AP-2/Eps15 interaction is required for receptor-mediated endocytosis.

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.
Mesh Terms:
Adaptor Proteins, Vesicular Transport, Binding Sites, Calcium-Binding Proteins, Endocytosis, HeLa Cells, Humans, Intracellular Signaling Peptides and Proteins, Nerve Tissue Proteins, Phosphoproteins, Receptor, Epidermal Growth Factor, Receptors, Transferrin, Recombinant Fusion Proteins, Transferrin, Tumor Cells, Cultured
J. Cell Biol.
Date: Mar. 09, 1998
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