Endocytic adaptor complexes bind the C-terminal domain of CFTR

The cystic fibrosis transmembrane conductance regulator (CFTR) functions at the apical membrane of epithelial cells to regulate chloride permeability. Recent studies have shown that CFTR is rapidly and efficiently internalize d from the plasma membrane. We have shown that such internalization is medi ated solely by clathrin-coated pathways, and that other pathways, such as c aveolae, exclude CFTR. Moreover, CFTR co-precipitates with alpha -adaptin, a component of the endocytic adaptor complex (AP-2). The goal of our curren t studies was to elucidate further the molecular mechanisms that facilitate entry of CFTR into endocytic clathrin-coated vesicles. Protein-protein int eractions generated by incubation of full-length in-vitro-translated CFTR w ith partially purified bovine brain adaptor complexes were evaluated follow ing immunoprecipitation using an antibody against the alpha -adaptin subuni t of the AP-2 complex. Such studies revealed co-immunoprecipitation of alph a -adaptin with full-length but not partially translated CFTR, suggesting t hat the C-terminus of CFTR may be responsible for this interaction. To test this hypothesis a C-terminal GST fusion protein (amino acids 1404-1480; CF -GST) was used in a "pull-down" assay with purified adaptor complexes. CF-G ST sepharose was able to pull-down AP-2 endocytic adaptor complexes, as det ermined by immunoblot analyses of the precipitates using antibodies directe d against alpha -adaptin. In contrast, CF-GST sepharose was unable to pull- down gamma -adaptin, a component of the Golgi-derived AP-1 clathrin adaptor complex. Thus, we demonstrate that CFTR is endocytosed via clathrin-coated vesicles, and that targeting of CFTR to these structures is mediated by bi nding of the AP-2 adaptor complex to the C-terminal domain of CFTR.