosine kinase substrate-positive endosomes was impaired. Depletion of alpha-adaptin abolished almost completely the plasma membrane association of clathrin. Binding of Eps15 to membranes was strongly and that of CALM moderately reduced. Whereas the uptake of transferrin was efficiently blocked in alpha-adaptin knockdown cells, the internalization and sorting of the epidermal growth factor was not significantly impaired. Since neither clathrin nor AP-2 is essential for the internalization of EGF, we conclude that it is taken up by an alternative mechanism.

    The Journal of biological chemistry 2003;278;46;45160-70

  • 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

  • Molecular and functional characterization of clathrin- and AP-2-binding determinants within a disordered domain of auxilin.

    Scheele U, Alves J, Frank R, Duwel M, Kalthoff C and Ungewickell E

    Departments of Cell Biology and Biophysical Chemistry, Hannover Medical School, 30625 Hannover, Germany.

    Uncoating of clathrin-coated vesicles requires the J-domain protein auxilin for targeting hsc70 to the clathrin coats and for stimulating the hsc70 ATPase activity. This results in the release of hsc70-complexed clathrin triskelia and concomitant dissociation of the coat. To understand the complex role of auxilin in uncoating and clathrin assembly in more detail, we analyzed the molecular organization of its clathrin-binding domain (amino acids 547-813). CD spectroscopy of auxilin fragments revealed that the clathrin-binding domain is almost completely disordered in solution. By systematic mapping using synthetic peptides and by site-directed mutagenesis, we identified short peptide sequences involved in clathrin heavy chain and AP-2 binding and evaluated their significance for the function of auxilin. Some of the binding determinants, including those containing sequences 674DPF and 636WDW, showed dual specificity for both clathrin and AP-2. In contrast, the two DLL motifs within the clathrin-binding domain were exclusively involved in clathrin binding. Surprisingly, they interacted not only with the N-terminal domain of the heavy chain, but also with the distal domain. Moreover, both DLL peptides proved to be essential for clathrin assembly and uncoating. In addition, we found that the motif 726NWQ is required for efficient clathrin assembly activity. Auxilin shares a number of protein-protein interaction motifs with other endocytic proteins, including AP180. We demonstrate that AP180 and auxilin compete for binding to the alpha-ear domain of AP-2. Like AP180, auxilin also directly interacts with the ear domain of beta-adaptin. On the basis of our data, we propose a refined model for the uncoating mechanism of clathrin-coated vesicles.

    The Journal of biological chemistry 2003;278;28;25357-68

  • Exploring proteomes and analyzing protein processing by mass spectrometric identification of sorted N-terminal peptides.

    Gevaert K, Goethals M, Martens L, Van Damme J, Staes A, Thomas GR and Vandekerckhove J

    Department of Medical Protein Research, Flanders Interuniversity Institute for Biotechnology, Ghent University, A. Baertsoenkaai 3, B-9000 Ghent, Belgium. kris.gevaert@rug.ac.be

    Current non-gel techniques for analyzing proteomes rely heavily on mass spectrometric analysis of enzymatically digested protein mixtures. Prior to analysis, a highly complex peptide mixture is either separated on a multidimensional chromatographic system or it is first reduced in complexity by isolating sets of representative peptides. Recently, we developed a peptide isolation procedure based on diagonal electrophoresis and diagonal chromatography. We call it combined fractional diagonal chromatography (COFRADIC). In previous experiments, we used COFRADIC to identify more than 800 Escherichia coli proteins by tandem mass spectrometric (MS/MS) analysis of isolated methionine-containing peptides. Here, we describe a diagonal method to isolate N-terminal peptides. This reduces the complexity of the peptide sample, because each protein has one N terminus and is thus represented by only one peptide. In this new procedure, free amino groups in proteins are first blocked by acetylation and then digested with trypsin. After reverse-phase (RP) chromatographic fractionation of the generated peptide mixture, internal peptides are blocked using 2,4,6-trinitrobenzenesulfonic acid (TNBS); they display a strong hydrophobic shift and therefore segregate from the unaltered N-terminal peptides during a second identical separation step. N-terminal peptides can thereby be specifically collected for further liquid chromatography (LC)-MS/MS analysis. Omitting the acetylation step results in the isolation of non-lysine-containing N-terminal peptides from in vivo blocked proteins.

    Nature biotechnology 2003;21;5;566-9

  • Adaptor and clathrin exchange at the plasma membrane and trans-Golgi network.

    Wu X, Zhao X, Puertollano R, Bonifacino JS, Eisenberg E and Greene LE

    Laboratory of Cell Biology, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland 20892, USA.

    We previously demonstrated, using fluorescence recovery after photobleaching, that clathrin in clathrin-coated pits at the plasma membrane exchanges with free clathrin in the cytosol, suggesting that clathrin-coated pits are dynamic structures. We now investigated whether clathrin at the trans-Golgi network as well as the clathrin adaptors AP2 and AP1 in clathrin-coated pits at the plasma membrane and trans-Golgi network, respectively, also exchange with free proteins in the cytosol. We found that when the budding of clathrin-coated vesicle is blocked without significantly affecting the structure of clathrin-coated pits, both clathrin and AP2 at the plasma membrane and clathrin and AP1 at the trans-Golgi network exchange rapidly with free proteins in the cytosol. In contrast, when budding of clathrin-coated vesicles was blocked at the plasma membrane or trans-Golgi network by hypertonic sucrose or K(+) depletion, conditions that markedly affect the structure of clathrin-coated pits, clathrin exchange was blocked but AP2 at the plasma membrane and both AP1 and the GGA1 adaptor at the trans-Golgi network continue to rapidly exchange. We conclude that clathrin-coated pits are dynamic structures with rapid exchange of both clathrin and adaptors and that adaptors are able to exchange independently of clathrin when clathrin exchange is blocked.

    Molecular biology of the cell 2003;14;2;516-28

  • ARH is a modular adaptor protein that interacts with the LDL receptor, clathrin, and AP-2.

    He G, Gupta S, Yi M, Michaely P, Hobbs HH and Cohen JC

    McDermott Center for Human Growth and Development, the Howard Hughes Medical Institute, University of Texas Southwestern Medical Center at Dallas, 75290, USA.

    Mutations in the phosphotyrosine binding domain protein ARH cause autosomal recessive hypercholesterolemia, a disorder caused by defective internalization of low density lipoprotein receptors (LDLR) in the liver. To examine the function of ARH, we used pull-down experiments to test for interactions between ARH, the LDLR, and proteins involved in clathrin-mediated endocytosis. The phosphotyrosine binding domain of ARH interacted with the internalization sequence (NPVY) in the cytoplasmic tail of LDLR in a sequence-specific manner. Mutations in the NPVY sequence that were previously shown to decrease LDLR internalization abolished in vitro binding to ARH. Recombinant ARH bound purified bovine clathrin with high affinity (K(D), approximately 44 nm). The interaction between ARH and clathrin was mapped to a canonical clathrin box sequence (LLDLE) in ARH and to the N-terminal domain of the clathrin heavy chain. A highly conserved 20-amino acid sequence in the C-terminal region of ARH bound the beta(2)-adaptin subunit of AP-2. Mutation of a glutamic acid residue in the appendage domain of beta(2)-adaptin that is required for interaction with the adapter protein beta-arrestin markedly reduced binding to ARH. These data are consistent with the hypothesis that ARH functions as an adaptor protein that couples LDLR to the endocytic machinery.

    Funded by: NHLBI NIH HHS: HL20948, HL53917

    The Journal of biological chemistry 2002;277;46;44044-9

  • Clathrin light and heavy chain interface: alpha-helix binding superhelix loops via critical tryptophans.

    Chen CY, Reese ML, Hwang PK, Ota N, Agard D and Brodsky FM

    Department of Microbiology and Immunology, Graduate Group in Biophysics, The G.W.Hooper Foundation, University of California, San Francisco, CA 94143-0552, USA.

    Clathrin light chain subunits (LCa and LCb) contribute to regulation of coated vesicle formation to sort proteins during receptor-mediated endocytosis and organelle biogenesis. LC binding to clathrin heavy chain (HC) was characterized by genetic and structural approaches. The core interactions were mapped to HC residues 1267-1522 (out of 1675) and LCb residues 90-157 (out of 228), using yeast two-hybrid assays. The C-termini of both subunits also displayed interactions extending beyond the core domains. Mutations to helix breakers within the LCb core disrupted HC association. Further suppressor mutagenesis uncovered compensatory mutations in HC (K1415E or K1326E) capable of rescuing the binding defects of LCb mutations W127R or W105R plus W138R, thereby pinpointing contacts between HC and LCb. Mutant HC K1415E also rescued loss of binding by LCa W130R, indicating that both LCs interact similarly with HC. Based on circular dichroism data, mapping and mutagenesis, LCa and LCb were represented as alpha-helices, aligned along the HC and, using molecular dynamics, a structural model of their interaction was generated with novel implications for LC control of clathrin assembly.

    Funded by: NIGMS NIH HHS: GM 08284, GM 38093, GM 55143, R01 GM038093, T32 GM008284

    The EMBO journal 2002;21;22;6072-82

  • Clint: a novel clathrin-binding ENTH-domain protein at the Golgi.

    Kalthoff C, Groos S, Kohl R, Mahrhold S and Ungewickell EJ

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

    We have characterized a novel clathrin-binding 68-kDa epsin N-terminal homology domain (ENTH-domain) protein that we name clathrin interacting protein localized in the trans-Golgi region (Clint). It localizes predominantly to the Golgi region of epithelial cells as well as to more peripheral vesicular structures. Clint colocalizes with AP-1 and clathrin only in the perinuclear area. Recombinantly expressed Clint interacts directly with the gamma-appendage domain of AP-1, with the clathrin N-terminal domain through the peptide motif (423)LFDLM, with the gamma-adaptin ear homology domain of Golgi-localizing, gamma-adaptin ear homology domain 2, with the appendage domain of beta2-adaptin and to a lesser extent with the appendage domain of alpha-adaptin. Moreover, the Clint ENTH-domain asssociates with phosphoinositide-containing liposomes. A significant amount of Clint copurifies with rat liver clathrin-coated vesicles. In rat kidney it is preferentially expressed in the apical region of epithelial cells that line the collecting duct. Clathrin and Clint also colocalize in the apical region of enterocytes along the villi of the small intestine. Apart from the ENTH-domain Clint has no similarities with the epsins AP180/CALM or Hip1/1R. A notable feature of Clint is a carboxyl-terminal methionine-rich domain (Met(427)-Met(605)), which contains >17% methionine. Our results suggest that Clint might participate in the formation of clathrin-coated vesicles at the level of the trans-Golgi network and remains associated with the vesicles longer than clathrin and adaptors.

    Molecular biology of the cell 2002;13;11;4060-73

  • Mixed lineage kinase 2 interacts with clathrin and influences clathrin-coated vesicle trafficking.

    Akbarzadeh S, Ji H, Frecklington D, Marmy-Conus N, Mok YF, Bowes L, Devereux L, Linsenmeyer M, Simpson RJ and Dorow DS

    Trescowthick Research Laboratories, Peter MacCallum Cancer Institute, Melbourne 8006, Victoria, Australia.

    Mixed lineage kinase 2 (MLK2) is a protein kinase that signals in the stress-activated Jun N-terminal kinase signal transduction pathway. We used immunoprecipitation and mass spectrometric analysis to identify MLK2-binding proteins in cell lines with inducible expression of green fluorescent protein-tagged MLK2. Here we report the identification of clathrin as a binding partner for MLK2 in both cultured cells and mammalian brain. We demonstrate that clathrin binding requires a motif (LLDMD) located near the MLK2 C terminus, which is similar to "clathrin box" motifs important for binding of clathrin coat assembly and accessory proteins to the clathrin heavy chain. A C-terminal fragment of MLK2 containing this motif binds strongly to clathrin, and mutation of the LLDMD sequence to LAAAD completely abrogates clathrin binding. We isolated clathrin-coated vesicles from green fluorescent protein-MLK2-expressing cells and from mouse brain lysates and found that MLK2 is enriched along with clathrin in these vesicles. In addition, we demonstrated that endogenous MLK2 co-immunoprecipitates with clathrin heavy chain from the vesicle-enriched fraction of mouse brain lysate. Furthermore, overexpression of MLK2 in cultured cells inhibits accumulation of labeled transferrin in recycling endosomes during receptor-mediated endocytosis. These findings suggest a role for MLK2 and the stress-signaling pathway at sites of clathrin activity in vesicle formation or trafficking.

    The Journal of biological chemistry 2002;277;39;36280-7

  • Tyrosine phosphorylation of clathrin heavy chain under oxidative stress.

    Ihara Y, Yasuoka C, Kageyama K, Wada Y and Kondo T

    Department of Biochemistry and Molecular Biology in Disease, Atomic Bomb Disease Institute, Nagasaki University School of Medicine, 1-12-4 Sakamoto, Japan.

    In mouse pancreatic insulin-producing betaTC cells, oxidative stress due to H(2)O(2) causes tyrosine phosphorylation in various proteins. To identify proteins bearing phosphotyrosine under stress, the proteins were affinity purified using an anti-phosphotyrosine antibody-conjugated agarose column. A protein of 180kDa was identified as clathrin heavy chain (CHC) by electrophoresis and mass spectrometry. Immunoprecipitated CHC showed tyrosine phosphorylation upon H(2)O(2) treatment and the phosphorylation was suppressed by the Src kinase inhibitor, PP2. The phosphorylation status of CHC affected the intracellular localization of CHC and the clathrin-dependent endocytosis of transferrin under oxidative stress. In conclusion, CHC is a protein that is phosphorylated at tyrosine by H(2)O(2) and this phosphorylation status is implicated in the intracellular localization and functions of CHC under oxidative stress. The present study demonstrates that oxidative stress affects intracellular vesicular trafficking via the alteration of clathrin-dependent vesicular trafficking.

    Biochemical and biophysical research communications 2002;297;2;353-60

  • HIP1 and HIP12 display differential binding to F-actin, AP2, and clathrin. Identification of a novel interaction with clathrin light chain.

    Legendre-Guillemin V, Metzler M, Charbonneau M, Gan L, Chopra V, Philie J, Hayden MR and McPherson PS

    Department of Neurology and Neurosurgery, Montreal Neurological Institute, McGill University, Montreal, Quebec H3A 2B4, Canada.

    Huntingtin-interacting protein 1 (HIP1) and HIP12 are orthologues of Sla2p, a yeast protein with essential functions in endocytosis and regulation of the actin cytoskeleton. We now report that HIP1 and HIP12 are major components of the clathrin coat that interact but differ in their ability to bind clathrin and the clathrin adaptor AP2. HIP1 contains a clathrin-box and AP2 consensus-binding sites that display high affinity binding to the terminal domain of the clathrin heavy chain and the ear domain of the AP2 alpha subunit, respectively. These consensus sites are poorly conserved in HIP12 and correspondingly, HIP12 does not bind to AP2 nor does it demonstrate high affinity clathrin binding. Moreover, HIP12 co-sediments with F-actin in contrast to HIP1, which exhibits no interaction with actin in vitro. Despite these differences, both proteins efficiently stimulate clathrin assembly through their central helical domain. Interestingly, in both HIP1 and HIP12, this domain binds directly to the clathrin light chain. Our data suggest that HIP1 and HIP12 play related yet distinct functional roles in clathrin-mediated endocytosis.

    The Journal of biological chemistry 2002;277;22;19897-904

  • Regulation of arrestin-3 phosphorylation by casein kinase II.

    Kim YM, Barak LS, Caron MG and Benovic JL

    Department of Microbiology and Immunology, Kimmel Cancer Center, Thomas Jefferson University, Philadelphia, Pennsylvania 19107, USA.

    Arrestins play an important role in regulating the function of G protein-coupled receptors including receptor desensitization, internalization, down-regulation, and signaling via nonreceptor tyrosine kinases and mitogen-activated protein kinases. Previous studies have revealed that arrestins themselves are also subject to regulation. In the present study, we focused on identifying potential mechanisms involved in regulating the function of arrestin-3. Using metabolic labeling, phosphoamino acid analysis, and mutagenesis studies, we found that arrestin-3 is constitutively phosphorylated at Thr-382 and becomes dephosphorylated upon beta(2)-adrenergic receptor activation in COS-1 cells. Casein kinase II (CKII) appears to be the major kinase mediating arrestin-3 phosphorylation, since 1) Thr-382 is contained within a canonical consensus sequence for CKII phosphorylation and 2) wild type arrestin-3 but not a T382A mutant is phosphorylated by CKII in vitro. Functional analysis reveals that mutants mimicking the phosphorylated (T382E) and dephosphorylated (T382A or T382V) states of arrestin-3 promote beta(2)-adrenergic receptor internalization and bind clathrin, beta-adaptin, and Src to comparable levels as wild type arrestin-3. This suggests that the phosphorylation of arrestin-3 does not directly regulate interaction with endocytic (clathrin, beta-adaptin) or signaling (Src) components and is in contrast to arrestin-2, where phosphorylation appears to regulate interaction with clathrin and Src. However, additional analysis reveals that arrestin-3 phosphorylation may regulate formation of a large arrestin-3-containing protein complex. Differences between the regulatory roles of arrestin-2 and -3 phosphorylation may contribute to the different cellular functions of these proteins in G protein-coupled receptor signaling and regulation.

    The Journal of biological chemistry 2002;277;19;16837-46

  • Identification of Ca2+-dependent binding partners for the neuronal calcium sensor protein neurocalcin delta: interaction with actin, clathrin and tubulin.

    Ivings L, Pennington SR, Jenkins R, Weiss JL and Burgoyne RD

    The Physiological Laboratory, University of Liverpool, Crown Street, Liverpool, L69 3BX, U.K.

    The neuronal calcium sensors are a family of EF-hand-containing Ca(2+)-binding proteins expressed predominantly in retinal photoreceptors and neurons. One of the family members is neurocalcin delta, the function of which is unknown. As an approach to elucidating the protein interactions made by neurocalcin delta, we have identified brain cytosolic proteins that bind to neurocalcin delta in a Ca(2+)-dependent manner. We used immobilized recombinant myristoylated neurocalcin delta combined with protein identification using MS. We demonstrate a specific interaction with clathrin heavy chain, alpha- and beta-tubulin, and actin. These interactions were dependent upon myristoylation of neurocalcin delta indicating that the N-terminal myristoyl group may be important for protein-protein interactions in addition to membrane association. Direct binding of neurocalcin delta to clathrin, tubulin and actin was confirmed using an overlay assay. These interactions were also demonstrated for endogenous neurocalcin delta by co-immunoprecipitation from rat brain cytosol. When expressed in HeLa cells, neurocalcin delta was cytosolic at resting Ca(2+) levels but translocated to membranes, including a perinuclear compartment (trans-Golgi network) where it co-localized with clathrin, following Ca(2+) elevation. These data suggest the possibility that neurocalcin delta functions in the control of clathrin-coated vesicle traffic.

    The Biochemical journal 2002;363;Pt 3;599-608

  • The Cdc42 target ACK2 interacts with sorting nexin 9 (SH3PX1) to regulate epidermal growth factor receptor degradation.

    Lin Q, Lo CG, Cerione RA and Yang W

    Department of Molecular Medicine and Chemistry and Chemical Biology, Cornell University, Ithaca, New York 14853, USA.

    Activated Cdc42-associated kinase-2 (ACK2) is a non-receptor tyrosine kinase that serves as a specific effector for Cdc42, a Rho family small G-protein. Recently, we have found that ACK2 directly interacts with clathrin heavy chain through a clathrin-binding motif that is conserved in all endocytic adaptor proteins and regulates clathrin assembly, suggesting that ACK2 plays a role in clathrin-coated vesicle endocytosis (Yang, W., Lo, C. G., Dispenza, T., and Cerione, R. A. (2001) J. Biol. Chem. 276, 17468-17473). Here we report the identification of another binding partner for ACK2 that has previously been implicated in endocytosis, namely the sorting nexin protein SH3PX1 (sorting nexin 9). The interaction occurs between a proline-rich domain of ACK2 and the Src homology 3 domain (SH3) of SH3PX1. Co-immunoprecipitation studies indicate that ACK2, clathrin, and SH3PX1 form a complex in cells. Epidermal growth factor (EGF) stimulated the tyrosine phosphorylation of SH3PX1, whereas co-transfection of ACK2 with SH3PX1 resulted in the constitutive phosphorylation of SH3PX1. However, co-transfection of the kinase-dead mutant ACK2(K158R) with SH3PX1 blocked EGF-induced tyrosine phosphorylation of SH3PX1, indicating that the EGF-stimulated phosphorylation of SH3PX1 is mediated by ACK2. EGF receptor levels were significantly decreased following EGF stimulation of cells co-expressing ACK2 and SH3PX1, thus highlighting a novel role for ACK2, working together with SH3PX1 to promote the degradation of the EGF receptor.

    Funded by: NIGMS NIH HHS: GM40654, GM47458

    The Journal of biological chemistry 2002;277;12;10134-8

  • Huntingtin interacting protein 1 Is a clathrin coat binding protein required for differentiation of late spermatogenic progenitors.

    Rao DS, Chang JC, Kumar PD, Mizukami I, Smithson GM, Bradley SV, Parlow AF and Ross TS

    Division of Hematology and Oncology, Department of Internal Medicine, University of Michigan Medical School, Ann Arbor, 48109-0936, USA.

    Huntingtin-interacting protein 1 (HIP1) interacts with huntingtin, the protein whose gene is mutated in Huntington's disease. In addition, a fusion between HIP1 and platelet-derived growth factor beta receptor causes chronic myelomonocytic leukemia. The HIP1 proteins, including HIP1 and HIP1-related (HIP1r), have an N-terminal polyphosphoinositide-interacting epsin N-terminal homology, domain, which is found in proteins involved in clathrin-mediated endocytosis. HIP1 and HIP1r also share a central leucine zipper and an actin binding TALIN homology domain. Here we show that HIP1, like HIP1r, colocalizes with clathrin coat components. We also show that HIP1 physically associates with clathrin and AP-2, the major components of the clathrin coat. To further understand the putative biological role(s) of HIP1, we have generated a targeted deletion of murine HIP1. HIP1(-/-) mice developed into adulthood, did not develop overt neurologic symptoms in the first year of life, and had normal peripheral blood counts. However, HIP1-deficient mice exhibited testicular degeneration with increased apoptosis of postmeiotic spermatids. Postmeiotic spermatids are the only cells of the seminiferous tubules that express HIP1. These findings indicate that HIP1 is required for differentiation, proliferation, and/or survival of spermatogenic progenitors. The association of HIP1 with clathrin coats and the requirement of HIP1 for progenitor survival suggest a role for HIP1 in the regulation of endocytosis.

    Funded by: NCI NIH HHS: K08 CA76025-01, R01 CA082363, R01 CA82363-01A1

    Molecular and cellular biology 2001;21;22;7796-806

  • HIP1 functions in clathrin-mediated endocytosis through binding to clathrin and adaptor protein 2.

    Metzler M, Legendre-Guillemin V, Gan L, Chopra V, Kwok A, McPherson PS and Hayden MR

    Centre for Molecular Medicine and Therapeutics, Department of Medical Genetics, University of British Columbia, Vancouver, British Columbia V5Z 4H4, Canada.

    Polyglutamine expansion in huntingtin is the underlying mutation leading to neurodegeneration in Huntington disease. This mutation influences the interaction of huntingtin with different proteins, including huntingtin-interacting protein 1 (HIP1), in which affinity to bind to mutant huntingtin is profoundly reduced. Here we demonstrate that HIP1 colocalizes with markers of clathrin-mediated endocytosis in neuronal cells and is highly enriched on clathrin-coated vesicles (CCVs) purified from brain homogenates. HIP1 binds to the clathrin adaptor protein 2 (AP2) and the terminal domain of the clathrin heavy chain, predominantly through a small fragment encompassing amino acids 276-335. This region, which contains consensus clathrin- and AP2-binding sites, functions in conjunction with the coiled-coil domain to target HIP1 to CCVs. Expression of various HIP1 fragments leads to a potent block of clathrin-mediated endocytosis. Our findings demonstrate that HIP1 is a novel component of the endocytic machinery.

    The Journal of biological chemistry 2001;276;42;39271-6

  • Multiple interactions of auxilin 1 with clathrin and the AP-2 adaptor complex.

    Scheele U, Kalthoff C and Ungewickell E

    Department of Cell Biology, Center of Anatomy, Hannover Medical School, D-30125 Hannover, Germany.

    The removal of the clathrin coat is essential for vesicle fusion with acceptor membranes. Disassembly of the coat involves hsc70, which is specifically recruited by members of the auxilin protein family to clathrin lattices. In vitro, this function of auxilin does not require the globular amino-terminal domain of the clathrin heavy chain, which is known to play a prominent role in the interaction of clathrin with adaptors and numerous endocytic accessory proteins. Here we report the unexpected finding that the neuron-specific form of auxilin (auxilin 1) can also associate with the clathrin amino-terminal domain. This interaction is mediated through tandemly arranged sites within the auxilin 1 carboxyl-terminal segment 547-910. The overlapping auxilin 1 fragments 547-714 and 619-738 bind the clathrin terminal domain with high affinity, whereas auxilin 1-(715-901) interacts only poorly with it. All three fragments also associate with the clathrin distal domain and the alpha-appendage domain of AP-2. Moreover, they support efficient assembly of clathrin triskelia into regular cages. A novel uncoating assay was developed to demonstrate that auxilin 1-(715-901) functions efficiently as a cofactor for hsc70 in the uncoating of clathrin-coated vesicles. The multiple protein-protein interactions of auxilin 1 suggest that its function in endocytic trafficking may be more complex than previously anticipated.

    The Journal of biological chemistry 2001;276;39;36131-8

  • Hrs recruits clathrin to early endosomes.

    Raiborg C, Bache KG, Mehlum A, Stang E and Stenmark H

    Department of Biochemistry, Institute for Cancer Research, The Norwegian Radium Hospital, Montebello, N-0310 Oslo, Norway.

    The hepatocyte growth factor-regulated tyrosine kinase substrate, Hrs, has been implicated in intracellular trafficking and signal transduction. Hrs contains a phosphatidylinositol 3-phosphate-binding FYVE domain that contributes to its endosomal targeting. Here we show that Hrs and EEA1, a FYVE domain protein involved in endocytic membrane fusion, are localized to different regions of early endosomes. We demonstrate that Hrs co-localizes with clathrin, and that the C-terminus of Hrs contains a functional clathrin box motif that interacts directly with the terminal beta-propeller domain of clathrin heavy chain. A massive recruitment of clathrin to early endosomes was observed in cells transfected with Hrs, but not with Hrs lacking the C-terminus. Furthermore, the phosphatidylinositol 3-kinase inhibitor wortmannin caused the dissociation of both Hrs and clathrin from endosomes. While overexpression of Hrs did not affect endocytosis and recycling of transferrin, endocytosed epidermal growth factor and dextran were retained in early endosomes. These results provide a molecular mechanism for the recruitment of clathrin onto early endosomes and suggest a function for Hrs in trafficking from early to late endosomes.

    The EMBO journal 2001;20;17;5008-21

  • Association of insulin-like growth factor 1 receptor with EHD1 and SNAP29.

    Rotem-Yehudar R, Galperin E and Horowitz M

    Department of Cell Research and Immunology, Tel-Aviv University, Ramat-Aviv, 69978, Tel-Aviv, Israel.

    Ligand-induced receptor-mediated endocytosis plays a central role in regulating signaling conveyed by tyrosine kinase receptors. This process depends on the recruitment of the adaptor protein 2 (AP-2) complex, clathrin, dynamin, and other accessory proteins to the ligand-bound receptor. We show here that besides AP-2 and clathrin, two other proteins participate in the endocytic process of the insulin-like growth factor receptor (IGF-1R); they are EHD1, an Eps15 homology (EH) domain-containing protein 1, and SNAP29, a synaptosomal-associated protein. EHD1 and SNAP29 form complexes with alpha-adaptin of AP-2 and co-localize in endocytic vesicles, indicating a role for them in endocytosis. EHD1 and SNAP29 interact directly with each other and are present in complexes with IGF-1R. After IGF-1 induction, EHD1 and IGF-1R co-localize intracellularly. Overexpression of EHD1 in Chinese hamster ovary cells represses IGF-1-mediated signaling, as measured by mitogen-activated protein kinase phosphorylation and Akt phosphorylation, indicating that EHD1 plays a role as a down-regulator in IGF-1 signaling pathway.

    The Journal of biological chemistry 2001;276;35;33054-60

  • The huntingtin interacting protein HIP1 is a clathrin and alpha-adaptin-binding protein involved in receptor-mediated endocytosis.

    Waelter S, Scherzinger E, Hasenbank R, Nordhoff E, Lurz R, Goehler H, Gauss C, Sathasivam K, Bates GP, Lehrach H and Wanker EE

    Max-Planck-Institut für Molekulare Genetik, Ihnestrasse 73, D-14195 Berlin (Dahlem), Germany.

    The huntingtin interacting protein (HIP1) is enriched in membrane-containing cell fractions and has been implicated in vesicle trafficking. It is a multidomain protein containing an N-terminal ENTH domain, a central coiled-coil forming region and a C-terminal actin-binding domain. In the present study we have identified three HIP1 associated proteins, clathrin heavy chain and alpha-adaptin A and C. In vitro binding studies revealed that the central coiled-coil domain is required for the interaction of HIP1 with clathrin, whereas DPF-like motifs located upstream to this domain are important for the binding of HIP1 to the C-terminal 'appendage' domain of alpha-adaptin A and C. Expression of full length HIP1 in mammalian cells resulted in a punctate cytoplasmic immunostaining characteristic of clathrin-coated vesicles. In contrast, when a truncated HIP1 protein containing both the DPF-like motifs and the coiled-coil domain was overexpressed, large perinuclear vesicle-like structures containing HIP1, huntingtin, clathrin and endocytosed transferrin were observed, indicating that HIP1 is an endocytic protein, the structural integrity of which is crucial for maintenance of normal vesicle size in vivo.

    Human molecular genetics 2001;10;17;1807-17

  • Gamma subunit of the AP-1 adaptor complex binds clathrin: implications for cooperative binding in coated vesicle assembly.

    Doray B and Kornfeld S

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

    The heterotetrameric AP-1 adaptor complex is involved in the assembly of clathrin-coated vesicles originating from the trans-Golgi network (TGN). The beta 1 subunit of AP-1 is known to contain a consensus clathrin binding sequence, LLNLD (the so-called clathrin box motif), in its hinge segment through which the beta chain interacts with the N-terminal domains of clathrin trimers. Here, we report that the hinge region of the gamma subunit of human and mouse AP-1 contains two copies of a new variant, LLDLL, of the clathrin box motif that also bind to the terminal domain of the clathrin heavy chain. High-affinity binding of the gamma hinge to clathrin trimers requires both LLDLL sequences to be present and the spacing between them to be maintained. We also identify an independent clathrin-binding site within the appendage domain of the gamma subunit that interacts with a region of clathrin other than the N-terminal domain. Clathrin polymerization is promoted by glutathione S-transferase (GST)-gamma hinge, but not by GST-gamma appendage. However, the hinge and appendage domains of gamma function in a cooperative manner to recruit and polymerize clathrin, suggesting that clathrin lattice assembly at the TGN involves multivalent binding of clathrin by the gamma and beta1 subunits of AP-1.

    Funded by: NCI NIH HHS: R01 CA-08759, R37 CA008759

    Molecular biology of the cell 2001;12;7;1925-35

  • Binding of GGA2 to the lysosomal enzyme sorting motif of the mannose 6-phosphate receptor.

    Zhu Y, Doray B, Poussu A, Lehto VP and Kornfeld S

    Department of Internal Medicine, Washington University School of Medicine, 660 South Euclid Avenue, St. Louis, MO 63110, USA.

    The GGAs are a multidomain protein family implicated in protein trafficking between the Golgi and endosomes. Here, the VHS domain of GGA2 was shown to bind to the acidic cluster-dileucine motif in the cytoplasmic tail of the cation-independent mannose 6-phosphate receptor (CI-MPR). Receptors with mutations in this motif were defective in lysosomal enzyme sorting. The hinge domain of GGA2 bound clathrin, suggesting that GGA2 could be a link between cargo molecules and clathrin-coated vesicle assembly. Thus, GGA2 binding to the CI-MPR is important for lysosomal enzyme targeting.

    Funded by: NCI NIH HHS: R01 CA-08759

    Science (New York, N.Y.) 2001;292;5522;1716-8

  • The tyrosine kinase ACK1 associates with clathrin-coated vesicles through a binding motif shared by arrestin and other adaptors.

    Teo M, Tan L, Lim L and Manser E

    Glaxo-IMCB Group, Institute of Molecular and Cell Biology, 30 Medical Drive, Singapore 117609, Japan.

    One target for the small GTPase Cdc42 is the nonreceptor tyrosine kinase activated Cdc42-associated kinase (ACK), which binds selectively to Cdc42.GTP. We report that ACK1 can associate directly with the heavy chain of clathrin. A central region in ACK1 containing a conserved motif behaves as a clathrin adaptor and competes with beta-arrestin for a common binding site on the clathrin N-terminal head domain. Overexpressed ACK1 perturbs clathrin distribution, an activity dependent on the presence of C-terminal "adaptor" sequences that are also present in the related nonkinase gene 33. ACK1 interacts with the adaptor Nck via SH3 interactions but does not form a trimeric complex with p21-activated serine/threonine kinase, which also binds Nck. Stable low level expression of green fluorescent protein-ACK1 in NIH 3T3 cells has been used to localize ACK1 to clathrin-containing vesicles. The co-localization of ACK1 in vivo with clathrin and AP-2 indicates that it participates in trafficking, underlying an ability to increase receptor-mediated transferrin uptake.

    The Journal of biological chemistry 2001;276;21;18392-8

  • The GGAs promote ARF-dependent recruitment of clathrin to the TGN.

    Puertollano R, Randazzo PA, Presley JF, Hartnell LM and Bonifacino JS

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

    The GGAs constitute a family of modular adaptor-related proteins that bind ADP-ribosylation factors (ARFs) and localize to the trans-Golgi network (TGN) via their GAT domains. Here, we show that binding of the GAT domain stabilizes membrane-bound ARF1.GTP due to interference with the action of GTPase-activating proteins. We also show that the hinge and ear domains of the GGAs interact with clathrin in vitro, and that the GGAs promote recruitment of clathrin to liposomes in vitro and to TGN membranes in vivo. These observations suggest that the GGAs could function to link clathrin to membrane-bound ARF.GTP.

    Cell 2001;105;1;93-102

  • 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

  • Three ways to make a vesicle.

    Kirchhausen T

    Harvard Medical School, 200 Longwood Avenue, Boston, Massachusetts 02115, USA. kirchhausen@crystal.harvard.edu

    Cargo molecules have to be included in carrier vesicles of different forms and sizes to be transported between organelles. During this process, a limited set of proteins, including the coat proteins COPI, COPII and clathrin, carries out a programmed set of sequential interactions that lead to the budding of vesicles. A general model to explain the formation of coated vesicles is starting to emerge but the picture is more complex than we had imagined.

    Nature reviews. Molecular cell biology 2000;1;3;187-98

  • Proteomic analysis of NMDA receptor-adhesion protein signaling complexes.

    Husi H, Ward MA, Choudhary JS, Blackstock WP and Grant SG

    Centre for Genome Research, Centre for Neuroscience, University of Edinburgh, West Mains Road, Edinburgh EH9 3JQ, UK.

    N-methyl-d-aspartate receptors (NMDAR) mediate long-lasting changes in synapse strength via downstream signaling pathways. We report proteomic characterization with mass spectrometry and immunoblotting of NMDAR multiprotein complexes (NRC) isolated from mouse brain. The NRC comprised 77 proteins organized into receptor, adaptor, signaling, cytoskeletal and novel proteins, of which 30 are implicated from binding studies and another 19 participate in NMDAR signaling. NMDAR and metabotropic glutamate receptor subtypes were linked to cadherins and L1 cell-adhesion molecules in complexes lacking AMPA receptors. These neurotransmitter-adhesion receptor complexes were bound to kinases, phosphatases, GTPase-activating proteins and Ras with effectors including MAPK pathway components. Several proteins were encoded by activity-dependent genes. Genetic or pharmacological interference with 15 NRC proteins impairs learning and with 22 proteins alters synaptic plasticity in rodents. Mutations in three human genes (NF1, Rsk-2, L1) are associated with learning impairments, indicating the NRC also participates in human cognition.

    Nature neuroscience 2000;3;7;661-9

  • Epsin binds to clathrin by associating directly with the clathrin-terminal domain. Evidence for cooperative binding through two discrete sites.

    Drake MT, Downs MA and Traub LM

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

    Epsin is a recently identified protein that appears to play an important role in clathrin-mediated endocytosis. The central region of epsin 1, the so-called DPW domain, binds to the heterotetrameric AP-2 adaptor complex by associating directly with the globular appendage of the alpha subunit. We have found that this central portion of epsin 1 also associates with clathrin. The interaction with clathrin is direct and not mediated by epsin-bound AP-2. Alanine scanning mutagenesis shows that clathrin binding depends on the sequence (257)LMDLADV located within the epsin 1 DPW domain. This sequence, related to the known clathrin-binding sequences in the adaptor beta subunits, amphiphysin, and beta-arrestin, facilitates the association of epsin 1 with the terminal domain of the clathrin heavy chain. Unexpectedly, inhibiting the binding of AP-2 to the GST-epsin DPW fusion protein by progressively deleting DPW triplets but leaving the LMDLADV sequence intact, diminishes the association of clathrin in parallel with AP-2. Because the beta subunit of the AP-2 complex also contains a clathrin-binding site, optimal association with soluble clathrin appears to depend on the presence of at least two distinct clathrin-binding sites, and we show that a second clathrin-binding sequence (480)LVDLD, located within the carboxyl-terminal segment of epsin 1, also interacts with clathrin directly. The LMDLADV and LVDLD sequences act cooperatively in clathrin recruitment assays, suggesting that they bind to different sites on the clathrin-terminal domain. The evolutionary conservation of similar clathrin-binding sequences in several metazoan epsin-like molecules suggests that the ability to establish multiple protein-protein contacts within a developing clathrin-coated bud is an important aspect of epsin function.

    Funded by: NIDDK NIH HHS: DK53249

    The Journal of biological chemistry 2000;275;9;6479-89

  • Secretory protein trafficking and organelle dynamics in living cells.

    Lippincott-Schwartz J, Roberts TH and Hirschberg K

    Cell Biology and Metabolism Branch, NICHD, National Institutes of Health, Bethesda, Maryland 20892, USA. jlippin@helix.nih.gov

    Green fluorescent protein chimerae acting as reporters for protein localization and trafficking within the secretory membrane system of living cells have been used in a wide variety of applications, including time-lapse imaging, double-labeling, energy transfer, quantitation, and photobleaching experiments. Results from this work are clarifying the steps involved in the formation, translocation, and fusion of transport intermediates; the organization and biogenesis of organelles; and the mechanisms of protein retention, sorting, and recycling in the secretory pathway. In so doing, they are broadening our thinking about the temporal and spatial relationships among secretory organelles and the membrane trafficking pathways that operate between them.

    Annual review of cell and developmental biology 2000;16;557-89

  • 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

  • Signaling via Src family kinases is required for normal internalization of the receptor c-Kit.

    Broudy VC, Lin NL, Liles WC, Corey SJ, O'Laughlin B, Mou S and Linnekin D

    Divisions of Hematology, Department of Medicine, University of Washington, Seattle, WA, USA. vcbroudy@u.washington.edu

    Stem cell factor (SCF) exerts its biological effects by binding to a specific receptor, the tyrosine kinase c-Kit, which is expressed on the cell surface. Although normal cellular trafficking of growth factor receptors may play a critical role in the modulation of receptor function, the mechanisms that regulate the distribution of c-Kit on the cell surface and the internalization of c-Kit have not been fully defined. We investigated whether signal transduction via Src family kinases is required for normal c-Kit trafficking. Treatment of the SCF-responsive human hematopoietic cell line MO7e with the inhibitor of Src family kinases PP1 blocked SCF-induced capping of c-Kit and internalization of c-Kit. c-Kit was able to associate with clathrin in the presence of PP1, suggesting that entry of c-Kit into clathrin-coated pits occurs independently of Src family kinases. SCF-induced internalization of c-Kit was also diminished in the D33-3 lymphoid cell line in which expression of Lyn kinase was disrupted by homologous recombination. These results indicate that Src family kinases play a role in ligand-induced trafficking of c-Kit.

    Funded by: NCI NIH HHS: CA31615; NIDDK NIH HHS: DK43719, DK44194; ...

    Blood 1999;94;6;1979-86

  • Clathrin assembly lymphoid myeloid leukemia (CALM) protein: localization in endocytic-coated pits, interactions with clathrin, and the impact of overexpression on clathrin-mediated traffic.

    Tebar F, Bohlander SK and Sorkin A

    Department of Pharmacology, University of Colorado Health Science Center, Denver, Colorado 80111, USA.

    The clathrin assembly lymphoid myeloid leukemia (CALM) gene encodes a putative homologue of the clathrin assembly synaptic protein AP180. Hence the biochemical properties, the subcellular localization, and the role in endocytosis of a CALM protein were studied. In vitro binding and coimmunoprecipitation demonstrated that the clathrin heavy chain is the major binding partner of CALM. The bulk of cellular CALM was associated with the membrane fractions of the cell and localized to clathrin-coated areas of the plasma membrane. In the membrane fraction, CALM was present at near stoichiometric amounts relative to clathrin. To perform structure-function analysis of CALM, we engineered chimeric fusion proteins of CALM and its fragments with the green fluorescent protein (GFP). GFP-CALM was targeted to the plasma membrane-coated pits and also found colocalized with clathrin in the Golgi area. High levels of expression of GFP-CALM or its fragments with clathrin-binding activity inhibited the endocytosis of transferrin and epidermal growth factor receptors and altered the steady-state distribution of the mannose-6-phosphate receptor in the cell. In addition, GFP-CALM overexpression caused the loss of clathrin accumulation in the trans-Golgi network area, whereas the localization of the clathrin adaptor protein complex 1 in the trans-Golgi network remained unaffected. The ability of the GFP-tagged fragments of CALM to affect clathrin-mediated processes correlated with the targeting of the fragments to clathrin-coated areas and their clathrin-binding capacities. Clathrin-CALM interaction seems to be regulated by multiple contact interfaces. The C-terminal part of CALM binds clathrin heavy chain, although the full-length protein exhibited maximal ability for interaction. Altogether, the data suggest that CALM is an important component of coated pit internalization machinery, possibly involved in the regulation of clathrin recruitment to the membrane and/or the formation of the coated pit.

    Funded by: NCI NIH HHS: CA-46934, P30 CA046934; NIDDK NIH HHS: DK-46817

    Molecular biology of the cell 1999;10;8;2687-702

  • Splice variants of intersectin are components of the endocytic machinery in neurons and nonneuronal cells.

    Hussain NK, Yamabhai M, Ramjaun AR, Guy AM, Baranes D, O'Bryan JP, Der CJ, Kay BK and McPherson PS

    Department of Neurology and Neurosurgery, Montreal Neurological Institute, McGill University, Montreal, QC, H3A 2B4, Canada.

    We recently identified and cloned intersectin, a protein containing two Eps15 homology (EH) domains and five Src homology 3 (SH3) domains. Using a newly developed intersectin antibody, we demonstrate that endogenous COS-7 cell intersectin localizes to clathrin-coated pits, and transfection studies suggest that the EH domains may direct this localization. Through alternative splicing in a stop codon, a long form of intersectin is generated with a C-terminal extension containing Dbl homology (DH), pleckstrin homology (PH), and C2 domains. Western blots reveal that the long form of intersectin is expressed specifically in neurons, whereas the short isoform is expressed at lower levels in glia and other nonneuronal cells. Immunofluorescence analysis of cultured hippocampal neurons reveals that intersectin is found at the plasma membrane where it is co-localized with clathrin. Ibp2, a protein identified based on its interactions with the EH domains of intersectin, binds to clathrin through the N terminus of the heavy chain, suggesting a mechanism for the localization of intersectin at clathrin-coated pits. Ibp2 also binds to the clathrin adaptor AP2, and antibodies against intersectin co-immunoprecipitate clathrin, AP2, and dynamin from brain extracts. These data suggest that the long and short forms of intersectin are components of the endocytic machinery in neurons and nonneuronal cells.

    The Journal of biological chemistry 1999;274;22;15671-7

  • Clathrin self-assembly is mediated by a tandemly repeated superhelix.

    Ybe JA, Brodsky FM, Hofmann K, Lin K, Liu SH, Chen L, Earnest TN, Fletterick RJ and Hwang PK

    The G. W. Hooper Foundation, Department of Microbiology and Immunology, University of California San Francisco, 94143, USA.

    Clathrin is a triskelion-shaped cytoplasmic protein that polymerizes into a polyhedral lattice on intracellular membranes to form protein-coated membrane vesicles. Lattice formation induces the sorting of membrane proteins during endocytosis and organelle biogenesis by interacting with membrane-associated adaptor molecules. The clathrin triskelion is a trimer of heavy-chain subunits (1,675 residues), each binding a single light-chain subunit, in the hub domain (residues 1,074-1,675). Light chains negatively modulate polymerization so that intracellular clathrin assembly is adaptor-dependent. Here we report the atomic structure, to 2.6 A resolution, of hub residues 1,210-1,516 involved in mediating spontaneous clathrin heavy-chain polymerization and light-chain association. The hub fragment folds into an elongated coil of alpha-helices, and alignment analyses reveal a 145-residue motif that is repeated seven times along the filamentous leg and appears in other proteins involved in vacuolar protein sorting. The resulting model provides a three-dimensional framework for understanding clathrin heavy-chain self-assembly, light-chain binding and trimerization.

    Nature 1999;399;6734;371-5

  • Paxillin LD4 motif binds PAK and PIX through a novel 95-kD ankyrin repeat, ARF-GAP protein: A role in cytoskeletal remodeling.

    Turner CE, Brown MC, Perrotta JA, Riedy MC, Nikolopoulos SN, McDonald AR, Bagrodia S, Thomas S and Leventhal PS

    Department of Anatomy and Cell Biology, State University of New York, Health Science Center, Syracuse, New York 13210, USA. turnerc@vax.cs.hscsyr.edu

    Paxillin is a focal adhesion adaptor protein involved in the integration of growth factor- and adhesion-mediated signal transduction pathways. Repeats of a leucine-rich sequence named paxillin LD motifs (Brown M.C., M.S. Curtis, and C.E. Turner. 1998. Nature Struct. Biol. 5:677-678) have been implicated in paxillin binding to focal adhesion kinase (FAK) and vinculin. Here we demonstrate that the individual paxillin LD motifs function as discrete and selective protein binding interfaces. A novel scaffolding function is described for paxillin LD4 in the binding of a complex of proteins containing active p21 GTPase-activated kinase (PAK), Nck, and the guanine nucleotide exchange factor, PIX. The association of this complex with paxillin is mediated by a new 95-kD protein, p95PKL (paxillin-kinase linker), which binds directly to paxillin LD4 and PIX. This protein complex also binds to Hic-5, suggesting a conservation of LD function across the paxillin superfamily. Cloning of p95PKL revealed a multidomain protein containing an NH2-terminal ARF-GAP domain, three ankyrin-like repeats, a potential calcium-binding EF hand, calmodulin-binding IQ motifs, a myosin homology domain, and two paxillin-binding subdomains (PBS). Green fluorescent protein- (GFP-) tagged p95PKL localized to focal adhesions/complexes in CHO.K1 cells. Overexpression in neuroblastoma cells of a paxillin LD4 deletion mutant inhibited lamellipodia formation in response to insulin-like growth fac- tor-1. Microinjection of GST-LD4 into NIH3T3 cells significantly decreased cell migration into a wound. These data implicate paxillin as a mediator of p21 GTPase-regulated actin cytoskeletal reorganization through the recruitment to nascent focal adhesion structures of an active PAK/PIX complex potentially via interactions with p95PKL.

    Funded by: NIGMS NIH HHS: GM47607, R01 GM047607

    The Journal of cell biology 1999;145;4;851-63

  • The beta2-adrenergic receptor/betaarrestin complex recruits the clathrin adaptor AP-2 during endocytosis.

    Laporte SA, Oakley RH, Zhang J, Holt JA, Ferguson SS, Caron MG and Barak LS

    Howard Hughes Medical Institute Laboratories and Department of Cell Biology, Duke University Medical Center, Durham, NC 27710, USA.

    betaarrestins mediate the desensitization of the beta2-adrenergic receptor (beta2AR) and many other G protein-coupled receptors (GPCRs). Additionally, betaarrestins initiate the endocytosis of these receptors via clathrin coated-pits and interact directly with clathrin. Consequently, it has been proposed that betaarrestins serve as clathrin adaptors for the GPCR family by linking these receptors to clathrin lattices. AP-2, the heterotetrameric clathrin adaptor protein, has been demonstrated to mediate the internalization of many types of plasma membrane proteins other than GPCRs. AP-2 interacts with the clathrin heavy chain and cytoplasmic domains of receptors such as those for epidermal growth factor and transferrin. In the present study we demonstrate the formation of an agonist-induced multimeric complex containing a GPCR, betaarrestin 2, and the beta2-adaptin subunit of AP-2. beta2-Adaptin binds betaarrestin 2 in a yeast two-hybrid assay and coimmunoprecipitates with betaarrestins and beta2AR in an agonist-dependent manner in HEK-293 cells. Moreover, beta2-adaptin translocates from the cytosol to the plasma membrane in response to the beta2AR agonist isoproterenol and colocalizes with beta2AR in clathrin-coated pits. Finally, expression of betaarrestin 2 minigene constructs containing the beta2-adaptin interacting region inhibits beta2AR endocytosis. These findings point to a role for AP-2 in GPCR endocytosis, and they suggest that AP-2 functions as a clathrin adaptor for the endocytosis of diverse classes of membrane receptors.

    Funded by: NHLBI NIH HHS: HL 03422; NINDS NIH HHS: NS 19576, R01 NS019576

    Proceedings of the National Academy of Sciences of the United States of America 1999;96;7;3712-7

  • Atomic structure of clathrin: a beta propeller terminal domain joins an alpha zigzag linker.

    ter Haar E, Musacchio A, Harrison SC and Kirchhausen T

    Department of Cell Biology and Center for Blood Research, Harvard Medical School, Boston, Massachusetts 02115-5701, USA.

    Clathrin triskelions form the lattice that organizes recruitment of proteins to coated pits and helps drive vesiculation of the lipid bilayer. We report the crystal structure at 2.6 A resolution of a 55 kDa N-terminal fragment from the 190 kDa clathrin heavy chain. The structure comprises the globular "terminal domain" and the linker that joins it to the end of a triskelion leg. The terminal domain is a seven-blade beta propeller, a structure well adapted to interaction with multiple partners, such as the AP-1 and AP-2 sorting adaptor complexes and the nonvisual arrestins. The linker is an alpha-helical zigzag emanating from the propeller domain. We propose that this simple motif may extend into the rest of the clathrin leg.

    Funded by: NIGMS NIH HHS: GM36548, R01 GM036548

    Cell 1998;95;4;563-73

  • Multiple amphiphysin II splice variants display differential clathrin binding: identification of two distinct clathrin-binding sites.

    Ramjaun AR and McPherson PS

    Department of Neurology and Neurosurgery, Montreal Neurological Institute, McGill University, Quebec, Canada.

    Amphiphysin I and II are nerve terminal-enriched proteins that display src homology 3 domain-mediated interactions with dynamin and synaptojanin. It has been demonstrated that the amphiphysins also bind to clathrin, and we have proposed that this interaction may help to target synaptojanin and dynamin to sites of synaptic vesicle endocytosis. To understand better this potential functional role, we have begun to characterize clathrin-amphiphysin interactions. Using PCR from adult human cortex cDNA, we have cloned a number of amphiphysin II splice variants. In in vitro binding assays, the amphiphysin II splice variants display differential clathrin binding and define a 44-amino acid region mediating the interaction. Amphiphysin II truncation and deletion mutants identify two distinct clathrin-binding domains within this region: one with the sequence LLDLDFDP, the second with the sequence PWDLW. Both domains are conserved in amphiphysin I, and saturation binding analysis demonstrates that both sites bind clathrin with approximately equal affinity. The elucidation of clathrin as a splice-specific binding partner for amphiphysin II begins to address the potential functional role(s) for the multiple amphiphysin II splice variants and further supports an important function for clathrin-amphiphysin interactions in protein targeting during endocytosis.

    Journal of neurochemistry 1998;70;6;2369-76

  • Association of the AP-3 adaptor complex with clathrin.

    Dell'Angelica EC, Klumperman J, Stoorvogel W and Bonifacino JS

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

    A heterotetrameric complex termed AP-3 is involved in signal-mediated protein sorting to endosomal-lysosomal organelles. AP-3 has been proposed to be a component of a nonclathrin coat. In vitro binding assays showed that mammalian AP-3 did associate with clathrin by interaction of the appendage domain of its beta3 subunit with the amino-terminal domain of the clathrin heavy chain. The beta3 appendage domain contained a conserved consensus motif for clathrin binding. AP-3 colocalized with clathrin in cells as observed by immunofluorescence and immunoelectron microscopy. Thus, AP-3 function in protein sorting may depend on clathrin.

    Science (New York, N.Y.) 1998;280;5362;431-4

  • Nef-mediated clathrin-coated pit formation.

    Foti M, Mangasarian A, Piguet V, Lew DP, Krause KH, Trono D and Carpentier JL

    Department of Morphology, Centre Médical Universitaire, University of Geneva, Switzerland.

    The sequence of events leading to clathrin-coated pit (CCP) nucleation on the cell surface and to the incorporation of receptors into these endocytic structures is still imperfectly understood. In particular, the question remains as to whether receptor tails initiate the assembly of the coat proteins or whether receptors migrate into preformed CCP. This question was approached through a dissection of the mechanisms implemented by Nef, an early protein of human and simian immunodeficiency virus (HIV and SIV, respectively), to accelerate the endocytosis of cluster of differentiation antigen type 4 (CD4), the major receptor for these viruses. Results collected showed that: (a) Nef promotes CD4 internalization via an increased association of CD4 with CCP; (b) the Nef-mediated increase of CD4 association with CCP is related to a doubling of the plasma membrane area occupied by clathrin-coated structures; (c) this increased CCP number at the plasma membrane has functional consequences preferentially on CD4 uptake and does not significantly affect transferrin receptor internalization or fluid-phase endocytosis; (d) the presence of a CD4 cytoplasmic tail including a critical dileucine motif is required to induce CCP formation via Nef; and (e) when directly anchored to the cytoplasmic side of the plasma membrane, Nef itself can promote CCP formation. Taken together, these observations lead us to propose that CD4 can promote CCP generation via the connector molecule Nef. In this model, Nef interacts on one side with CD4 through a dileucine-based motif present on CD4 cytoplasmic tail and on the other side with components of clathrin-coated surface domain (i.e., adaptins). These Nef-generated complexes would then initiate the nucleation of CCP.

    Funded by: FIC NIH HHS: R03 TW 00531; NIAID NIH HHS: R37 AI34306

    The Journal of cell biology 1997;139;1;37-47

  • Clathrin interacts specifically with amphiphysin and is displaced by dynamin.

    McMahon HT, Wigge P and Smith C

    Neurobiology Division, MRC-LMB, Cambridge, UK. hmm@mrc-lmb.cam.ac.uk

    Amphiphysin is an SH3 domain protein that has been implicated in synaptic vesicle endocytosis. We have recently cloned a second amphiphysin isoform, Amph2 (sequence submitted to GenBank, Y13380). Proteins capable of forming a complex with amphiphysin were isolated from rat brain by using recombinant GST-Amph2 for binding experiments. As well as interacting with dynamin I, the full-length protein bound to a weaker 180-kDa band. Immunoblotting demonstrated this protein to be clathrin. To address whether this is a direct interaction, the clathrin binding to amphiphysin was reconstituted in vitro with purified proteins. The N-terminal domain of Amph2 is sufficient for clathrin binding. Dynamin, which interacts with the SH3 domain of Amph2, displaces clathrin from the N-terminus. We propose a model that may explain how clathrin and dynamin are recruited to non-overlapping sites of the coated pit.

    FEBS letters 1997;413;2;319-22

  • Identification and characterization of a nerve terminal-enriched amphiphysin isoform.

    Ramjaun AR, Micheva KD, Bouchelet I and McPherson PS

    Department of Neurology and Neurosurgery, Montreal Neurological Institute, McGill University, Montreal, Québec H3A 2B4, Canada.

    Amphiphysin is a nerve terminal-enriched protein thought to function in synaptic vesicle endocytosis, in part through Src homology 3 (SH3) domain-mediated interactions with dynamin and synaptojanin. Here, we report the characterization of a novel amphiphysin isoform (termed amphiphysin II) that was identified through a homology search of the data base of expressed sequence tags. Antibodies specific to amphiphysin II recognize a 90-kDa protein on Western blot that is brain-specific and highly enriched in nerve terminals. Like amphiphysin (now referred to as amphiphysin I), amphiphysin II binds to dynamin and synaptojanin through its SH3 domain. Further, both proteins bind directly to clathrin in an SH3 domain-independent manner. Taken together, these data suggest that amphiphysin II may participate with amphiphysin I in the regulation of synaptic vesicle endocytosis.

    The Journal of biological chemistry 1997;272;26;16700-6

  • Arrestin/clathrin interaction. Localization of the arrestin binding locus to the clathrin terminal domain.

    Goodman OB, Krupnick JG, Gurevich VV, Benovic JL and Keen JH

    Department of Biochemistry, Kimmel Cancer Institute, Thomas Jefferson University, Philadelphia, Pennsylvania 19107, USA.

    Previously we demonstrated that nonvisual arrestins exhibit a high affinity interaction with clathrin, consistent with an adaptor function in the internalization of G protein-coupled receptors (Goodman, O. B., Jr., Krupnick, J. G., Santini, F., Gurevich, V. V., Penn, R. B., Gagnon, A. W., Keen, J. H., and Benovic, J. L. (1996) Nature 383, 447-450). In this report we show that a short sequence of highly conserved residues within the globular clathrin terminal domain is responsible for arrestin binding. Limited proteolysis of clathrin cages results in the release of terminal domains and concomitant abrogation of arrestin binding. The nonvisual arrestins, beta-arrestin and arrestin3, but not visual arrestin, bind specifically to a glutathione S-transferase-clathrin terminal domain fusion protein. Deletion analysis and alanine scanning mutagenesis localize the binding site to residues 89-100 of the clathrin heavy chain and indicate that residues 1-100 can function as an independent arrestin binding domain. Site-directed mutagenesis identifies an invariant glutamine (Glu-89) and two highly conserved lysines (Lys-96 and Lys-98) as residues critical for arrestin binding, complementing hydrophobic and acidic residues in arrestin3 which have been implicated in clathrin binding (Krupnick, J. G., Goodman, O. B., Jr., Keen, J. H., and Benovic, J. L. (1997) J. Biol. Chem. 272, 15011-15016). Despite exhibiting high affinity clathrin binding, arrestins do not induce coat assembly. The terminal domain is oriented toward the plasma membrane in coated pits, and its binding of both arrestins and AP-2 suggests that this domain is the anchor responsible for adaptor-receptor recruitment to the coated pit.

    Funded by: NIDDK NIH HHS: 5-T32-DK07705-03; NIGMS NIH HHS: GM-28526, GM-44944

    The Journal of biological chemistry 1997;272;23;15017-22

  • Characterization of a second human clathrin heavy chain polypeptide gene (CLH-22) from chromosome 22q11.

    Kedra D, Peyrard M, Fransson I, Collins JE, Dunham I, Roe BA and Dumanski JP

    Department of Molecular Medicine, Karolinska Hospital, Stockholm, Sweden.

    We report cloning and characterization of the second human clathrin heavy chain polypeptide gene (CLH-22) localized to chromosome 22q11. Hence H. sapiens is the first species for which two clathrin heavy chain genes have been reported. We provide 5470 bp cDNA sequence covering the entire open reading frame of the CLH-22 gene. The predicted polypeptide is composed of 1640 amino acids. Its 6 kb transcript is expressed in all of 16 tested human tissues, suggesting it is a housekeeping gene. Skeletal muscle, testis and heart show significantly higher expression levels. Compared to the previously characterized human clathrin heavy chain gene localized on chromosome 17 (CLH-17), CLH-22 shows different transcript size and expression profile in human tissues. Northern analysis of CLH-22 suggests that several alternatively spliced transcripts exist. A presumably single, 171 bp long alternatively spliced exon has been characterized. Amino acid sequence comparison between CLH-22 and CLH-17 shows an overall identify and similarity of 84.7 and 91.1%, respectively. At the nucleic acid level, identity between open reading frames of both genes is 74.3%. Sequence comparison with previously cloned genes in other species suggests that counterparts of the CLH-17 gene have been cloned in B. taurus and R. norvegicus, whereas presumptive mammalian homologues of the CLH-22 gene are yet to be characterized. Our Northern and Southern blot analyses of meningiomas clearly suggest the CLH-22 gene may be involved in the tumor development and can be considered as a candidate for a tumor suppressor.

    Funded by: Wellcome Trust

    Human molecular genetics 1996;5;5;625-31

  • Prediction of the coding sequences of unidentified human genes. I. The coding sequences of 40 new genes (KIAA0001-KIAA0040) deduced by analysis of randomly sampled cDNA clones from human immature myeloid cell line KG-1.

    Nomura N, Miyajima N, Sazuka T, Tanaka A, Kawarabayasi Y, Sato S, Nagase T, Seki N, Ishikawa K and Tabata S

    Institute of Gerontology, Nippon Medical School, Kanagawa, Japan.

    We established a protocol for the prediction of the coding sequences of unidentified human genes based on the double selection and sequence analysis of cDNA clones with inserts carrying unreported 5'-terminal sequences and with insert sizes corresponding to nearly full-length transcripts. By applying the protocol, cDNA clones with inserts longer than 2 kb were isolated from a cDNA library of human immature myeloid cell line KG-1, and the coding sequences of 40 new genes were predicted. A computer search of the sequences indicated that 20 genes contained sequences similar to known genes in the GenBank/EMBL databases. The sequences of the remaining 20 genes were entirely new, and characteristic protein motifs or domains were identified in 32 genes. Other sequence features noted were that the coding sequences of 23 genes were followed by relatively long stretches of 3'-untranslated sequences and that 5 genes contained repetitive sequences in their 3'-untranslated regions. The chromosomal location of these genes has been determined. By increasing the scale of the above analysis, the coding sequences of many unidentified genes can be predicted.

    DNA research : an international journal for rapid publication of reports on genes and genomes 1994;1;1;27-35

  • Prediction of the coding sequences of unidentified human genes. I. The coding sequences of 40 new genes (KIAA0001-KIAA0040) deduced by analysis of randomly sampled cDNA clones from human immature myeloid cell line KG-1 (supplement).

    Nomura N, Miyajima N, Sazuka T, Tanaka A, Kawarabayasi Y, Sato S, Nagase T, Seki N, Ishikawa K and Tabata S

    Institute of Gerontology, Nippon Medical School, Kanagawa, Japan.

    DNA research : an international journal for rapid publication of reports on genes and genomes 1994;1;1;47-56

  • Interaction of vinculin with the clathrin heavy chain.

    Fausser JL, Ungewickell E, Ruch JV and Lesot H

    Institut de Biologie Médicale, Faculté de Médecine, Strasbourg, France.

    To further document the interaction of vinculin with the clathrin heavy chain (CHC) which was observed by using gel overlay, co-sedimentation experiments were performed and attempts were made to localize the domains involved on both molecules. The binding properties of proteolytic fragments of vinculin were investigated after cleavage with V8 protease. Neither the isolated globular domain, nor the C-terminal rod domain were able to interact with the CHC. Either the interaction involved the portion of vinculin which links these two domains, or the region of vinculin mediating the interaction was present on one of the two major fragments, but the cleavage itself resulted in conformational changes which abolished the binding. The first hypothesis could be ruled out using alpha-chymotrypsin generated fragments of vinculin, suggesting that the native conformation of vinculin might play an important role. Proteolytic cleavage of CHC with trypsin demonstrated that the interaction with vinculin is mediated by the proximal or distal segment of the CHC. Presence of clathrin light chain (CLC) associated with the CHC did not affect its interaction with vinculin. Vinculin did not interact with the CLC.

    Journal of biochemistry 1993;114;4;498-503

  • Recognition sites for clathrin-associated proteins AP-2 and AP-3 on clathrin triskelia.

    Murphy JE and Keen JH

    Department of Biochemistry, Temple University School of Medicine, Philadelphia, Pennsylvania 19140.

    AP-2 and AP-3 are cellular proteins that drive the in vitro polymerization of clathrin triskelia into cage structures. The interaction of these two types of assembly proteins (APs) with preassembled clathrin cages has been studied in order to identify the sites on the triskelia required for binding. Comparing binding of the APs to intact or to proteolytically clipped cages, we attempted to distinguish between binding to the terminal domain, the globular end of the heavy chain, and binding to the hub of the clathrin triskelia, the portion that remains assembled after trypsin treatment. AP-3 binds to intact clathrin cages but not to those that were treated with trypsin. AP-3 also bound to cages consisting solely of clathrin heavy chains; proteolysis of these cages also eliminated AP-3 binding. In addition, AP-3 did not bind to either isolated hubs or terminal domains that had been immobilized on Sepharose. These data indicate that clathrin light chains are not required for binding of AP-3, and that neither terminal domain nor hubs alone will suffice. However, an intact heavy chain is both necessary and sufficient for the binding of AP-3. Previous work has demonstrated one binding site for AP-2 on proteolyzed cages containing only clathrin hubs; the existence of a second binding site associated with the terminal domain was hypothesized. Here we provide direct evidence for recognition by AP-2 of isolated terminal domains immobilized on Sepharose and show that the core of the AP-2 molecule is responsible for this interaction. These results provide the first demonstration of a functional role for the conserved terminal domain of the clathrin heavy chain.

    Funded by: NIGMS NIH HHS: GM-28526

    The Journal of biological chemistry 1992;267;15;10850-5

  • Spinal cord repair: future directions.

    Fawcett JW

    Physiological Laboratory, Cambridge, England.

    The main cause of disability following spinal injury is failure of axons to regenerate and reconnect the spinal cord with the brain. If patients with cord lesions are ever to make a full recovery some means will have to be found to restore ascending sensory and descending motor connections. Until the last few years there has been a very limited understanding of the reasons why axons in the central nervous system (CNS) fail to regenerate, but as a result of recent work the picture is now much clearer.

    Paraplegia 1992;30;2;83-5

  • Human clathrin heavy chain (CLTC): partial molecular cloning, expression, and mapping of the gene to human chromosome 17q11-qter.

    Dodge GR, Kovalszky I, McBride OW, Yi HF, Chu ML, Saitta B, Stokes DG and Iozzo RV

    Department of Pathology and Cell Biology, Thomas Jefferson University, Philadelphia, Pennsylvania 19107.

    The nucleotide sequence of a 916-bp human cDNA clone isolated from a human colon lambda gt11 cDNA library was determined. Sequence analysis showed this cDNA to have 88% homology to the nucleotide sequence of the heavy chain of rat clathrin. The deduced amino acid sequence was 98.7% identical to the rat sequence, a change of only four amino acids. The mRNA identified in both human and rat cells with the human clathrin clone revealed transcripts of approximately 6.5 kb, which is consistent with the predicted 180 kDa molecular weight of the clathrin heavy chain. Southern analysis of human/rodent somatic cell hybrids localized the human clathrin heavy chain gene (CLTC) to chromosome 17. Additional analyses using panels of human/rodent somatic cell hybrids with specific chromosomal translocations and deletions mapped the human clathrin heavy chain gene locus to 17q11-qter.

    Funded by: NCI NIH HHS: CA-39481, CA-47282

    Genomics 1991;11;1;174-8

  • Analysis of clathrin light chain-heavy chain interactions using truncated mutants of rat liver light chain LCB3.

    Scarmato P and Kirchhausen T

    Department of Anatomy and Cellular Biology, Harvard Medical School, Boston, Massachusetts 02115.

    Clathrin light chains are extended molecules located along the proximal segment of each of the three heavy chain legs of a clathrin trimer. All mammalian light chains share a central segment with 10 repeated heptad motifs believed to mediate the interaction with clathrin heavy chains. In order to test this model in more detail, we have expressed intact rat liver clathrin light chain LCB3 in Escherichia coli and find that it binds tightly to calf clathrin heavy chains. Using a set of expressed truncated mutants of LCB3, we show that the presence of seven to eight heptads is indeed necessary for a successful interaction. More extensive deletions of the central segment completely abolish the ability to bind to heavy chains. Neither the amino- nor the carboxyl-terminal domain is essential for binding, but competition experiments show that the presence of the carboxyl-terminal domain does enhance the interaction with heavy chains.

    Funded by: NIGMS NIH HHS: R01 GM36548-01

    The Journal of biological chemistry 1990;265;7;3661-8

  • Insulin stimulates the assembly of cytosolic clathrin onto adipocyte plasma membranes.

    Corvera S

    Program in Molecular Medicine, University of Massachusetts Medical School, Worcester 01655.

    The effects of insulin on the subcellular distribution of the heavy chain of clathrin and on the insulin-like growth factor II (IGF-II) mannose 6-phosphate receptor were investigated in isolated rat adipocytes. Plasma membranes, intracellular membranes, and cytosol were separated by differential centrifugation, and the concentration of clathrin and receptor in each fraction was quantified by sequential immunoblotting with monoclonal and polyclonal antibodies against these proteins. A 3-fold increase in the amount of clathrin heavy chain associated with isolated plasma membranes was found after treatment of cells with low concentrations of insulin. This effect was complete within 2 min of stimulation at 37 degrees C and was abolished at 5-10 degrees C. The insulin-mediated increase in the cell surface concentration of receptors for IGF-II/mannose 6-phosphate displayed a similar time course and temperature dependence. A concomitant decrease in the concentration of IGF-II/mannose 6-phosphate receptors in intracellular membranes was observed. In contrast, no significant changes in the concentration of clathrin in this fraction could be detected. Instead, a marked decrease in the level of unassembled cytosolic clathrin was observed in insulin-treated cells compared with controls. These results suggest that insulin induces an increase in the assembly of cytosolic clathrin onto the plasma membrane in conjunction with its ability to increase the concentration of receptors on the cell surface.

    Funded by: NIDDK NIH HHS: DK-40330

    The Journal of biological chemistry 1990;265;5;2413-6

  • The association of clathrin fragments with coated vesicle membranes.

    Hanspal M, Luna E and Branton D

    The association between clathrin triskelions and the clathrin-stripped membranes of coated vesicles has been investigated using a filter assay to separate bound from unbound clathrin. The filter assay is more sensitive and less cumbersome than a sedimentation assay used previously (1). While confirming the high affinity interaction between clathrin and the vesicle membrane, our results yield Scatchard plots that are curvilinear and consistent with a positively cooperative interaction between clathrin and the vesicle membranes. Controlled digestion with trypsin removes the distal portions of the triskelion legs leaving the proximal 31 nm portions that form the hub of the triskelions. These hubs are trimers of large 112,000- and 124,000-dalton fragments of clathrin heavy chains. They competitively inhibit the binding of 125I-labeled intact triskelions to stripped vesicles with a KI identical to the KD for the association of 125I-labeled intact triskelions to stripped vesicles. Furthermore, these large fragment trimers bind to stripped vesicles with approximately the same high affinity as do intact triskelions and also show evidence of a positively cooperative interaction. It is concluded that clathrin binds to coated vesicles by an interaction that is mediated by the proximal 112,000-dalton fragment of the clathrin heavy chains.

    Funded by: NIGMS NIH HHS: GM 31579

    The Journal of biological chemistry 1984;259;17;11075-82

Gene lists (11)

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
L00000011 G2C Homo sapiens Human clathrin Human orthologues of mouse clathrin coated vesicle genes adapted from Collins et al (2006) 150
L00000012 G2C Homo sapiens Human Synaptosome Human orthologues of mouse synaptosome adapted from Collins et al (2006) 152
L00000013 G2C Homo sapiens Human mGluR5 Human orthologues of mouse mGluR5 complex adapted from Collins et al (2006) 52
L00000015 G2C Homo sapiens Human NRC Human orthologues of mouse NRC adapted from Collins et al (2006) 186
L00000016 G2C Homo sapiens Human PSP Human orthologues of mouse PSP adapted from Collins et al (2006) 1121
L00000049 G2C Homo sapiens TAP-PSD-95-CORE TAP-PSD-95 pull-down core list (ortho) 120
L00000059 G2C Homo sapiens BAYES-COLLINS-HUMAN-PSD-CONSENSUS Human cortex PSD consensus 748
L00000061 G2C Homo sapiens BAYES-COLLINS-MOUSE-PSD-CONSENSUS Mouse cortex PSD consensus (ortho) 984
L00000069 G2C Homo sapiens BAYES-COLLINS-HUMAN-PSD-FULL Human cortex biopsy PSD full list 1461
L00000071 G2C Homo sapiens BAYES-COLLINS-MOUSE-PSD-FULL Mouse cortex PSD full list (ortho) 1556
© G2C 2014. The Genes to Cognition Programme received funding from The Wellcome Trust and the EU FP7 Framework Programmes:
EUROSPIN (FP7-HEALTH-241498), SynSys (FP7-HEALTH-242167) and GENCODYS (FP7-HEALTH-241995).

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