ASSOCIATION OF EPIDERMAL GROWTH-FACTOR RECEPTORS WITH COATED PIT ADAPTINS VIA A TYROSINE PHOSPHORYLATION-REGULATED MECHANISM

We investigated the mechanism by which ligand-activated epidermal grow th factor receptors (EGFR) associate with coated pit adaptor protein ( AP) complexes, In vivo association, assayed by coimmunoprecipitation o f AP with mutant EGFR, required tyrosine kinase activity, intact autop hosphorylation sites, and the regulatory carboxyl terminus of EGFR, Th e role of autophosphorylation of EGFR in interaction with AP was exami ned in vitro using a BIAcoreTM instrument. Purified active EGFR, immob ilized on the biosensor surface, was reversibly autophosphorylated or dephosphorylated by treatment with ATP or phosphatase. Autophosphoryla tion of EGFR significantly increased AP binding, Once formed, EGFR . A P complexes were resistant to disassembly by dephosphorylation of EGFR or competition with phosphotyrosine, indicating that phosphorylated t yrosine residues do not directly participate in AP binding, Induction of conformational changes in EGFR by treatment with urea increased AP binding up to 10-fold in the absence of EGFR autophosphorylation, A re combinant EGFR carboxyl terminus specifically bound the AP complex and each of the isolated alpha- and beta-subunits of AP2. We conclude tha t tyrosine autophosphorylation of EGFR exposes structural motif(s) in the carboxyl terminus of EGFR that interact specifically with AP2.