STOICHIOMETRIC INTERACTION OF THE EPIDERMAL GROWTH-FACTOR RECEPTOR WITH THE CLATHRIN-ASSOCIATED PROTEIN COMPLEX AP-2

Plasma membrane clathrin-associated protein complexes (AP-2) have been shown to co-immunoprecipitate with the epidermal growth factor (EGF) receptor (Sorkin A., and Carpenter, G. (1993) Science 261, 612-615). Hence, we analyzed the stoichiometry of the EGF receptor interaction with AP-2 using a new antibody that efficiently immunoprecipitates native AP-2. EGF receptor AP-2 complexes were isolated from S-35-labeled cells treated with EGF by EGF receptor affinity chroma tography followed by precipitation with the antibody to AP-2. Quantitation of the relative molar concentrations of the proteins found in the complex revealed that 1 mol of AP-2 was associated with approximately 1.1 mol of EGF receptor. No other proteins were present in significant molar concentrations relative to AP-2. indicating that other proteins are not stoichiometrically involved in the interaction of EGF receptors and AP-2 in vitro. Co-immunoprecipitation experiments in cells expressing a mutant EGF receptor demonstrated that the cyto plasmic carboxyl-terminal 214 residues of the EGF receptor are essential for interaction with AP-2.