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eps15 and eps15R Are Essential Components of the Endocytic Pathway¹

Department of Experimental Oncology, European Institute of Oncology, 20141 Milan, Italy [R. C., S. F., G. I., P. G. P., P. P. D. F.]; Dipartimento di Medicina Sperimentale e Patologia, Università di Roma La Sapienza, 00161 Rome, Italy [F. B., P. M., M. R. T.]; Istituto di Patologia Speciale Medica, University of Parma, 43100 Parma, Italy [P. G. P.]; and Istituto di Microbiologia, University of Bari, 70124 Bari, Italy [P. P. D. F.]

eps15 and eps15R are substrates of the epidermal growth factor (EGF) receptor kinase that are characterized by the presence of a protein:protein interaction domain, the EH domain, and by their ability to bind to the clathrin adaptor protein complex adaptor protein 2. Indirect evidence suggests that eps15 and eps15R are involved in endocytosis. Here we show that microinjection of antibodies against eps15 and eps15R inhibits internalization of EGF and transferrin. In addition, fragments of eps15 (encompassing its EH domains or the COOH-terminal region that binds to adaptor protein 2) inhibit EGF internalization or endocytosis of Sindbis virus. These results demonstrate that eps15 and eps15R are essential components of the endocytic machinery.

¹ Supported in part by grants from Ministero dell'Università e Ricerca Scientifica e Tecnologica (to M. R. T.) and by grants from Associazione Italiana per la Ricerca sul Cancro and from Progetto Finalizzato Applicazioni Cliniche della Ricerca Oncologica from Consiglio Nazionale delle Ricerche, Italy (to P. P. D. F., P. G. P., and M. R. T.). Support from the European Community BIOMED-2 Programme (to P. P. D. F. and P. G. P.) and from the Ferrero Foundation (to P. P. D. F.) is also acknowledged. G. I. is the recipient of an Associazione Italiana per la Ricerca sul Cancro fellowship. R. C. and S. F. contributed equally to this work

² To whom requests for reprints should be addressed, at Istituto Europeo di Oncologia, Via Ripamonti 435, 20141 Milan, Italy. Phone: 39-2-57489855; Fax: 39-2-57489851; E-mail: pdifiore@ieo.cilea.it.

Received 11/ 5/97. Accepted 11/13/97.

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