Ap-2/Eps15 interaction is required for receptor-mediated endocytosis

Alexandre Benmerah, Christophe Lamaze, Bernadette Bègue, Sandra L. Schmid, Alice Dautry-Varsat, Nadine Cerf-Bensussan

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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.

Original languageEnglish (US)
Pages (from-to)1055-1062
Number of pages8
JournalJournal of Cell Biology
Volume140
Issue number5
DOIs
StatePublished - Mar 9 1998

ASJC Scopus subject areas

  • Cell Biology

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    Benmerah, A., Lamaze, C., Bègue, B., Schmid, S. L., Dautry-Varsat, A., & Cerf-Bensussan, N. (1998). Ap-2/Eps15 interaction is required for receptor-mediated endocytosis. Journal of Cell Biology, 140(5), 1055-1062. https://doi.org/10.1083/jcb.140.5.1055