EPIDERMAL GROWTH-FACTOR RECEPTOR INTERACTION WITH CLATHRIN ADAPTERS IS MEDIATED BY THE TYR(974)-CONTAINING INTERNALIZATION MOTIF

The carboxyl-terminal regulatory domain of the epidermal growth factor (EGF) receptor is essential for its endocytosis and interaction with the clathrin-associated protein complex AP-2. To identify AP-2 binding motif in the receptor, several single and multiple-point mutations wi thin the region between residues 966 and 977 of the human EGF receptor were made, and the mutant receptors were expressed in NIH3T3 cells. M utation of tyrosine 974 alone or together with surrounding residues an d the deletion of residues 973-975 essentially eliminated AP-2 co-immu noprecipitation with the EGF receptor, Furthermore, a synthetic peptid e corresponding to receptor residues 964-978 blocked AP-2 association with the wild-type EGF receptor, These data suggest that AP-2 has only one high-affinity binding site in the EGF receptor composed of Tyr(97 4)-containing motif. Receptor mutants that did not bind AP-2 displayed a lower rate of internalization, down-regulation, and turnover compar ed to wild type receptors when expressed at high levels. However, simi lar receptor mutants expressed at low levels were internalized and dow n-regulated as efficiently as wild type receptors, Internalization of the mutant receptors lacking the high-affinity binding site for AP-2 w as inhibited by K+-depletion of the cells, indicating that their endoc ytosis required intact coated pits, We suggest that whereas one mechan ism of EGF receptor recruitment into coated pits involves high-affinit y binding of AP-2 to Tyr(974)-containing motif, another pathway may be mediated by weak receptor/AP-2 interactions or by proteins other than AP-2.