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*
- Gianluca Cestra‡,
- Luisa Castagnoli‡,
- Luciana Dente‡,
- Olga Minenkova‡,
- Annalisa Petrelli§,
- Nicola Migone§,
- Ulrich Hoffmüller¶,
- Jens Schneider-Mergener¶ and
- Gianni Cesareni‡‖
- From the ‡Dipartimento di Biologia, Universitàdi Roma Tor Vergata, Rome 00133, Italy, the¶Universtitätsklinikum Charité, Humboldt-Universität zu Berlin, Berlin 10117, Germany, and the§Dipartimento di Genetica, Biologia e Biochimica, Università di Torino, Turin 10126, Italy
Abstract
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.
Footnotes
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↵* This work was supported by grants from the “Associazione Italiana per la Ricerca sul Cancro,” from the Biotechnology Target projects of CNR (law 95/96), from the European Union, and from MURST.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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↵‖ To whom correspondence should be addressed: Dept. of Biology, University of Rome, Tor Vergata, Via Della Ricerca Scientifica, 00133 Rome, Italy. Tel.: 39-6-72594315; Fax: 39-6-2023500; E-mail: Cesareni@uniroma2.it.
- Abbreviations:
- PRD
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proline-rich domains
- GST
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glutathione S-transferase
- ELISA
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enzyme-linked immunosorbent assay
- PBS
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phosphate-buffered saline
- PCR
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polymerase chain reaction
- Syn
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synaptojanin
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- Received April 21, 1999.
- Revision received August 12, 1999.
- The American Society for Biochemistry and Molecular Biology, Inc.
