Amphiphysin Heterodimers: Potential Role in Clathrin-mediated Endocytosis

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.

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αII-Spectrin interacts with Tes and EVL, two actin-binding proteins located at cell contacts

Biochemical Journal ◽

10.1042/bj20041502 ◽

2005 ◽

Vol 388 (2) ◽

pp. 631-638 ◽

Cited By ~ 35

Author(s):

Björn ROTTER ◽

Odile BOURNIER ◽

Gael NICOLAS ◽

Didier DHERMY ◽

Marie-Christine LECOMTE

Keyword(s):

Binding Protein ◽

Focal Adhesions ◽

Membrane Skeleton ◽

Actin Dynamics ◽

Actin Binding ◽

Actin Binding Protein ◽

Src Homology 3 ◽

Src Homology 3 Domain ◽

Src Homology

The spectrin-based membrane skeleton, a multi-protein scaffold attached to diverse cellular membranes, is presumed to be involved in the stabilization of membranes, the establishment of membrane domains as well as in vesicle trafficking and nuclear functions. Spectrin tetramers made of α- and β-subunits are linked to actin microfilaments, forming a network that binds a multitude of proteins. The most prevalent α-spectrin subunit in non-erythroid cells, αII-spectrin, contains two particular spectrin repeats in its central region, α9 and α10, which host an Src homology 3 domain, a tissue-specific spliced sequence of 20 residues, a calmodulin-binding site and major cleavage sites for caspases and calpains. Using yeast two-hybrid screening of kidney libraries, we identified two partners of the α9-α10 repeats: the potential tumour suppressor Tes, an actin-binding protein mainly located at focal adhesions; and EVL (Ena/vasodilator-stimulated phosphoprotein-like protein), another actin-binding protein, equally recruited at focal adhesions. Interactions between spectrin and overexpressed Tes and EVL were confirmed by co-immunoprecipitation. In vitro studies showed that the interaction between Tes and spectrin is mediated by a LIM (Lin-11, Isl-1 and Mec3) domain of Tes and by the α10 repeat of αII-spectrin whereas EVL interacts with the Src homology 3 domain located within the α9 repeat. Moreover, we describe an in vitro interaction between Tes and EVL, and a co-localization of these two proteins at focal adhesions. These interactions between αII-spectrin, Tes and EVL indicate new functions for spectrin in actin dynamics and focal adhesions.

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Src Homology 3-Domain Growth Factor Receptor-Bound 2-Like (Endophilin) Interacting Protein 1, a Novel Neuronal Protein that Regulates Energy Balance

Endocrinology ◽

10.1210/en.2005-0282 ◽

2005 ◽

Vol 146 (9) ◽

pp. 3757-3764 ◽

Cited By ~ 23

Author(s):

James Trevaskis ◽

Ken Walder ◽

Victoria Foletta ◽

Lyndal Kerr-Bayles ◽

Janine McMillan ◽

...

Keyword(s):

Body Weight ◽

Growth Factor ◽

Growth Factor Receptor ◽

Domain Growth ◽

Interacting Protein ◽

Src Homology 3 ◽

Src Homology 3 Domain ◽

Src Homology ◽

The Brain ◽

Growth Factor Receptor Bound

Abstract To identify genes involved in the central regulation of energy balance, we compared hypothalamic mRNA from lean and obese Psammomys obesus, a polygenic model of obesity, using differential display PCR. One mRNA transcript was observed to be elevated in obese, and obese diabetic, P. obesus compared with lean animals and was subsequently found to be increased 4-fold in the hypothalamus of lethal yellow agouti (Ay/a) mice, a murine model of obesity and diabetes. Intracerebroventricular infusion of antisense oligonucleotide targeted to this transcript selectively suppressed its hypothalamic mRNA levels and resulted in loss of body weight in both P. obesus and Sprague Dawley rats. Reductions in body weight were mediated by profoundly reduced food intake without a concomitant reduction in metabolic rate. Yeast two-hybrid screening, and confirmation in mammalian cells by bioluminescence resonance energy transfer analysis, demonstrated that the protein it encodes interacts with endophilins, mediators of synaptic vesicle recycling and receptor endocytosis in the brain. We therefore named this transcript Src homology 3-domain growth factor receptor-bound 2-like (endophilin) interacting protein 1 (SGIP1). SGIP1 encodes a large proline-rich protein that is expressed predominantly in the brain and is highly conserved between species. Together these data suggest that SGIP1 is an important and novel member of the group of neuronal molecules required for the regulation of energy homeostasis.

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The Small 11-kDa Protein from B19 Parvovirus Binds Growth Factor Receptor-Binding Protein 2 in Vitro in a Src Homology 3 Domain/Ligand-Dependent Manner

Virology ◽

10.1006/viro.2001.1217 ◽

2001 ◽

Vol 291 (2) ◽

pp. 285-291 ◽

Cited By ~ 22

Author(s):

Mannie M.Y. Fan ◽

Lillian Tamburic ◽

Cynthia Shippam-Brett ◽

Darren B. Zagrodney ◽

Caroline R. Astell

Keyword(s):

Growth Factor ◽

Receptor Binding ◽

Growth Factor Receptor ◽

Dependent Manner ◽

Src Homology 3 ◽

B19 Parvovirus ◽

Src Homology 3 Domain ◽

Src Homology ◽

Receptor Binding Protein

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Characterization of the Mouse Src Homology 3 Domain GeneSh3d2con Chr 7 Demonstrates Coexpression with Huntingtin in the Brain and Identifies the Processed PseudogeneSh3d2c-ps1on Chr 2

Genomics ◽

10.1006/geno.1998.5584 ◽

1998 ◽

Vol 54 (3) ◽

pp. 505-510 ◽

Cited By ~ 5

Author(s):

U. Zechner ◽

S. Scheel ◽

M. Hemberger ◽

M. Hopp ◽

T. Haaf ◽

...

Keyword(s):

Src Homology 3 ◽

Src Homology 3 Domain ◽

Src Homology ◽

The Brain

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Mutations inDrosophilaEnabled and Rescue by Human Vasodilator-stimulated Phosphoprotein (VASP) Indicate Important Functional Roles for Ena/VASP Homology Domain 1 (EVH1) and EVH2 Domains

Molecular Biology of the Cell ◽

10.1091/mbc.9.8.2157 ◽

1998 ◽

Vol 9 (8) ◽

pp. 2157-2171 ◽

Cited By ~ 86

Author(s):

Shawn M. Ahern-Djamali ◽

Allen R. Comer ◽

Christiane Bachmann ◽

Andrew S. Kastenmeier ◽

Srinevas K. Reddy ◽

...

Keyword(s):

Cultured Cells ◽

Physiological Activity ◽

Focal Adhesions ◽

Functional Roles ◽

Src Homology 3 ◽

Abelson Tyrosine Kinase ◽

Src Homology 3 Domain ◽

Src Homology

Drosophila Enabled (Ena) was initially identified as a dominant genetic suppressor of mutations in the Abelson tyrosine kinase and, more recently, as a member of the Ena/human vasodilator-stimulated phosphoprotein (VASP) family of proteins. We have used genetic, biochemical, and cell biological approaches to demonstrate the functional relationship between Ena and human VASP. In addition, we have defined the roles of Ena domains identified as essential for its activity in vivo. We have demonstrated that VASP rescues the embryonic lethality associated with loss of Ena function inDrosophila and have shown that Ena, like VASP, is associated with actin filaments and focal adhesions when expressed in cultured cells. To define sequences that are central to Ena function, we have characterized the molecular lesions present in two lethalena mutant alleles that affected the Ena/VASP homology domain 1 (EVH1) and EVH2. A missense mutation that resulted in an amino acid substitution in the EVH1 domain eliminated in vitro binding of Ena to the cytoskeletal protein zyxin, a previously reported binding partner of VASP. A nonsense mutation that resulted in a C-terminally truncated Ena protein lacking the EVH2 domain failed to form multimeric complexes and exhibited reduced binding to zyxin and the Abelson Src homology 3 domain. Our analysis demonstrates that Ena and VASP are functionally homologous and defines the conserved EVH1 and EVH2 domains as central to the physiological activity of Ena.

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Faculty Opinions recommendation of The Src homology 3 domain of the beta-subunit of voltage-gated calcium channels promotes endocytosis via dynamin interaction.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1064854.517791 ◽

2007 ◽

Author(s):

Brett Adams

Keyword(s):

Calcium Channels ◽

Beta Subunit ◽

Voltage Gated ◽

Src Homology 3 ◽

Voltage Gated Calcium Channels ◽

Src Homology 3 Domain ◽

Src Homology

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Faculty Opinions recommendation of Identification of a link between the SAMP repeats of adenomatous polyposis coli tumor suppressor and the Src homology 3 domain of DDEF.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1141847.598934 ◽

2009 ◽

Author(s):

Chloe Bulinski ◽

Andy Tran

Keyword(s):

Tumor Suppressor ◽

Adenomatous Polyposis Coli ◽

Adenomatous Polyposis ◽

Polyposis Coli ◽

Src Homology 3 ◽

Src Homology 3 Domain ◽

Src Homology

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Four proline-rich sequences of the guanine-nucleotide exchange factor C3G bind with unique specificity to the first Src homology 3 domain of Crk

Journal of Biological Chemistry ◽

10.1016/s0021-9258(20)30059-4 ◽

1994 ◽

Vol 269 (52) ◽

pp. 32781-32787

Author(s):

B S Knudsen ◽

S M Feller ◽

H Hanafusa

Keyword(s):

Guanine Nucleotide Exchange Factor ◽

Guanine Nucleotide ◽

Nucleotide Exchange ◽

Exchange Factor ◽

Src Homology 3 ◽

Guanine Nucleotide Exchange ◽

Nucleotide Exchange Factor ◽

Src Homology 3 Domain ◽

Src Homology ◽

Unique Specificity

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Myosin 1E coordinates actin assembly and cargo trafficking during clathrin-mediated endocytosis

Molecular Biology of the Cell ◽

10.1091/mbc.e11-04-0383 ◽

2012 ◽

Vol 23 (15) ◽

pp. 2891-2904 ◽

Cited By ~ 57

Author(s):

Jackie Cheng ◽

Alexandre Grassart ◽

David G. Drubin

Keyword(s):

Mammalian Cells ◽

Actin Polymerization ◽

Type I ◽

Actin Assembly ◽

Significant Delay ◽

Src Homology 3 ◽

Integral Role ◽

Src Homology 3 Domain ◽

Src Homology ◽

Endocytic Vesicles

Myosin 1E (Myo1E) is recruited to sites of clathrin-mediated endocytosis coincident with a burst of actin assembly. The recruitment dynamics and lifetime of Myo1E are similar to those of tagged actin polymerization regulatory proteins. Like inhibition of actin assembly, depletion of Myo1E causes reduced transferrin endocytosis and a significant delay in transferrin trafficking to perinuclear compartments, demonstrating an integral role for Myo1E in these actin-mediated steps. Mistargeting of GFP-Myo1E or its src-homology 3 domain to mitochondria results in appearance of WIP, WIRE, N-WASP, and actin filaments at the mitochondria, providing evidence for Myo1E's role in actin assembly regulation. These results suggest for mammalian cells, similar to budding yeast, interdependence in the recruitment of type I myosins, WIP/WIRE, and N-WASP to endocytic sites for Arp2/3 complex activation to assemble F-actin as endocytic vesicles are being formed.

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