AP-2/Eps15 Interaction Is Required for Receptor-mediated Endocytosis

Author:

Benmerah Alexandre11,Lamaze Christophe1,Bègue Bernadette1,Schmid Sandra L.1,Dautry-Varsat Alice1,Cerf-Bensussan Nadine1

Affiliation:

1. Développement Normal et Pathologique du Système Immunitaire, Institut National de la Santé et de la Recherche Médicale U 429, Hôpital Necker-Enfants Malades, 75743 Paris Cedex 15, France; Unité de Biologie des Interactions Cellulaires, URA-Centre National de la Recherche Scientifique 1960, Institut Pasteur, 75724 Paris Cedex 15, France; and Department of Cell Biology, The Scripps Research Institu

Abstract

We have previously shown that the protein Eps15 is constitutively associated with the plasma membrane adaptor complex, AP-2, suggesting its possible role in endocytosis. To explore the role of Eps15 and the function of AP-2/Eps15 association in endocytosis, the Eps15 binding domain for AP-2 was precisely delineated. The entire COOH-terminal domain of Eps15 or a mutant form lacking all the AP-2–binding sites was fused to the green fluorescent protein (GFP), and these constructs were transiently transfected in HeLa cells. Overexpression of the fusion protein containing the entire COOH-terminal domain of Eps15 strongly inhibited endocytosis of transferrin, whereas the fusion protein in which the AP-2–binding sites had been deleted had no effect. These results were confirmed in a cell-free assay that uses perforated A431 cells to follow the first steps of coated vesicle formation at the plasma membrane. Addition of Eps15-derived glutathione-S-transferase fusion proteins containing the AP-2–binding site in this assay inhibited not only constitutive endocytosis of transferrin but also ligand-induced endocytosis of epidermal growth factor. This inhibition could be ascribed to a competition between the fusion protein and endogenous Eps15 for AP-2 binding. Altogether, these results show that interaction of Eps15 with AP-2 is required for efficient receptor-mediated endocytosis and thus provide the first evidence that Eps15 is involved in the function of plasma membrane–coated pits.

Publisher

Rockefeller University Press

Subject

Cell Biology

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