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© The Rockefeller University Press, 0021-9525/1998//1055 $5.00
The Journal of Cell Biology, Volume 140, Number 5, , 1998 1055-1062

Article

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



Alexandre Benmerah*,{ddagger}, Christophe Lamaze{ddagger}, Bernadette Bègue*, Sandra L. Schmid§, Alice Dautry-Varsat{ddagger}, and Nadine Cerf-Bensussan*

* 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; {ddagger} 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 Institute, La Jolla, California 92037

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.

Abbreviations used in this paper: B-EGF, biotinylated epidermal growth factor; B-SS-Tf, disulfide bond–biotinylated transferrin; B-Tf, biotinylated transferrin; EGF and EGF-R, epidermal growth factor and receptor; GFP, green fluorescent protein; GST, glutathione-S-transferase; Tf, transferrin; Tf-R, transferrin receptor; VSV, vesicular stomatitis virus.

A. Benmerah and C. Lamaze contributed equally to this work.

Address all correspondence to Dr. N. Cerf-Bensussan, Institut National de la Santé et de la Recherche Médicale, U 429. Hôpital Necker-Enfants Malades, 149, Rue de Sèvres, 75743 Paris Cedex 15, France. Tel.: 33-1-44-49-50-82. Fax: 33-1-40-61-32-38. E-mail: cerf{at}necker.fr


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