Eps15 is a component of clathrin-coated pits and vesicles and is located at the rim of coated pits

Eps15, a phosphorylation substrate of the epidermal growth factor (EGF) receptor kinase, has been shown to bind to the alpha-subunit of the clathrin-associated protein complex AP-2. Here we report that in cells, virtually all Eps15 interacts with the cytosol and membrane-bound forms of AP-2. This association is not affected by the treatment of cells with EGF. Immunofluorescence microscopy reveals nearly absolute co-localization of Eps15 with AP-2 and clathrin, and analysis by immunoelectron microscopy shows that the localization of membrane-associated Eps15 is restricted to the profiles corresponding to endocytic coated pits and vesicles. Unexpectedly, Eps15 was found at the edge of forming coated pits and at the rim of budding coated vesicles. This asymmetric distribution is in sharp contrast to the localization of AP-2 that shows an even distribution along the same types of clathrin-coated structures. These findings suggest several possible regulatory roles of Eps15 during the formation of coated pits.