THE EAR OF ALPHA-ADAPTIN INTERACTS WITH THE COOH-TERMINAL DOMAIN OF THE EPS15 PROTEIN

The role of Eps15 in clathrin-mediated endocytosis is supported by two observations. First, it interacts specifically and constitutively wit h the plasma membrane adaptor AP-2. Second, its NH2 terminus shows sig nificant homology to the NH2 terminus of yeast End3p, necessary for en docytosis of Lu-factor. To gain further insight into the role of Eps15 -AP-2 association, we have now delineated their sites of interactions. AP-2 binds to a domain of 72 amino acids (767-739) present in the COO H terminus of Fps15. This domain contains 4 of the 15 DPF repeats char acteristic of the COOH-terminal domain of Eps15 and shares no homology with known proteins, including the related Fps15r protein. Precipitat ion of proteolytic fragments of AP-2 with Eps15-derived fusion protein s containing the binding site for AP-2 showed that Eps15 binds specifi cally to a 40-kDa fragment corresponding to the ear of alpha-adaptin, a result confirmed by precipitation of Eps15 by alpha-adaptin-derived fusion proteins. Our data indicate that this specific part of AP-2 bin ds to a cellular component and provide the tools for investigating the function(s) of the association between AP-2 and Eps15.