mu 2 binding directs the cystic fibrosis transmembrane conductance regulator to the clathrin-mediated endocytic pathway

The cystic fibrosis transmembrane conductance regulator (CFTR) contains a c onserved tyrosine-based internalization motif, (DSI)-D-1424y, which interac ts with the endocytic clathrin adaptor complex, AP-2, and is required for i ts efficient endocytosis. Although direct interactions between several endo cytic sequences and the medium chain and endocytic clathrin adaptor complex es 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 ph ysiologically relevant interaction between CFTR and the tt subunit of AP-2. Cross-linking experiments were performed using photoreactive peptides cont aining the YDSI motif and purified adaptor complexes. CFTR peptides cross-l inked a 50-kDa subunit of purified AP-2 complexes, the apparent molecular m ass of mu2. Furthermore, isolated mu2 bound to the sorting motif, YDSI, bot h in cross-linking experiments and glutathione S-transferase pull-down expe riments, confirming that mu2 mediates the interaction between CFTR and A-P- 2 complexes. Inducible overexpression of dominant-negative mu2 in HeLa cell s results in AP-2 complexes that fail to interact with CFTR. Moreover, inte rnalization of CFTR in mutant cells is greatly reduced compared with wild t ype HeLa cells. These results indicate that the AP-2 endocytic complex sele ctively interacts with the conserved tyrosine-based internalization signal in the carboxyl terminus of CFTR, YDSI. Furthermore, this interaction is me diated by the mu2 subunit of AP-2 and mutations in mu2 that block its inter action with YDSI inhibit the incorporation of CFTR into the clathrin-mediat ed endocytic pathway.