THE TYROSINE KINASE SUBSTRATE EPS15 IS CONSTITUTIVELY ASSOCIATED WITHTHE PLASMA-MEMBRANE ADAPTER AP-2

The ubiquitous eps15 protein was initially described as a substrate of the EGF receptor kinase. Its functions are not yet delineated and thi s work provides evidence for its possible role in endocytosis. A novel anti-eps15 antibody, 6G4, coimmunoprecipitated proteins of molecular mass 102 kD. In human cells, these proteins were identified as the alp ha- and beta-adaptins of the AP-2 complex on the basis of their NH2-te rminal sequence and their immunoreactivity with anti-alpha- and anti-b eta-adaptin antibodies but not with anti-gamma-adaptin antibody. In ad dition, the anti-eps15 antibody coimmunoprecipitated metabolically lab eled polypeptides with molecular mass of 50 and 17 kD, comparable to t hose of the two other components of the AP-2 complex, mu 2 and sigma 2 . Constitutive association of eps15 with AP-2 was confirmed by two set s of experiments, First, eps15 was detected in immunoprecipitates of a nti-alpha- and anti-beta-adaptin antibodies. Second, alpha- and beta- but not gamma-adaptins were precipitated by a glutathione-S-transferas e eps15 fusion protein. The association of eps15 with AP-2 was ubiquit ous and conserved between species, since it was observed in human lymp hocytes and epithelial cells and in murine NIH3T3 fibroblasts. Our res ults are in keeping with a recent study showing homology between the N H2-terminal domains of eps15 and the product of the gene END3, involve d in clathrin-mediated endocytosis of the pheromone alpha factor in Sa ccharomyces cerevisiae, and suggest a possible role for eps15 in clath rin-mediated endocytosis in mammals.