G2Cdb::Gene report

Gene id
G00001924
Gene symbol
AMPH (HGNC)
Species
Homo sapiens
Description
amphiphysin
Orthologue
G00000675 (Mus musculus)

Databases (8)

Curated Gene
OTTHUMG00000023725 (Vega human gene)
Gene
ENSG00000078053 (Ensembl human gene)
273 (Entrez Gene)
1080 (G2Cdb plasticity & disease)
AMPH (GeneCards)
Literature
600418 (OMIM)
Marker Symbol
HGNC:471 (HGNC)
Protein Sequence
P49418 (UniProt)

Literature (41)

Pubmed - other

  • Characterization of domain-peptide interaction interface: a case study on the amphiphysin-1 SH3 domain.

    Hou T, Zhang W, Case DA and Wang W

    Department of Chemistry and Biochemistry, University of California at San Diego, La Jolla, CA 92093, USA.

    Many important protein-protein interactions are mediated by peptide recognition modular domains, such as the Src homology 3 (SH3), SH2, PDZ, and WW domains. Characterizing the interaction interface of domain-peptide complexes and predicting binding specificity for modular domains are critical for deciphering protein-protein interaction networks. Here, we propose the use of an energetic decomposition analysis to characterize domain-peptide interactions and the molecular interaction energy components (MIECs), including van der Waals, electrostatic, and desolvation energy between residue pairs on the binding interface. We show a proof-of-concept study on the amphiphysin-1 SH3 domain interacting with its peptide ligands. The structures of the human amphiphysin-1 SH3 domain complexed with 884 peptides were first modeled using virtual mutagenesis and optimized by molecular mechanics (MM) minimization. Next, the MIECs between domain and peptide residues were computed using the MM/generalized Born decomposition analysis. We conducted two types of statistical analyses on the MIECs to demonstrate their usefulness for predicting binding affinities of peptides and for classifying peptides into binder and non-binder categories. First, combining partial least squares analysis and genetic algorithm, we fitted linear regression models between the MIECs and the peptide binding affinities on the training data set. These models were then used to predict binding affinities for peptides in the test data set; the predicted values have a correlation coefficient of 0.81 and an unsigned mean error of 0.39 compared with the experimentally measured ones. The partial least squares-genetic algorithm analysis on the MIECs revealed the critical interactions for the binding specificity of the amphiphysin-1 SH3 domain. Next, a support vector machine (SVM) was employed to build classification models based on the MIECs of peptides in the training set. A rigorous training-validation procedure was used to assess the performances of different kernel functions in SVM and different combinations of the MIECs. The best SVM classifier gave satisfactory predictions for the test set, indicated by average prediction accuracy rates of 78% and 91% for the binding and non-binding peptides, respectively. We also showed that the performance of our approach on both binding affinity prediction and binder/non-binder classification was superior to the performances of the conventional MM/Poisson-Boltzmann solvent-accessible surface area and MM/generalized Born solvent-accessible surface area calculations. Our study demonstrates that the analysis of the MIECs between peptides and the SH3 domain can successfully characterize the binding interface, and it provides a framework to derive integrated prediction models for different domain-peptide systems.

    Journal of molecular biology 2008;376;4;1201-14

  • Modeling and prediction of binding affinities between the human amphiphysin SH3 domain and its peptide ligands using genetic algorithm-Gaussian processes.

    Zhou P, Tian F, Chen X and Shang Z

    Department of Chemistry, Zhejiang University, Hangzhou 310027, China.

    In this article, we discuss the application of the Gaussian process (GP) and other statistical methods (PLS, ANN, and SVM) for the modeling and prediction of binding affinities between the human amphiphysin SH3 domain and its peptide ligands. Divided physicochemical property scores of amino acids, involving significant hydrogen bond, electronic, hydrophobic, and steric properties, was used to characterize the peptide structures, and quantitative structure-affinity relationship models were then constructed by PLS, ANN, SVM, and GP coupled with genetic algorithm-variable selection. The results show that: (i) since the significant flexibility and high complexity possessed in polypeptide structures, linear PLS method was incapable of fulfilling a satisfying behavior on SH3 domain binding peptide dataset; (ii) the overfitting involved in training process has decreased the predictive power of ANN model to some extent; (iii) both SVM and GP have a good performance for SH3 domain binding peptide dataset. Moreover, by combining linear and nonlinear terms in the covariance function, the GP is capable of handling linear and nonlinear-hybrid relationship, and which thus obtained a more stable and predictable model than SVM. Analyses of GP models showed that diversified properties contribute remarkable effect to the interactions between the SH3 domain and the peptides. Particularly, steric property and hydrophobicity of P(2), electronic property of P(0), and electronic property and hydrogen bond property of P(-3) in decapeptide (P(4)P(3)P(2)P(1)P(0)P(-1)P(-2)P(-3)P(-4)P(-5)) significantly contribute to the binding affinities of SH3 domain-peptide interactions.

    Biopolymers 2008;90;6;792-802

  • Phosphorylation of amphiphysin I by minibrain kinase/dual-specificity tyrosine phosphorylation-regulated kinase, a kinase implicated in Down syndrome.

    Murakami N, Xie W, Lu RC, Chen-Hwang MC, Wieraszko A and Hwang YW

    Department of Molecular Biology, New York State Institute for Basic Research in Developmental Disabilities, Staten Island, New York 10314, USA. Noriko.Murakami@omr.state.ny.us

    Minibrain kinase/dual-specificity tyrosine phosphorylation-regulated kinase (Mnb/Dyrk1A) is a proline-directed serine/threonine kinase encoded in the Down syndrome critical region of human chromosome 21. This kinase has been shown to phosphorylate dynamin 1 and synaptojanin 1. Here we report that amphiphysin I (Amph I) is also a Mnb/Dyrk1A substrate. This kinase phosphorylated native Amph I in rodent brains and recombinant human Amph I expressed in Escherichia coli. Serine 293 (Ser-293) was identified as the major site, whereas serine 295 and threonine 310 were found as minor kinase sites. In cultured cells, recombinant Amph I was phosphorylated at Ser-293 by endogenous kinase(s). Because mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) has been suggested to phosphorylate Amph I at Ser-293, our efforts addressed whether Ser-293 is phosphorylated in vivo by MAPK/ERK or by Mnb/Dyrk1A. Overnight serum-withdrawal inactivated MAPK/ERK; nonetheless, Ser-293 was phosphorylated in Chinese hamster ovary and SY5Y cells. Epigallocatechin-3-gallate, a potent Mnb/Dyrk1A inhibitor in vitro, apparently reduced the phosphorylation at Ser-293, whereas PD98059, a potent MAPK/ERK inhibitor, did not. High frequency stimulation of mouse hippocampal slices reduced the phosphorylation at Ser-293, albeit in the midst of MAPK/ERK activation. The endophilin binding in vitro was inhibited by phosphorylating Amph I with Mnb/Dyrk1A. However, phosphorylation at Ser-293 did not appear to alter cellular distribution patterns of the protein. Our results suggest that Mnb/Dyrk1A, not MAPK/ERK, is responsible for in vivo phosphorylation of Amph I at Ser-293 and that phosphorylation changes the recruitment of endophilin at the endocytic sites.

    Funded by: NICHD NIH HHS: HD38295

    The Journal of biological chemistry 2006;281;33;23712-24

  • Antiamphiphysin-positive stiff-person syndrome associated with small cell lung cancer.

    Nguyen-Huu BK, Urban PP, Schreckenberger M, Dieterich M and Werhahn KJ

    Department of Neurology, University of Mainz, Mainz, Germany.

    The paraneoplastic amphiphysin(+) stiff-person syndrome (SPS) has so far only been described in women with breast adenocarcinoma. Here, we describe the rare case of a female patient with antiamphiphysin(+) SPS due to small cell cancer of the lung.

    Movement disorders : official journal of the Movement Disorder Society 2006;21;8;1285-7

  • Two mechanistically distinct forms of endocytosis in adrenal chromaffin cells: Differential effects of SH3 domains and amphiphysin antagonism.

    Elhamdani A, Azizi F, Solomaha E, Palfrey HC and Artalejo CR

    Department of Pharmacology, Wayne State University School of Medicine, Detroit, MI 48201, USA. aelhamda@med.wayne.edu

    We previously identified two forms of endocytosis using capacitance measurements in chromaffin cells: rapid endocytosis (RE), dynamin-1 dependent but clathrin-independent and slow endocytosis (SE), dynamin-2 and clathrin-dependent. Various recombinant SH3 domains that interact with the proline-rich domain of dynamin were introduced into single cells via the patch pipette. GST-SH3 domains of amphiphysin-1, intersectin-IC, and endophilin-I inhibited SE but had no effect on RE. Grb2-SH3 (N-terminal) or a mutant of amphiphysin-1-SH3 was inactive on either process. These data confirm that dynamin-1 dependent RE is independent of clathrin and show that amphiphysin is exclusively associated with clathrin and dynamin-2-dependent SE.

    Funded by: NIDDK NIH HHS: DK-58921; NIGMS NIH HHS: GM-56396; NIMH NIH HHS: MH-47181

    FEBS letters 2006;580;13;3263-9

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

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

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

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

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

    Nature 2005;437;7062;1173-8

  • Kinetics of Src homology 3 domain association with the proline-rich domain of dynamins: specificity, occlusion, and the effects of phosphorylation.

    Solomaha E, Szeto FL, Yousef MA and Palfrey HC

    Department of Neurobiology, Pharamacology, and Physiology, University of Chicago, IL 60637, USA.

    Dynamin function is mediated in part through association of its proline-rich domain (PRD) with the Src homology 3 (SH3) domains of several putative binding proteins. To assess the specificity and kinetics of this process, we undertook surface plasmon resonance studies of the interaction between isolated PRDs of dynamin-1 and -2 and several purified SH3 domains. Glutathione S-transferase-linked SH3 domains bound with high affinity (K(D) approximately 10 nm to 1 microm) to both dynamin-1 and -2. The simplest interaction appeared to take place with the amphiphysin-SH3 domain; this bound to a single high affinity site (K(D) approximately 10 nm) in the C terminus of dynamin-1 PRD, as predicted by previous studies. Binding to the dynamin-2 PRD was also monophasic but with a slightly lower affinity (K(D) approximately 25 nm). Endophilin-SH3 binding to both dynamin-1 and -2 PRDs was biphasic, with one high affinity site (K(D) approximately 14 nm) in the N terminus of the PRD and another lower affinity site (K(D) approximately 60 nm) in the C terminus of dynamin-1. The N-terminal site in dynamin-2 PRD had a 10-fold lower affinity for endophilin-SH3. Preloading of dynamin-1 PRD with the amphiphysin-SH3 domain partially occluded binding of the endophilin-SH3 domain, indicating overlap between the binding sites in the C terminus, but endophilin was still able to interact with the high affinity N-terminal site. This shows that more than one SH3 domain can simultaneously bind to the PRD and suggests that competition probably occurs in vivo between different SH3-containing proteins for the limited number of PXXP motifs. Endophilin-SH3 binding to the high affinity site was disrupted when dynamin-1 PRD was phosphorylated with Cdk5, indicating that this site overlaps the phosphorylation sites, but amphiphysin-SH3 binding was unaffected. Other SH3 domains showed similarly complex binding characteristics, and substantial differences were noted between the PRDs from dynamin-1 and -2. For example, SH3 domains from c-Src, Grb2, and intersectin bound only to the C-terminal half of dynamin-2 PRD but to both the N- and C-terminal portions of dynamin-1 PRD. Thus, differential binding of SH3 domain-containing proteins to dynamin-1 and -2 may contribute to the distinct functions performed by these isoforms.

    The Journal of biological chemistry 2005;280;24;23147-56

  • Phosphoproteomic analysis of the developing mouse brain.

    Ballif BA, Villén J, Beausoleil SA, Schwartz D and Gygi SP

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

    Proper development of the mammalian brain requires the precise integration of numerous temporally and spatially regulated stimuli. Many of these signals transduce their cues via the reversible phosphorylation of downstream effector molecules. Neuronal stimuli acting in concert have the potential of generating enormous arrays of regulatory phosphoproteins. Toward the global profiling of phosphoproteins in the developing brain, we report here the use of a mass spectrometry-based methodology permitting the first proteomic-scale phosphorylation site analysis of primary animal tissue, identifying over 500 protein phosphorylation sites in the developing mouse brain.

    Funded by: NHGRI NIH HHS: HG00041

    Molecular & cellular proteomics : MCP 2004;3;11;1093-101

  • 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

  • Regulation of synaptojanin 1 by cyclin-dependent kinase 5 at synapses.

    Lee SY, Wenk MR, Kim Y, Nairn AC and De Camilli P

    Department of Cell Biology and Howard Hughes Medical Institute, Yale University School of Medicine, 295 Congress Avenue, New Haven, CT 06510, USA.

    Synaptojanin 1 is a polyphosphoinositide phosphatase concentrated in presynaptic nerve terminals, where it dephosphorylates a pool of phosphatidylinositol 4,5-bisphosphate implicated in synaptic vesicle recycling. Like other proteins with a role in endocytosis, synaptojanin 1 undergoes constitutive phosphorylation in resting synapses and stimulation-dependent dephosphorylation by calcineurin. Here, we show that cyclin-dependent kinase 5 (Cdk5) phosphorylates synaptojanin 1 and regulates its function both in vitro and in intact synaptosomes. Cdk5 phosphorylation inhibited the inositol 5-phosphatase activity of synaptojanin 1, whereas dephosphorylation by calcineurin stimulated such activity. The activity of synaptojanin 1 was also stimulated by its interaction with endophilin 1, its major binding partner at the synapse. Notably, Cdk5 phosphorylated serine 1144, which is adjacent to the endophilin binding site. Mutation of serine 1144 to aspartic acid to mimic phosphorylation by Cdk5 inhibited the interaction of synaptojanin 1 with endophilin 1. These results suggest that Cdk5 and calcineurin may have an antagonistic role in the regulation of synaptojanin 1 recruitment and activity, and therefore in the regulation of phosphatidylinositol 4,5-bisphosphate turnover at synapses.

    Funded by: NCI NIH HHS: CA46128, P01 CA046128; NIDA NIH HHS: DA10044, P01 DA010044; NINDS NIH HHS: NS36251, R01 NS036251, R37 NS036251

    Proceedings of the National Academy of Sciences of the United States of America 2004;101;2;546-51

  • A putative role for intramolecular regulatory mechanisms in the adaptor function of amphiphysin in endocytosis.

    Farsad K, Slepnev V, Ochoa G, Daniell L, Haucke V, De Camilli P and Hauke V

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

    Amphiphysin 1 is a brain-specific protein enriched at the synapse and a major binding partner of several components of the clathrin-mediated endocytic machinery (Proc Natl Acad Sci USA 93 (1996) 331). It interacts with clathrin-coat proteins, dynamin, and membranes (Nat Cell Biol 1 (1999) 33; JBC). A role of amphiphysin in synaptic vesicle recycling is supported by both acute and chronic perturbation studies (Science 276 (1997) 259; Neuron 33 (2002) 789). Here we show that amphiphysin directly stimulates clathrin recruitment onto liposomes in an in vitro assay. Amphiphysin-dependent clathrin-coat recruitment is enhanced by the interaction of amphiphysin with dynamin. We also show that the amphiphysin SH3 domain binds full-length amphiphysin, likely via an internal poly-proline region, and that clathrin recruitment onto liposomes by amphiphysin is enhanced in the presence of the isolated amphiphysin SH3 domain. Expression of a mutant amphiphysin harboring two amino acid substitutions in the SH3 domain, and therefore unable to bind proline-containing motifs, induces an accumulation of large intracellular aggregates including amphiphysin, clathrin, AP-2, and other endocytic proteins, as well as a concomitant block of transferrin endocytosis. Thus, putative intramolecular interactions between the amphiphysin COOH-terminal SH3 domain and its internal poly-proline region may regulate clathrin recruitment onto membranes.

    Neuropharmacology 2003;45;6;787-96

  • The DNA sequence of human chromosome 7.

    Hillier LW, Fulton RS, Fulton LA, Graves TA, Pepin KH, Wagner-McPherson C, Layman D, Maas J, Jaeger S, Walker R, Wylie K, Sekhon M, Becker MC, O'Laughlin MD, Schaller ME, Fewell GA, Delehaunty KD, Miner TL, Nash WE, Cordes M, Du H, Sun H, Edwards J, Bradshaw-Cordum H, Ali J, Andrews S, Isak A, Vanbrunt A, Nguyen C, Du F, Lamar B, Courtney L, Kalicki J, Ozersky P, Bielicki L, Scott K, Holmes A, Harkins R, Harris A, Strong CM, Hou S, Tomlinson C, Dauphin-Kohlberg S, Kozlowicz-Reilly A, Leonard S, Rohlfing T, Rock SM, Tin-Wollam AM, Abbott A, Minx P, Maupin R, Strowmatt C, Latreille P, Miller N, Johnson D, Murray J, Woessner JP, Wendl MC, Yang SP, Schultz BR, Wallis JW, Spieth J, Bieri TA, Nelson JO, Berkowicz N, Wohldmann PE, Cook LL, Hickenbotham MT, Eldred J, Williams D, Bedell JA, Mardis ER, Clifton SW, Chissoe SL, Marra MA, Raymond C, Haugen E, Gillett W, Zhou Y, James R, Phelps K, Iadanoto S, Bubb K, Simms E, Levy R, Clendenning J, Kaul R, Kent WJ, Furey TS, Baertsch RA, Brent MR, Keibler E, Flicek P, Bork P, Suyama M, Bailey JA, Portnoy ME, Torrents D, Chinwalla AT, Gish WR, Eddy SR, McPherson JD, Olson MV, Eichler EE, Green ED, Waterston RH and Wilson RK

    Genome Sequencing Center, Washington University School of Medicine, Campus Box 8501, 4444 Forest Park Avenue, St Louis, Missouri 63108, USA.

    Human chromosome 7 has historically received prominent attention in the human genetics community, primarily related to the search for the cystic fibrosis gene and the frequent cytogenetic changes associated with various forms of cancer. Here we present more than 153 million base pairs representing 99.4% of the euchromatic sequence of chromosome 7, the first metacentric chromosome completed so far. The sequence has excellent concordance with previously established physical and genetic maps, and it exhibits an unusual amount of segmentally duplicated sequence (8.2%), with marked differences between the two arms. Our initial analyses have identified 1,150 protein-coding genes, 605 of which have been confirmed by complementary DNA sequences, and an additional 941 pseudogenes. Of genes confirmed by transcript sequences, some are polymorphic for mutations that disrupt the reading frame.

    Nature 2003;424;6945;157-64

  • Human chromosome 7: DNA sequence and biology.

    Scherer SW, Cheung J, MacDonald JR, Osborne LR, Nakabayashi K, Herbrick JA, Carson AR, Parker-Katiraee L, Skaug J, Khaja R, Zhang J, Hudek AK, Li M, Haddad M, Duggan GE, Fernandez BA, Kanematsu E, Gentles S, Christopoulos CC, Choufani S, Kwasnicka D, Zheng XH, Lai Z, Nusskern D, Zhang Q, Gu Z, Lu F, Zeesman S, Nowaczyk MJ, Teshima I, Chitayat D, Shuman C, Weksberg R, Zackai EH, Grebe TA, Cox SR, Kirkpatrick SJ, Rahman N, Friedman JM, Heng HH, Pelicci PG, Lo-Coco F, Belloni E, Shaffer LG, Pober B, Morton CC, Gusella JF, Bruns GA, Korf BR, Quade BJ, Ligon AH, Ferguson H, Higgins AW, Leach NT, Herrick SR, Lemyre E, Farra CG, Kim HG, Summers AM, Gripp KW, Roberts W, Szatmari P, Winsor EJ, Grzeschik KH, Teebi A, Minassian BA, Kere J, Armengol L, Pujana MA, Estivill X, Wilson MD, Koop BF, Tosi S, Moore GE, Boright AP, Zlotorynski E, Kerem B, Kroisel PM, Petek E, Oscier DG, Mould SJ, Döhner H, Döhner K, Rommens JM, Vincent JB, Venter JC, Li PW, Mural RJ, Adams MD and Tsui LC

    Department of Genetics and Genomic Biology, The Hospital for Sick Children, Toronto, Ontario, Canada, M5G 1X8. steve@genet.sickkids.on.ca

    DNA sequence and annotation of the entire human chromosome 7, encompassing nearly 158 million nucleotides of DNA and 1917 gene structures, are presented. To generate a higher order description, additional structural features such as imprinted genes, fragile sites, and segmental duplications were integrated at the level of the DNA sequence with medical genetic data, including 440 chromosome rearrangement breakpoints associated with disease. This approach enabled the discovery of candidate genes for developmental diseases including autism.

    Funded by: Canadian Institutes of Health Research: 38103; NIGMS NIH HHS: P01 GM061354

    Science (New York, N.Y.) 2003;300;5620;767-72

  • Amphiphysin1 inhibits vitronectin-mediated cell adhesion, spreading, and migration in vitro.

    Otsuka A, Hirose K, Kilimann MW and Kamata T

    Department of Molecular Biology and Biochemistry, Shinshu University School of Medicine, Asahi 3-1-1, Matsumoto, Nagano, Japan.

    To investigate the regulatory mechanism of cell adhesion, we have searched for cellular inhibitory factors which prevent cell adhesion. The brain cytosol was found to inhibit the adhesion of various transformed cells to the substratum. An inhibitory 120-kDa protein was purified by sequential column chromatography. Peptide sequencing revealed that the protein is identical to amphiphysin1. GST-amphiphysin1 suppressed the attachment of HeLa cells to the plate when cells were cultured in the serum-containing medium. Vitronectin, a major cell-adhesive protein in serum and a ligand to alpha(v)beta3 integrin, was responsible for this cell attachment, and the vitronectin action was blocked by GST-amphiphysin1. GST-amphiphysin1 also inhibited the vitronectin-mediated spreading and migration of malignant melanoma cells. Furthermore, GST-amphiphysin1 bound directly to vitronectin. These findings point to the interesting possibility that amphiphysin1 could be a useful tool to inhibit cell-adhesive vitronectin.

    Biochemical and biophysical research communications 2003;301;3;769-75

  • Characterization of Endophilin B1b, a brain-specific membrane-associated lysophosphatidic acid acyl transferase with properties distinct from endophilin A1.

    Modregger J, Schmidt AA, Ritter B, Huttner WB and Plomann M

    Center for Biochemistry II, Medical Faculty, Joseph-Stelzmann-Strasse 52, University of Cologne, Germany.

    We have characterized mammalian endophilin B1, a novel member of the endophilins and a representative of their B subgroup. The endophilins B show the same domain organization as the endophilins A, which contain an N-terminal domain responsible for lipid binding and lysophosphatidic acid acyl transferase activity, a central coiled-coil domain for oligomerization, a less conserved linker region, and a C-terminal Src homology 3 (SH3) domain. The endophilin B1 gene gives rise to at least three splice variants, endophilin B1a, which shows a widespread tissue distribution, and endophilins B1b and B1c, which appear to be brain-specific. Endophilin B1, like endophilins A, binds to palmitoyl-CoA, exhibits lysophosphatidic acid acyl transferase activity, and interacts with dynamin, amphiphysins 1 and 2, and huntingtin. However, in contrast to endophilins A, endophilin B1 does not bind to synaptojanin 1 and synapsin 1, and overexpression of its SH3 domain does not inhibit transferrin endocytosis. Consistent with this, immunofluorescence analysis of endophilin B1b transfected into fibroblasts shows an intracellular reticular staining, which in part overlaps with that of endogenous dynamin. Upon subcellular fractionation of brain and transfected fibroblasts, endophilin B1 is largely recovered in association with membranes. Together, our results suggest that the action of the endophilins is not confined to the formation of endocytic vesicles from the plasma membrane, with endophilin B1 being associated with, and presumably exerting a functional role at, intracellular membranes.

    The Journal of biological chemistry 2003;278;6;4160-7

  • Specific interactions of neuronal focal adhesion kinase isoforms with Src kinases and amphiphysin.

    Messina S, Onofri F, Bongiorno-Borbone L, Giovedì S, Valtorta F, Girault JA and Benfenati F

    Department of Experimental Medicine, Section of Human Physiology, University of Genova, Italy.

    Focal adhesion kinase (FAK) is a non-receptor tyrosine kinase that activates Src family kinases via SH2- and SH3-mediated interactions. Specific FAK isoforms (FAK+), responsive to depolarization and neurotransmitters, are enriched in neurons. We analyzed the interactions of endogenous FAK+ and recombinant FAK+ isoforms containing amino acid insertions (boxes 6,7,28) with an array of SH3 domains and the c-Src SH2/SH3 domain tandem. Endogenous FAK+ bound specifically to the SH3 domains of c-Src (but not n-Src), Fyn, Yes, phosphtidylinositol-3 kinase, amphiphysin II, amphiphysin I, phospholipase Cgamma and NH2-terminal Grb2. The inclusion of boxes 6,7 was associated with a significant decrease in the binding of FAK+ to the c-Src and Fyn SH3 domains, and a significant increase in the binding to the Src SH2 domain, as a consequence of the higher phosphorylation of Tyr-397. The novel interaction with the amphiphysin SH3 domain, involving the COOH-terminal proline-rich region of FAK, was confirmed by coimmunoprecipitation of the two proteins and a closely similar response to stimuli affecting the actin cytoskeleton. Moreover, an impairment of endocytosis was observed in synaptosomes after internalization of a proline-rich peptide corresponding to the site of interaction. The data account for the different subcellular distribution of FAK and Src kinases and the specific regulation of the transduction pathways linked to FAK activation in the brain and implicate FAK in the regulation of membrane trafficking in nerve terminals.

    Funded by: Telethon: 1131

    Journal of neurochemistry 2003;84;2;253-65

  • Accessory protein recruitment motifs in clathrin-mediated endocytosis.

    Brett TJ, Traub LM and Fremont DH

    Department of Pathology and Immunology, Washington University School of Medicine, St. Louis, MO 63110, USA.

    Clathrin-mediated endocytosis depends upon the interaction of accessory proteins with the alpha-ear of the AP-2 adaptor. We present structural characterization of these regulatory interactions. DPF and DPW motif peptides derived from eps15 and epsin bind in type I beta turn conformations to a conserved pocket on the alpha-ear platform. We show evidence for a second binding site that is DPW motif specific. The structure of a complex with an AP-2 binding segment from amphiphysin reveals a novel binding motif that we term FxDxF, which is engaged in an extended conformation by a unique surface of the platform domain. The FxDxF motif is also used by AP180 and the 170 kDa isoform of synaptojanin and can be found in several potential endocytic proteins, including HIP1, CD2AP, and PLAP. A mechanism of clathrin assembly regulation is suggested by three different AP-2 engagement modes.

    Structure (London, England : 1993) 2002;10;6;797-809

  • Novel splice variants of amphiphysin I are expressed in retina.

    Terada Y, Tsutsui K, Sano K, Hosoya O, Ohtsuki H, Tokunaga A and Tsutsui K

    Department of Ophthalmology, Okayama University Graduate School of Medicine and Dentistry, 2-5-1 Shikata-cho, Okayama, Japan.

    Using RT-PCR-based cDNA cloning, we identified novel splice variants of amphiphysin I, termed amph Ir, that are expressed specifically in retina. In comparison with the prototype amphiphysin I, amph Ir contained two novel insertions (inserts A and B) and one deletion. Insert A is only 9 bp in length but appears to be a determinant for the retina-specific expression. In contrast, insert B is a large domain of 1740 bp and two shorter transcripts with 3'-truncated insert B were also expressed. All the insert sequences were present as unidentified exons in the amphiphysin I gene on human chromosome 7. Western blot analysis of various rat tissues with anti-insert B antibody confirmed the presence and tissue specificity of the variant proteins.

    FEBS letters 2002;519;1-3;185-90

  • Dynamin is a minibrain kinase/dual specificity Yak1-related kinase 1A substrate.

    Chen-Hwang MC, Chen HR, Elzinga M and Hwang YW

    Molecular Biology Department, New York State Institute for Basic Research in Developmental Disabilities, Staten Island, New York 10314, USA.

    The minibrain kinase (Mnbk)/dual specificity Yak 1-related kinase 1A (Dyrk1A) gene is implicated in the mental retardation associated with Down's syndrome. It encodes a proline-directed serine/threonine kinase whose function has yet to be defined. We have used a solid-phase Mnbk/Dyrk1A kinase assay to aid in the search for the cellular Mnbk/Dyrk1A substrates. The assay revealed that rat brain contains two cytosolic proteins, one with a molecular mass of 100 kDa and one with a molecular mass of 140 kDa, that were prominently phosphorylated by Mnbk/Dyrk1A. The 100-kDa protein was purified and identified as dynamin 1. The conclusion was further supported by evidence that a recombinant glutathione S-transferase fusion protein containing dynamin isoform 1aa was phosphorylated by Mnbk/Dyrk1A. In addition to isoform 1aa, Mnbk/Dyrk1A also phosphorylated isoforms 1ab and 2aa but not human MxA protein when analyzed by the solid-phase kinase assay. Upon Mnbk/Dyrk1A phosphorylation, the interaction of dynamin 1 with the Src homology 3 domain of amphiphysin 1 was reduced. However, when Mnbk/Dyrk1A phosphorylation was allowed to proceed more extensively, the phosphorylation enhanced rather than reduced the binding of dynamin 1 to amphiphysin 1. The result suggests that Mnbk/Dyrk1A can play a dual role in regulating the interaction of dynamin 1 with amphiphysin 1. Mnbk/Dyrk1A phosphorylation also reduced the interaction of dynamin with endophilin 1, whereas the same phosphorylation enhanced the binding of dynamin 1 to Grb2. Nevertheless, the dual function of Mnbk/Dyrk1A phosphorylation was not observed for the interaction of dynamin 1 with endophilin 1 or Grb2. The interactions of dynamin with amphiphysin and endophilin are essential for the formation of endocytic complexes; our results suggest that Mnbk/Dyrk1A may function as a regulator controlling the assembly of endocytic apparatus.

    Funded by: NICHD NIH HHS: HD35870, HD38295

    The Journal of biological chemistry 2002;277;20;17597-604

  • Amphiphysin 1 binds the cyclin-dependent kinase (cdk) 5 regulatory subunit p35 and is phosphorylated by cdk5 and cdc2.

    Floyd SR, Porro EB, Slepnev VI, Ochoa GC, Tsai LH and De Camilli P

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

    Amphiphysin 1 is a phosphoprotein expressed at high levels in neurons, where it participates in synaptic vesicle endocytosis and neurite outgrowth. It is a substrate for cyclin-dependent kinase (cdk) 5, a member of the cyclin-dependent protein kinase family, which has been functionally linked to neuronal migration and neurite outgrowth via its action on the actin cytoskeleton. The yeast homologue of amphiphysin, Rvs167, functions in endocytosis and actin dynamics, is phosphorylated by the cdk5 homologue Pho85, and binds the Pho85 regulatory subunit Pcl2. We show here that amphiphysin 1 interacts with the cdk5-activating subunit p35 and that this interaction is mediated by the conserved NH2-terminal region of amphiphysin. Amphiphysin 1 colocalizes with p35 in the growth cones of neurons and at actin-rich peripheral lamellipodia in transfected fibroblasts. Amphiphysin is phosphorylated by cdk5 in a region including serines 272, 276, and 285. Amphiphysin 1 is also phosphorylated by the cdc2/cyclin B kinase complex in the same region and undergoes mitotic phosphorylation in dividing cells. These data indicate that phosphorylation by members of the cyclin-dependent kinase family is a conserved property of amphiphysin and suggest that this phosphorylation may play an important physiological role both in mitosis and in differentiated cells.

    Funded by: NCI NIH HHS: CA46128; NINDS NIH HHS: NS36251, NS37007

    The Journal of biological chemistry 2001;276;11;8104-10

  • The calcineurin-binding protein cain is a negative regulator of synaptic vesicle endocytosis.

    Lai MM, Luo HR, Burnett PE, Hong JJ and Snyder SH

    Department of Neuroscience, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA.

    During neurotransmitter release, exocytosed neurotransmitter vesicles are recycled by endocytosis, which involves the assembly of a complex of endocytic proteins. Assembly of endocytic proteins into a functional complex depends on their dephosphorylation by calcineurin, a calcium-sensitive protein phosphatase and the inhibitory target of immunosuppressive drugs cyclosporin A and FK506. Cain is a recently identified protein inhibitor of calcineurin. We now provide evidence that cain is a component of the endocytic protein complex. The proline-rich region of cain forms a stable association with the SH3 domain of amphiphysin 1. Using a transferrin uptake assay, we found that overexpression of cain in HEK293 cells blocks endocytosis as potently as expression of a dominant negative dynamin 1 construct. The use of other calcineurin inhibitors such as cyclosporin A and FK506 also blocks endocytosis. Since binding of cain to amphiphysin 1 does not affect amphiphysin's interaction with other endocytic proteins, our results suggest that cain negatively regulates synaptic vesicle endocytosis by inhibiting calcineurin activity, rather than sterically interfering with the assembly of the endocytic protein complex.

    Funded by: NIDA NIH HHS: DA-00074; NIGMS NIH HHS: GM-07309; NIMH NIH HHS: MH-18501

    The Journal of biological chemistry 2000;275;44;34017-20

  • Specificity of the binding of synapsin I to Src homology 3 domains.

    Onofri F, Giovedi S, Kao HT, Valtorta F, Bongiorno Borbone L, De Camilli P, Greengard P and Benfenati F

    Department of Experimental Medicine, Section of Physiology, University of Genova, Via Benedetto XV 3, I-16132 Genova, Italy.

    Synapsins are synaptic vesicle-associated phosphoproteins involved in synapse formation and regulation of neurotransmitter release. Recently, synapsin I has been found to bind the Src homology 3 (SH3) domains of Grb2 and c-Src. In this work we have analyzed the interactions between synapsins and an array of SH3 domains belonging to proteins involved in signal transduction, cytoskeleton assembly, or endocytosis. The binding of synapsin I was specific for a subset of SH3 domains. The highest binding was observed with SH3 domains of c-Src, phospholipase C-gamma, p85 subunit of phosphatidylinositol 3-kinase, full-length and NH(2)-terminal Grb2, whereas binding was moderate with the SH3 domains of amphiphysins I/II, Crk, alpha-spectrin, and NADPH oxidase factor p47(phox) and negligible with the SH3 domains of p21(ras) GTPase-activating protein and COOH-terminal Grb2. Distinct sites in the proline-rich COOH-terminal region of synapsin I were found to be involved in binding to the various SH3 domains. Synapsin II also interacted with SH3 domains with a partly distinct binding pattern. Phosphorylation of synapsin I in the COOH-terminal region by Ca(2+)/calmodulin-dependent protein kinase II or mitogen-activated protein kinase modulated the binding to the SH3 domains of amphiphysins I/II, Crk, and alpha-spectrin without affecting the high affinity interactions. The SH3-mediated interaction of synapsin I with amphiphysins affected the ability of synapsin I to interact with actin and synaptic vesicles, and pools of synapsin I and amphiphysin I were shown to associate in isolated nerve terminals. The ability to bind multiple SH3 domains further implicates the synapsins in signal transduction and protein-protein interactions at the nerve terminal level.

    Funded by: NIA NIH HHS: AG15072; NIMH NIH HHS: MH39327; Telethon: 1131

    The Journal of biological chemistry 2000;275;38;29857-67

  • Inhibition of phospholipase D by amphiphysins.

    Lee C, Kim SR, Chung JK, Frohman MA, Kilimann MW and Rhee SG

    Laboratory of Cell Signaling, NHLBI, National Institutes of Health, Bethesda, Maryland 20892-0320, USA.

    Two distinct proteins inhibiting phospholipase D (PLD) activity in rat brain cytosol were previously purified and identified as synaptojanin and AP180, which are specific to nerve terminals and associate with the clathrin coat. Two additional PLD-inhibitory proteins have now been purified and identified as the amphiphysins I and II, which forms a heterodimer that also associates with the clathrin coat. Bacterially expressed recombinant amphiphysins inhibited both PLD1 and PLD2 isozymes in vitro with a potency similar to that of brain amphiphysin (median inhibitory concentration of approximately 15 nm). Expressions of either amphiphysin in COS-7 cells reduced activity of endogenous PLD as well as exogenously expressed PLD1 and PLD2. Coprecipitation experiments suggested that the inhibitory effect of amphiphysins results from their direct interaction with PLDs. The NH(2) terminus of amphiphysin I was critical for both inhibition of and binding to PLD. Phosphatidic acid formed by signal-induced PLD is thought to be required for the assembly of clathrin-coated vesicles during endocytosis. Thus, the inhibition of PLD by amphiphysins, synaptojanin, and AP180 might play an important role in synaptic vesicle trafficking.

    The Journal of biological chemistry 2000;275;25;18751-8

  • Tandem arrangement of the clathrin and AP-2 binding domains in amphiphysin 1 and disruption of clathrin coat function by amphiphysin fragments comprising these sites.

    Slepnev VI, Ochoa GC, Butler MH and De Camilli P

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

    Amphiphysin 1 and 2 are proteins implicated in the recycling of synaptic vesicles in nerve terminals. They interact with dynamin and synaptojanin via their COOH-terminal SH3 domain, whereas their central regions contain binding sites for clathrin and for the clathrin adaptor AP-2. We have defined here amino acids of amphiphysin 1 crucial for binding to AP-2 and clathrin. Overexpression in Chinese hamster ovary cells of an amphiphysin 1 fragment that binds both AP-2 and clathrin resulted in a segregation of clathrin, which acquired a diffuse distribution, from AP-2, which accumulated at patches also positive for Eps15. These effects correlated with a block in clathrin-mediated endocytosis. A fragment selectively interacting with clathrin produced a similar effect. These results can be explained by the binding of amphiphysin to the NH(2)-terminal domain of clathrin and by a competition with the binding of this domain to the beta-subunit of AP-2 and AP180. The interaction of amphiphysin 1 with either clathrin or AP-2 did not prevent its interaction with dynamin, supporting the existence of tertiary complexes between these proteins. Together with previous evidence indicating a direct interaction between amphiphysin and membrane lipids, these findings support a model in which amphiphysin acts as a multifunctional adaptor linking the membrane to coat proteins and coat proteins to dynamin and synaptojanin.

    Funded by: NCI NIH HHS: CA46128; NINDS NIH HHS: NS36251

    The Journal of biological chemistry 2000;275;23;17583-9

  • The SH3 domains of endophilin and amphiphysin bind to the proline-rich region of synaptojanin 1 at distinct sites that display an unconventional binding specificity.

    Cestra G, Castagnoli L, Dente L, Minenkova O, Petrelli A, Migone N, Hoffmüller U, Schneider-Mergener J and Cesareni G

    Dipartimento di Biologia, Università di Roma Tor Vergata, Rome 00133, Italy.

    The proline-rich domain of synaptojanin 1, a synaptic protein with phosphatidylinositol phosphatase activity, binds to amphiphysin and to a family of recently discovered proteins known as the SH3p4/8/13, the SH3-GL, or the endophilin family. These interactions are mediated by SH3 domains and are believed to play a regulatory role in synaptic vesicle recycling. We have precisely mapped the target peptides on human synaptojanin that are recognized by the SH3 domains of endophilins and amphiphysin and proven that they are distinct. By a combination of different approaches, selection of phage displayed peptide libraries, substitution analyses of peptides synthesized on cellulose membranes, and a peptide scan spanning a 252-residue long synaptojanin fragment, we have concluded that amphiphysin binds to two sites, PIRPSR and PTIPPR, whereas endophilin has a distinct preferred binding site, PKRPPPPR. The comparison of the results obtained by phage display and substitution analysis permitted the identification of proline and arginine at positions 4 and 6 in the PIRPSR and PTIPPR target sequence as the major determinants of the recognition specificity mediated by the SH3 domain of amphiphysin 1. More complex is the structural rationalization of the preferred endophilin ligands where SH3 binding cannot be easily interpreted in the framework of the "classical" type I or type II SH3 binding models. Our results suggest that the binding repertoire of SH3 domains may be more complex than originally predicted.

    The Journal of biological chemistry 1999;274;45;32001-7

  • Role of phosphorylation in regulation of the assembly of endocytic coat complexes.

    Slepnev VI, Ochoa GC, Butler MH, Grabs D and De Camilli P

    Howard Hughes Medical Institute and Department of Cell Biology, Yale University School of Medicine, 295 Congress Avenue, New Haven, CT 06510, USA.

    Clathrin-mediated endocytosis involves cycles of assembly and disassembly of clathrin coat components and their accessory proteins. Dephosphorylation of rat brain extract was shown to promote the assembly of dynamin 1, synaptojanin 1, and amphiphysin into complexes that also included clathrin and AP-2. Phosphorylation of dynamin 1 and synaptojanin 1 inhibited their binding to amphiphysin, whereas phosphorylation of amphiphysin inhibited its binding to AP-2 and clathrin. Thus, phosphorylation regulates the association and dissociation cycle of the clathrin-based endocytic machinery, and calcium-dependent dephosphorylation of endocytic proteins could prepare nerve terminals for a burst of endocytosis.

    Funded by: NCI NIH HHS: CA46128; NINDS NIH HHS: NS36251

    Science (New York, N.Y.) 1998;281;5378;821-4

  • 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

  • Expression of amphiphysin I, an autoantigen of paraneoplastic neurological syndromes, in breast cancer.

    Floyd S, Butler MH, Cremona O, David C, Freyberg Z, Zhang X, Solimena M, Tokunaga A, Ishizu H, Tsutsui K and De Camilli P

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

    Amphiphysin I is a 128 kD protein highly concentrated in nerve terminals, where it has a putative role in endocytosis. It is a dominant autoantigen in patients with stiff-man syndrome associated with breast cancer, as well as in other paraneoplastic autoimmune neurological disorders. To elucidate the connection between amphiphysin I autoimmunity and cancer, we investigated its expression in breast cancer tissue. We report that amphiphysin I was expressed as two isoforms of 128 and 108 kD in the breast cancer of a patient with anti-amphiphysin I antibodies and paraneoplastic sensory neuronopathy. Amphiphysin I was also detectable at variable levels in several other human breast cancer tissues and cell lines and at low levels in normal mammary tissue and a variety of other non-neuronal tissues. The predominant amphiphysin I isoform expressed outside the brain in humans is the 108 kD isoform which represents an alternatively spliced variant of neuronal amphiphysin I missing a 42 amino acid insert. Our study suggests a link between amphiphysin I expression in cancer and amphiphysin I autoimmunity. The enhanced expression of amphiphysin I in some forms of cancer supports the hypothesis that amphiphysin family members may play a role in the biology of cancer cells.

    Funded by: NCI NIH HHS: CA46128, P50 CA58183; NINDS NIH HHS: NS36251; Telethon: D.061

    Molecular medicine (Cambridge, Mass.) 1998;4;1;29-39

  • Synaptojanin forms two separate complexes in the nerve terminal. Interactions with endophilin and amphiphysin.

    Micheva KD, Kay BK and McPherson PS

    Department of Neurology and Neurosurgery, Montreal Neurological Institute, McGill University, 3801 Rue University, Montreal, Quebec H3A 2B4, Canada.

    Endophilin is a recently discovered src homology 3 domain-containing protein that is a major in vitro binding partner for synaptojanin. To further characterize endophilin, we generated an antipeptide antibody. Endophilin is enriched in the brain, and immunofluorescence analysis reveals a high concentration of the protein in synaptic terminals, where it colocalizes with synaptojanin. In vitro binding assays demonstrate that endophilin binds through its src homology 3 domain to synaptojanin, and immunoprecipitation analysis with the antiendophilin antibody reveals that endophilin is stably associated with synaptojanin in the nerve terminal. Immunoprecipitation with an antibody against amphiphysin I and II, which interact through their src homology 3 domains with dynamin and synaptojanin at sites distinct from those for endophilin, reveals a second stable complex, which includes dynamin and synaptojanin but excludes endophilin. These data demonstrate that synaptojanin is present in two separate complexes in the nerve terminal and support an important role for endophilin in the regulation of synaptojanin function.

    The Journal of biological chemistry 1997;272;43;27239-45

  • Amphiphysin heterodimers: potential role in clathrin-mediated endocytosis.

    Wigge P, Köhler K, Vallis Y, Doyle CA, Owen D, Hunt SP and McMahon HT

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

    Amphiphysin (Amph) is a src homology 3 domain-containing protein that has been implicated in synaptic vesicle endocytosis as a result of its interaction with dynamin. In a screen for novel members of the amphiphysin family, we identified Amph2, an isoform 49% identical to the previously characterized Amph1 protein. The subcellular distribution of this isoform parallels Amph1, both being enriched in nerve terminals. Like Amph1, a role in endocytosis at the nerve terminal is supported by the rapid dephosphorylation of Amph2 on depolarization. Importantly, the two isoforms can be coimmunoprecipitated from the brain as an equimolar complex, suggesting that the two isoforms act in concert. As determined by cross-linking of brain extracts, the Amph1-Amph2 complex is a 220- to 250-kDa heterodimer. COS cells transfected with either Amph1 or Amph2 show greatly reduced transferrin uptake, but coexpression of the two proteins rescues this defect, supporting a role for the heterodimer in clathrin-mediated endocytosis. Although the src homology 3 domains of both isoforms interact with dynamin, the heterodimer can associate with multiple dynamin molecules in vitro and activates dynamin's GTPase activity. We propose that it is an amphiphysin heterodimer that drives the recruitment of dynamin to clathrin-coated pits in endocytosing nerve terminals.

    Molecular biology of the cell 1997;8;10;2003-15

  • SH3 domain-dependent interactions of endophilin with amphiphysin.

    Micheva KD, Ramjaun AR, Kay BK and McPherson PS

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

    Amphiphysin I and II are nerve terminal-enriched proteins thought to function in synaptic vesicle endocytosis. In addition to a C-terminal SH3 domain, the proteins contain a highly conserved putative SH3 binding site and numerous consensus phosphorylation sites. We now demonstrate that amphiphysin I but not amphiphysin II is a phosphoprotein which undergoes dephosphorylation during nerve terminal depolarization. Further, both amphiphysin I and II interact with the SH3 domain of endophilin, a synaptically enriched protein implicated in synaptic vesicle endocytosis. The interaction is direct and mediated through a 43 amino acid region of amphiphysin containing the putative SH3 binding site. These data further support a role for amphiphysin I, II and endophilin in synaptic vesicle endocytosis.

    FEBS letters 1997;414;2;308-12

  • 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

  • Amphiphysin II (SH3P9; BIN1), a member of the amphiphysin/Rvs family, is concentrated in the cortical cytomatrix of axon initial segments and nodes of ranvier in brain and around T tubules in skeletal muscle.

    Butler MH, David C, Ochoa GC, Freyberg Z, Daniell L, Grabs D, Cremona O and De Camilli P

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

    Amphiphysin (amphiphysin I), a dominant autoantigen in paraneoplastic Stiff-man syndrome, is a neuronal protein highly concentrated in nerve terminals, where it has a putative role in endocytosis. The yeast homologue of amphiphysin, Rvs167, has pleiotropic functions, including a role in endocytosis and in actin dynamics, suggesting that amphiphysin may also be implicated in the function of the presynaptic actin cytoskeleton. We report here the characterization of a second mammalian amphiphysin gene, amphiphysin II (SH3P9; BIN1), which encodes products primarily expressed in skeletal muscle and brain, as differentially spliced isoforms. In skeletal muscle, amphiphysin II is concentrated around T tubules, while in brain it is concentrated in the cytomatrix beneath the plasmamembrane of axon initial segments and nodes of Ranvier. In both these locations, amphiphysin II is colocalized with splice variants of ankyrin3 (ankyrinG), a component of the actin cytomatrix. In the same regions, the presence of clathrin has been reported. These findings support the hypothesis that, even in mammalian cells, amphiphysin/Rvs family members have a role both in endocytosis and in actin function and suggest that distinct amphiphysin isoforms contribute to define distinct domains of the cortical cytoplasm. Since amphiphysin II (BIN1) was reported to interact with Myc, it may also be implicated in a signaling pathway linking the cortical cytoplasm to nuclear function.

    Funded by: NCI NIH HHS: CA46128, P01 CA046128

    The Journal of cell biology 1997;137;6;1355-67

  • The SH3 domain of amphiphysin binds the proline-rich domain of dynamin at a single site that defines a new SH3 binding consensus sequence.

    Grabs D, Slepnev VI, Songyang Z, David C, Lynch M, Cantley LC and De Camilli P

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

    Amphiphysin is an SH3 domain-containing neuronal protein that is highly concentrated in nerve terminals where it interacts via its SH3 domain with dynamin I, a GTPase implicated in synaptic vesicle endocytosis. We show here that the SH3 domain of amphiphysin, but not a mutant SH3 domain, bound with high affinity to a single site in the long proline-rich region of human dynamin I, that this site was distinct from the binding sites for other SH3 domains, and that the mutation of two adjacent amino acids in dynamin I was sufficient to abolish binding. The dynamin I sequence critically required for amphiphysin binding (PSRPNR) fits in the novel SH3 binding consensus identified for the SH3 domain of amphiphysin via a combinatorial peptide library approach: PXRPXR(H)R(H). Our data demonstrate that the long proline-rich stretch present in dynamin I contained multiple SH3 domain binding sites that recognize interacting proteins with high specificity.

    Funded by: NIGMS NIH HHS: R01 GM056203

    The Journal of biological chemistry 1997;272;20;13419-25

  • A role of amphiphysin in synaptic vesicle endocytosis suggested by its binding to dynamin in nerve terminals.

    David C, McPherson PS, Mundigl O and de Camilli P

    Department of Cell Biology, Yale University School of Medicine, New Haven, CT 06510, USA.

    Amphiphysin, a major autoantigen in paraneoplastic Stiff-Man syndrome, is an SH3 domain-containing neuronal protein, concentrated in nerve terminals. Here, we demonstrate a specific, SH3 domain-mediated, interaction between amphiphysin and dynamin by gel overlay and affinity chromatography. In addition, we show that the two proteins are colocalized in nerve terminals and are coprecipitated from brain extracts consistent with their interactions in situ. We also report that a region of amphiphysin distinct from its SH3 domain mediates its binding to the alpha c subunit of AP2 adaptin, which is also concentrated in nerve terminals. These findings support a role of amphiphysin in synaptic vesicle endocytosis.

    Funded by: NCI NIH HHS: CA46128

    Proceedings of the National Academy of Sciences of the United States of America 1996;93;1;331-5

  • Primary structure of human amphiphysin, the dominant autoantigen of paraneoplastic stiff-man syndrome, and mapping of its gene (AMPH) to chromosome 7p13-p14.

    Yamamoto R, Li X, Winter S, Francke U and Kilimann MW

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

    Amphiphysin is a protein peripherally associated with synaptic vesicles. It is expressed in many neurons, certain endocrine cell types, and spermatocytes. Autoantibodies against amphiphysin occur in patients afflicted with a rare neurologic autoimmune disease, paraneoplastic Stiff-Man syndrome. To provide a basis for the understanding of anti-amphiphysin autoimmunity, we have cloned cDNAs and determined the primary structure of human amphiphysin. Comparison with chicken amphiphysin defines domains of low and high amino acid sequence conservation. As a candidate for heritable disorders of the nervous system, endocrine tissues or male fertility, the human amphiphysin gene was mapped to chromosome 7, region p13-p14.

    Funded by: NHGRI NIH HHS: HG00298

    Human molecular genetics 1995;4;2;265-8

  • Autoimmunity in stiff-Man syndrome with breast cancer is targeted to the C-terminal region of human amphiphysin, a protein similar to the yeast proteins, Rvs167 and Rvs161.

    David C, Solimena M and De Camilli P

    Department of Cell Biology, Yale University School of Medicine, Boyer Center for Molecular Medicine, New Haven, CT 06510.

    Amphiphysin, a neuronal protein first identified in chicken synaptic membranes, is the autoantigen of Stiff-Man Syndrome (SMS) associated with breast cancer. We have now cloned human amphiphysin and found the N- and C-terminal domains of the protein to be highly conserved between chicken and human. Patient autoantibodies have a distinct pattern of reactivity with amphiphysin, and the dominant autoepitope is located in its C-terminal region, which contains an SH3 domain. Portions of chicken and human amphiphysin are also homologous to portions of Rvs167 and Rvs161, two yeast proteins which are involved in cell entry into stationary phase upon exposure to unfavourable growth conditions.

    Funded by: NIAID NIH HHS: AI30248; NIDDK NIH HHS: DK43078

    FEBS letters 1994;351;1;73-9

  • The synaptic vesicle-associated protein amphiphysin is the 128-kD autoantigen of Stiff-Man syndrome with breast cancer.

    De Camilli P, Thomas A, Cofiell R, Folli F, Lichte B, Piccolo G, Meinck HM, Austoni M, Fassetta G, Bottazzo G, Bates D, Cartlidge N, Solimena M, Kilimann MW et al.

    Department of Cell Biology, Yale University School of Medicine, New Haven, Connecticut 06510.

    Stiff-Man syndrome (SMS) is a rare disease of the central nervous system (CNS) characterized by progressive rigidity of the body musculature with superimposed painful spasms. An autoimmune origin of the disease has been proposed. In a caseload of more than 100 SMS patients, 60% were found positive for autoantibodies directed against the GABA-synthesizing enzyme glutamic acid decarboxylase (GAD). Few patients, all women affected by breast cancer, were negative for GAD autoantibodies but positive for autoantibodies directed against a 128-kD synaptic protein. We report here that this antigen is amphiphysin. GAD and amphiphysin are nonintrinsic membrane proteins that are concentrated in nerve terminals, where a pool of both proteins is associated with the cytoplasmic surface of synaptic vesicles. GAD and amphiphysin are the only two known targets of CNS autoimmunity with this distribution. This finding suggests a possible link between autoimmunity directed against cytoplasmic proteins associated with synaptic vesicles and SMS.

    The Journal of experimental medicine 1993;178;6;2219-23

  • Amphiphysin, a novel protein associated with synaptic vesicles.

    Lichte B, Veh RW, Meyer HE and Kilimann MW

    Institut für Physiologische Chemie, Ruhr-Universität Bochum, FRG.

    To obtain access to novel proteins of the neuronal synapse, we have raised antisera against proteins of synaptic plasma membranes and used them for immunoscreening brain cDNA expression libraries. One of the newly isolated cDNAs encodes an acidic protein of 75 kDa with a distinct architecture of structural domains and multiple potential phosphorylation sites. Light and electron microscopy employing monospecific antisera raised against the expression product indicate a synapse-specific, presynaptic localization of this protein in many synapses of the chicken and rat nervous system. Its overall distribution in brain is very similar to that of synaptophysin, a ubiquitous protein of synaptic vesicles. In addition to brain, the protein or its mRNA is expressed in adrenal gland and anterior and posterior pituitary, but was not detected in a variety of other tissues. In controlled pore glass chromatography the native protein copurifies with synaptic vesicles and largely remains associated with them under various washing conditions. However, its amino acid sequence is very hydrophilic and it segregates into the aqueous phase in detergent phase partition. An earlier step of synaptic vesicle purification, sucrose cushion centrifugation, separates a vesicle-bound fraction of this protein from an unbound fraction. This seems to be a new, perhaps peripheral, protein of synaptic vesicles for which we propose the name, amphiphysin.

    The EMBO journal 1992;11;7;2521-30

Gene lists (8)

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
L00000016 G2C Homo sapiens Human PSP Human orthologues of mouse PSP adapted from Collins et al (2006) 1121
L00000059 G2C Homo sapiens BAYES-COLLINS-HUMAN-PSD-CONSENSUS Human cortex PSD consensus 748
L00000061 G2C Homo sapiens BAYES-COLLINS-MOUSE-PSD-CONSENSUS Mouse cortex PSD consensus (ortho) 984
L00000069 G2C Homo sapiens BAYES-COLLINS-HUMAN-PSD-FULL Human cortex biopsy PSD full list 1461
L00000071 G2C Homo sapiens BAYES-COLLINS-MOUSE-PSD-FULL Mouse cortex PSD full list (ortho) 1556
© G2C 2014. The Genes to Cognition Programme received funding from The Wellcome Trust and the EU FP7 Framework Programmes:
EUROSPIN (FP7-HEALTH-241498), SynSys (FP7-HEALTH-242167) and GENCODYS (FP7-HEALTH-241995).

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