μ2 Binding Directs the Cystic Fibrosis Transmembrane Conductance Regulator to the Clathrin-mediated Endocytic Pathway

Abstract

The cystic fibrosis transmembrane conductance regulator (CFTR) contains a conserved tyrosine-based internalization motif, 1424YDSI, which interacts with the endocytic clathrin adaptor complex, AP-2, and is required for its efficient endocytosis. Although direct interactions between several endocytic sequences and the medium chain and endocytic clathrin adaptor complexes have been shown by protein-protein interaction assays, whether all these interactions occur in vivo or are physiologically important has not always been addressed. Here we show, using both in vitro and in vivo assays, a physiologically relevant interaction between CFTR and the μ subunit of AP-2. Cross-linking experiments were performed using photoreactive peptides containing the YDSI motif and purified adaptor complexes. CFTR peptides cross-linked a 50-kDa subunit of purified AP-2 complexes, the apparent molecular mass of μ2. Furthermore, isolated μ2 bound to the sorting motif, YDSI, both in cross-linking experiments and glutathione S-transferase pull-down experiments, confirming that μ2 mediates the interaction between CFTR and AP-2 complexes. Inducible overexpression of dominant-negative μ2 in HeLa cells results in AP-2 complexes that fail to interact with CFTR. Moreover, internalization of CFTR in mutant cells is greatly reduced compared with wild type HeLa cells. These results indicate that the AP-2 endocytic complex selectively interacts with the conserved tyrosine-based internalization signal in the carboxyl terminus of CFTR, YDSI. Furthermore, this interaction is mediated by the μ2 subunit of AP-2 and mutations in μ2 that block its interaction with YDSI inhibit the incorporation of CFTR into the clathrin-mediated endocytic pathway.