title: Endocytic adaptor complexes bind the C-terminal domain of CFTR.
abstract: 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 internalized from the plasma membrane. We have shown that such internalization is mediated solely by clathrin-coated pathways, and that other pathways, such as caveolae, exclude CFTR. Moreover, CFTR co-precipitates with alpha-adaptin, a component of the endocytic adaptor complex (AP-2). The goal of our current studies was to elucidate further the molecular mechanisms that facilitate entry of CFTR into endocytic clathrin-coated vesicles. Protein-protein interactions generated by incubation of full-length in-vitro-translated CFTR with partially purified bovine brain adaptor complexes were evaluated following immunoprecipitation using an antibody against the alpha-adaptin subunit of the AP-2 complex. Such studies revealed co-immunoprecipitation of alpha-adaptin with full-length but not partially translated CFTR, suggesting that 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-GST sepharose was able to pull-down AP-2 endocytic adaptor complexes, as determined by immunoblot analyses of the precipitates using antibodies directed 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 binding of the AP-2 adaptor complex to the C-terminal domain of CFTR.
authors.affiliation: Cystic Fibrosis Research Centre, S306 BST South, Department of Cell Biology and Physiology, 3500 Terrace Street, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261, USA.
authors.forename: K M
authors.initials: KM
authors.lastname: Weixel
authors.forename: N A
authors.initials: NA
authors.lastname: Bradbury
mesh_terms: D033965:Adaptor Protein Complex alpha Subunits; D033966:Adaptor Protein Complex beta Subunits; D033982:Adaptor Protein Complex gamma Subunits; D000818:Animals; D001665:Binding Sites / Q000502:physiology; D002352:Carrier Proteins / Q000378:metabolism*; D002417:Cattle; D002462:Cell Membrane / Q000378:metabolism; D022163:Clathrin-Coated Vesicles / Q000378:metabolism; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000737:chemistry* / Q000378:metabolism*; D004705:Endocytosis / Q000502:physiology; D008565:Membrane Proteins / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D011817:Rabbits
chemical_list: D033965:Adaptor Protein Complex alpha Subunits; D033966:Adaptor Protein Complex beta Subunits; D033982:Adaptor Protein Complex gamma Subunits; D002352:Carrier Proteins; D008565:Membrane Proteins; D019005:Cystic Fibrosis Transmembrane Conductance Regulator