Mapping of the Molecular Determinants Involved in the Interaction between eps15 and AP-2

eps15, a substrate for the epidermal growth factor receptor and other receptor tyrosine kinases, possesses a discrete domain structure with protein-binding properties. It interacts with a number of cellular proteins through an evolutionarily conserved protein-binding domain, the eps15 homology domain, located in its NH2-terminal region. In addition, a proline-rich region, located in the COOH-terminal portion of eps15, can bind to the Src homology 3 domain of the crk proto-oncogene product in vitro . Recently, coimmunoprecipitation between eps15 and AP-2, a major component of coated pits, was reported. Here, we characterize the molecular determinants of the eps15/AP-2 interaction. The AP-2 binding region of eps15 is localized in its COOH-terminal region and spans ∼80 amino acids. At least three molecular determinants, located at residues 650–660, 680–690, and 720–730, are involved in the binding. AP-2 binds to eps15 through its α subunit (α-adaptin); in particular, the COOH-terminal region of α-adaptin, the so-called α-ear, contains the eps15 binding region.