Inhibition of the receptor-binding function of clathrin adaptor protein AP-2 by dominant-negative mutant mu 2 subunit and its effects on endocytosis

Although interactions between the mu 2 subunit of the clathrin adaptor prot ein complex AP-2 and tyrosine-based internalization motifs have been implic ated in the selective recruitment of cargo molecules into coated pits, the functional significance of this interaction for endocytosis of many types o f membrane proteins remains unclear. To analyze the function of mu 2-recept or interactions, we constructed an epitope-tagged mu 2 that incorporates in to AP-2 and is targeted to coated pits. Mutational analysis revealed that A sp176 and Trp421 of mu 2 are involved in the interaction with internalizati on motifs of TGN38 and epidermal growth factor (EGF) receptor, Inducible ov erexpression of mutant mu 2, in which these two residues were changed to al anines, resulted in metabolic replacement of endogenous mu 2 in AP-2 comple xes and complete abrogation of AP-2 interaction with the tyrosine-based int ernalization motifs. As a consequence, endocytosis of the transferrin recep tor was severely impaired, In contrast, internalization of the EGF receptor was not affected. These results demonstrate the potential usefulness of th e dominant-interfering approach for functional analysis of the adaptor prot ein family, and indicate that clathrin-mediated endocytosis may proceed in both a mu 2-dependent and -independent manner.