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Mapping of the Molecular Determinants Involved in the Interaction between eps15 and AP-2¹

Department of Experimental Oncology, European Institute of Oncology, Milan-20141, Italy [G. I., A. E. S., P. G. P., P. P. D. F.]; Istituto di Patologia Medica, Facolta' di Medicina e Chirurgia, Parma-43100, Italy [P. G. P.]; Istituto di Microbiologia, Facolta' di Medicina e Chirurgia, Bari-70124, Italy [P. P. D. F.]; Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, TN 37232 [J. B., G. C.]; and the Kimmel Cancer Institute, Thomas Jefferson University, Philadelphia, Pennsylvania 19107 [I. G., O. B. G., J. H. K.]

eps15, a substrate for the epidermal growth factor receptor and other receptor tyrosine kinases, possesses a discrete domain structure with protein-binding properties. It interacts with a number of cellular proteins through an evolutionarily conserved protein-binding domain, the eps15 homology domain, located in its NH₂-terminal region. In addition, a proline-rich region, located in the COOH-terminal portion of eps15, can bind to the Src homology 3 domain of the crk proto-oncogene product in vitro. Recently, coimmunoprecipitation between eps15 and AP-2, a major component of coated pits, was reported. Here, we characterize the molecular determinants of the eps15/AP-2 interaction. The AP-2 binding region of eps15 is localized in its COOH-terminal region and spans 80 amino acids. At least three molecular determinants, located at residues 650–660, 680–690, and 720–730, are involved in the binding. AP-2 binds to eps15 through its subunit (-adaptin); in particular, the COOH-terminal region of -adaptin, the so-called -ear, contains the eps15 binding region.

¹ This work was supported in part by grants from Consiglio Nazionale delle Ricerche and Associazione Italiana Ricerca sul Cancro (to P. P. D. F. and P. G. P.) and by NIH Grants GM-49217 (to J. H. K.) and CA-24071 (to G. C.). G. I. was supported by an Associazione Italiana Ricerca sul Cancro fellowship and European Community, project BIOMED-2. O. G. was supported by NIH Training Grant 5-T32-DK07705. J. B. was supported by a fellowship from the Spanish Ministerio de Education y Ciencia.

² To whom requests for reprints should be addressed, at the Kimmel Cancer Institute, Thomas Jefferson University, 233 South 10th Street, BLSB/915, Philadelphia, PA 19107. Phone: (215) 503-4624; Fax: (215) 923-1098; E-mail: j_keen@lac.jci.tju.edu.

Received 6/27/96. Accepted 11/18/96.

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