Eps15R Is a Tyrosine Kinase Substrate with Characteristics of a Docking Protein Possibly Involved in Coated Pits-mediated Internalization

doi:10.1074/jbc.273.5.3003

Eps15R Is a Tyrosine Kinase Substrate with Characteristics of a Docking Protein Possibly Involved in Coated Pits-mediated Internalization*

From the ^‡Department of Experimental Oncology, European Institute of Oncology, 20141 Milan, Italy, the ^¶Department of Pharmacology, University of Colorado Health Sciences Center, Denver, Colorado 80262, the ^**Istituto di Patologia Speciale Medica, University of Parma, Parma, 43100, Italy, and the ^‡Istituto di Microbiologia, University of Bari, Bari, 70124, Italy

Abstract

eps15R was identified because of its relatedness to eps15, a gene encoding a tyrosine kinase substrate bearing a novel protein-protein interaction domain, called EH. In this paper, we report a biochemical characterization of theeps15R gene product(s). In NIH-3T3 cells, three proteins of 125, 108, and 76 kDa were specifically recognized by anti-eps15R sera. The 125-kDa species is a bona fide product of the eps15Rgene, whereas p108 and p76 are most likely products of alternative splicing events. Eps15R protein(s) are tyrosine-phosphorylated following epidermal growth factor receptor activation in NIH-3T3 cells overexpressing the receptor, even at low levels of receptor occupancy, thus behaving as physiological substrates. A role for eps15R in clathrin-mediated endocytosis is suggested by its localization in plasma membrane-coated pits and in vivo association to the coated pits’ adapter protein AP-2. Finally, we demonstrate that a sizable fraction of eps15R exists in the cell as a complex with eps15 and that its EH domains exhibit binding specificities that are partially distinct from those of eps15. We propose that eps15 and eps15R are multifunctional binding proteins that serve pleiotropic functions within the cell.

Footnotes

↵* This work was supported in part by grants from the Associazione Italiana Ricerca sul Cancro, from the Consiglio Nazionale delle Ricerche (to P. P. D. F. and P. G. P.), and from the European Community (BIOMED-2 Program) (to P. P. D. F. and P. G. P.).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.
↵§ Recipient of a fellowship from the Fondazione Italiana Ricerca sul Cancro.
↵‖ Supported by National Institutes of Health Grant DK46817 and Grant SA039 from the Tobacco Research Association.
↵§§ To whom correspondence should be addressed: Istituto Europeo di Oncologia, Via Ripamonti 435, 20141 Milan, Italy. Tel.: 39-2-57489855; Fax: 39-2-57489851; E-mail: pdifiore{at}ieo.cilea.it.
↵1 The abbreviations used are: eps15R, eps15-related protein; eps15, epidermal growth factor receptor pathway substrate clone 15; EGFR, epidermal growth factor receptor; EGF, epidermal growth factor; EH, eps15 homology domain; GST, glutathioneS-transferase; AP-1, adapter protein 1; AP-2, adapter protein 2; Tyr(P), phosphotyrosine; a.u., arbitrary units; TnT, transcription/translation.
↵2 L. Coda, G. Pelicci, and P. P. Di Fiore, unpublished data.
- Received June 26, 1997.
- Revision received October 22, 1997.
The American Society for Biochemistry and Molecular Biology, Inc.