The Ear of -Adaptin Interacts with the COOH-terminal Domain of the Eps15 Protein

doi:10.1074/jbc.271.20.12111

The Ear of -Adaptin Interacts with the COOH-terminal Domain of the Eps15 Protein (*)

From the (1)Développement Normal et Pathologique du Système Immunitaire, INSERM U429, Hôpital Necker-Enfants Malades, 75743 Paris Cedex 15, France and
(2)Unité de Biologie des Interactions Cellulaires, URA-CNRS 1960, Institut Pasteur, 75724 Paris Cedex 15, France

^¶ To whom all correspondence should be addressed:
INSERM U429, Hôpital Necker-Enfants-Malades, 149 Rue de Sèvres, 75743 Paris Cedex 15, France.
Tel.: 33-1-44-49-50-82; Fax: 33-1-42-73-06-40.

Abstract

The role of Eps15 in clathrin-mediated endocytosis is supported by two observations. First, it interacts specifically and constitutively with the plasma membrane adaptor AP-2. Second, its NH terminus shows significant homology to the NH terminus of yeast End3p, necessary for endocytosis of α-factor. To gain further insight into the role of Eps15-AP-2 association, we have now delineated their sites of interactions. AP-2 binds to a domain of 72 amino acids (767-739) present in the COOH terminus of Eps15. This domain contains 4 of the 15 DPF repeats characteristic of the COOH-terminal domain of Eps15 and shares no homology with known proteins, including the related Eps15r protein. Precipitation of proteolytic fragments of AP-2 with Eps15-derived fusion proteins containing the binding site for AP-2 showed that Eps15 binds specifically to a 40-kDa fragment corresponding to the ear of α-adaptin, a result confirmed by precipitation of Eps15 by α-adaptin-derived fusion proteins. Our data indicate that this specific part of AP-2 binds to a cellular component and provide the tools for investigating the function(s) of the association between AP-2 and Eps15.

Footnotes

↵^§ Supported by a fellowship from the Association pour la Recherche contre le Cancer (ARC, Villejuif, France).
↵* This work was supported by INSERM and a grant from the Association pour la Recherche contre le Cancer (ARC Contract 6836). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵¹ The abbreviations used are:

EGF

epidermal growth factor

EGFR

EGF receptor

Eps15

EGFR pathway substrate clone 15

GST

glutathione S-transferase

mAb

monoclonal antibody

PAGE

polyacrylamide gel electrophoresis

EH

Eps15 homology.
↵²B. Bègue, A. Benmerah, and N. Cerf-Bensussan, unpublished results.
↵³G. Raposo, A. Benmerah, B. Bègue, A. Dautry-Varsat, and N. Cerf-Bensussan, unpublished results.
- Received February 12, 1996.
- Revision received March 7, 1996.