The carboxyl terminus of the cystic fibrosis transmembrane conductance regulator binds to AP-2 clathrin adaptors

The cystic fibrosis transmembrane conductance regulator (CFTR) undergoes ra pid and efficient endocytosis. Since functionally active CFTR is found in p urified clathrin-coated vesicles isolated from both cultured epithelial cel ls and intact epithelial tissues, we investigated the molecular mechanisms whereby CFTR could enter such endocytic clathrin-coated vesicles. In vivo c ross-linking and in vitro pull-down assays show that full-length CFTR binds to the endocytic adaptor complex AP-2. Fusion proteins containing the carb oxyl terminus of CFTR (amino acids 1404-1480) were also able to bind AP-2 b ut did not bind the Golgi-specific adaptor complex AP-1, Substitution of an alanine residue for tyrosine at position 1424 significantly reduced the ab ility of AP-2 to bind the carboxyl terminus of CFTR; however, mutation to a phenylalanine residue (an amino acid found at position 1424 in dogfish CFT R) did not perturb AP-2 binding. Secondary structure predictions suggest th at Tyr(1424) is present in a beta-turn conformation, a conformation disrupt ed by alanine but not phenylalanine. Together, these data suggest that the carboxyl terminus of CFTR contains a tyrosine-based internalization signal that interacts with the endocytic adaptor complex AP-2 to facilitate effici ent entry of CFTR into clathrin-coated vesicles.