Characterization of the internalization pathways for the cystic fibrosis transmembrane conductance regulator

Mutations in the gene encoding the cystic fibrosis (CF) transmembrane condu ctance regulator (CFTR) chloride channel give rise to the most common letha l genetic disease of Caucasian populations, CF. Although the function of CF TR is primarily related to the regulation of apical membrane chloride perme ability, biochemical, immunocytochemical, and functional studies indicate t hat CFTR is also present in endosomal and tl ans Golgi compartments. The mo lecular pathways by which CFTR is internalized into intracellular compartme nts are not fully understood. To define the pathways for CFTR internalizati on, we investigated the association of CFTR with two specialized domains of the plasma membrane, clathrin-coated pits and caveolae. Internalization of CFTR was monitored after cell surface biotinylation and quantitation of ce ll surface CFTR levels after elution of cell lysates from a monomeric avidi n column. Cell surface levels of CFTR were determined after disruption of c aveolae or clathrin-coated vesicle formation. Biochemical assays revealed t hat disrupting the formation of clathrin-coated vesicles inhibited the inte rnalization of CFTR from the plasma membrane, resulting in a threefold incr ease in the steady-state levels of cell surface CFTR. In contrast, the leve ls of cell surface CFTR after disruption of caveolae were not different fro m those in control cells. In addition, although our studies show the presen ce of caveolin at the apical membrane domain of human airway epithelial cel ls, we were unable to detect CFTR in purified caveolae. These results sugge st that CFTR is constitutively internalized from the apical plasma membrane via clathrin-coated pits and that CFTR is excluded from caveolae.