Eps15 Is Constitutively Oligomerized Due to Homophilic Interaction of Its Coiled-coil Region*
- Francesc Tebar‡,
- Stefano Confalonieri§,
- Royston E. Carter‡,
- Pier Paolo Di Fiore§¶ and
- Alexander Sorkin‡‖
- From the ‡Department of Pharmacology, University of Colorado Health Sciences Center, Denver, Colorado 80262, the§European Institute of Oncology, 20141 Milan, Italy, and the ¶Istituto di Microbiologia, Universitá di Bari, 70124 Bari, Italy
Abstract
Eps15 is a member of an emerging family of proteins containing a novel protein/protein interaction domain, the EH domain, of as yet unknown function. Recent findings of Eps15 association with clathrin adaptor complex AP-2 and its localization in clathrin-coated pits have implicated Eps15 in the regulation of vesicle trafficking. Here we show that Eps15 exists in several multimeric states in vivo. When purified recombinant Eps15 or lysates of NIH 3T3 cells were treated with cross-linking reagents, covalent dimers of Eps15 and larger covalent multimers were detected in high yield. Large Eps15 oligomers co-immunoprecipitated with AP-2 at an efficiency higher than that of Eps15 dimers. Furthermore, cross-linking of the membrane-bound fraction of Eps15 in mildly permeabilized cells was as efficient as that of the cytosolic fraction. Size-exclusion column chromatography of recombinantly produced Eps15 and of total cell lysates was performed to examine the equilibrium ratio of the monomersversus the aggregated forms of Eps15. These experiments showed that essentially all the Eps15 was aggregated, whereas monomers of Eps15 could be obtained only under strong denaturing conditions. To map the region of Eps15 responsible for dimerization, fusion proteins corresponding to the three structural domains of Eps15 were prepared. Cross-linking analysis revealed that the central portion of Eps15, which possesses a coiled-coil region (residues 321–520), serves as the interacting interface. The possibility that hetero-oligomeric complexes of Eps15 dimers and AP-2 function during the recruitment of proteins into coated pits is discussed.
Footnotes
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↵* This work was supported by Grant DK46817 from the National Institutes of Health (to A. S.); by Grant SA039 from the Council for Tobacco Research (to A. S. and R. E. C.); by a fellowship from the Ministerio de Educaci′n y Cultura, Spain (to F. T.); and by grants from the Associazione Italiana Ricerca sul Cancro and the Consiglio Nazionale delle Ricerche and by the European Community BIOMED-2 Program (to P. P. D. F.).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 Pharmacology, University of Colorado Health Science Center, 4200 East Ninth Ave., Denver, CO 80262. Tel.: 303-315-7252; Fax: 303-315-7097; E-mail: alexander.sorkin{at}uchsc.edu.
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↵1 The abbreviations used are: DPF, Asp-Pro-Phe; BS3, bis(sulfosuccinimidyl) suberate; GST, glutathioneS-transferase; FPLC, fast protein liquid chromatography; PAGE, polyacrylamide gel electrophoresis; CMF-PBS, Ca2+,Mg2+-free phosphate-buffered saline.
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↵2 Sequences of the primers utilized for the constructions are available upon request.
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- Received January 30, 1997.
- Revision received April 3, 1997.
