The SH3 Domain of Amphiphysin Binds the Proline-rich Domain of Dynamin at a Single Site That Defines a New SH3 Binding Consensus Sequence*
- Detlev Grabs‡§,
- Vladimir I. Slepnev‡,
- Zhou Songyang¶,
- Carol David‡‖,
- Mary Lynch¶,
- Lewis C. Cantley¶ and
- Pietro De Camilli‡‡
- From the ‡Department of Cell Biology and Howard Hughes Medical Institute, Yale University School of Medicine, Boyer Center for Molecular Medicine, New Haven, Connecticut 06510 and the¶Department of Cell Biology, Harvard Medical School, Boston, Massachusetts 02115
Abstract
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.
Footnotes
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↵* This work was supported by grants from the Donaghue Foundation, the Human Frontier Science Programme, and the National Institutes of Health (CA46128) (to P. D. C.) and by postdoctoral fellowships from the Deutscher Akademischer Austauschdienst (to D. G.), the Muscular Dystrophy Association (to V. I. S.), and the United States Army Medical Research and Development Command (to C. D.).
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↵§ Present address: Institut fuer Anatomie und Spezielle Embryologie, Universitaet Fribourg, CH-1700 Fribourg, Switzerland.
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↵‖ Present address: Dept. of Immunology, Weizmann Inst. of Science, Rehovot, Israel.
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↵‡ To whom correspondence should be addressed: Dept. of Cell Biology and Howard Hughes Medical Inst., Yale University School of Medicine, Boyer Center for Molecular Medicine, 295 Congress Ave., New Haven, CT 06510. Tel.: 203-737-4461; Fax: 203-737-1762; E-mail:pietro_decamilli{at}quickmail.yale.edu.
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↵1 The abbreviations used are: PLC, phospholipase C; GST, glutathione S-transferase; PAGE, polyacrylamide gel electrophoresis; PRD, proline-rich domain.
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- Received December 23, 1996.
- Revision received March 13, 1997.
