CONSTITUTIVE INTERNALIZATION OF CYSTIC-FIBROSIS TRANSMEMBRANE CONDUCTANCE REGULATOR OCCURS VIA CLATHRIN-DEPENDENT ENDOCYTOSIS AND IS REGULATED BY PROTEIN-PHOSPHORYLATION

Although the cystic fibrosis transmembrane conductance regulator (CFTR ) is primarily implicated in the regulation of plasma-membrane chlorid e permeability, immunolocalization and functional studies indicate the presence of CFTR in the endosomal compartment. The mechanism of CFTR delivery from the cell surface to endosomes is not understood, To deli neate the internalization pathway, both the rate and extent of CFTR ac cumulation in endosomes were monitored in stably transfected Chinese h amster ovary (CHO) cells. The role of clathrin-dependent endocytosis w as assessed in cells exposed to hypertonic medium, potassium depletion or intracellular acid-load. These treatments inhibited clathrin-depen dent endocytosis by > 90 %, as verified by measurements of I-125-trans ferrin uptake. Functional association of CFTR with newly formed endoso mes was determined by an endosomal pH dissipation protocol [Lukacs, Ch ang, Kartner, Rotstein, Riordan and Grinstein (1992) J. Biol. Chem. 26 7, 14568-14572]. As a second approach, endocytosis of CFTR was determi ned after cell-surface biotinylation with the cleavable nimidyl-2-(bio tinamido)ethyl-1,3-dithiopropionate. Both the biochemical and the func tional assays indicated that arresting the formation of clathrin-coate d vesicles inhibited the retrieval of the CFTR from the plasma membran e to endosomes. An overall arrest of membrane traffic cannot account f or the inhibition of CFTR internalization, since the fluid-phase endoc ytosis was not effected by the treatments used. Thus the efficient, co nstitutive internalization of surface CFTR (5% per min) occurs, predom inantly by clathrin-dependent endocytosis. Stimulation of protein phos phorylation by cAMP-dependent protein kinase A and by protein kinase C decreased the rate of internalization of cell-surface biotinylated CF TR, and contributed to a substantial diminution of the internal CFTR p ool compared with that of unstimulated cells. These results suggest th at the rate of CFTR internalization may participate in the determinati on of the CFTR channel density, and consequently, of the cAMP-stimulat ed chloride conductance of the plasma membrane.