AP-2 EPS15 INTERACTION IS REQUIRED FOR RECEPTOR-MEDIATED ENDOCYTOSIS/

We have previously shown that the protein Eps15 is constitutively asso ciated with the plasma membrane adaptor complex, AP-2, suggesting its possible role in endocytosis. To explore the role of Eps15 and the fun ction of AP-2/Eps15 association in endocytosis, the Eps15 binding doma in 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 tra nsiently transfected in HeLa cells, Overexpression of the fusion prote in containing the entire COOH-terminal domain of Eps15 strongly inhibi ted endocytosis of transferrin, whereas the fusion protein in which th e AP-2-binding sites had been deleted had no effect. These results wer e confirmed in a cell-free assay that uses perforated A431 cells to fo llow the first steps of coated vesicle formation at the plasma membran e, Addition of Eps15-derived glutathione-S-transferase fusion proteins containing the AP-2-binding site in this assay inhibited not only con stitutive endocytosis of transferrin but also ligand-induced endocytos is of epidermal growth factor, This inhibition could be ascribed to a competition between the fusion protein and endogenous Eps15 for AP-2 b inding. 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 p lasma membrane-coated pits.