Localization of cystic fibrosis transmembrane conductance regulator in chloride secretory epithelia.
Cystic fibrosis is caused by mutations in the gene coding for the cystic fibrosis transmembrane conductance regulator (CFTR). To further our understanding of CFTR's function and regulation, we used confocal immunofluorescence microscopy to localize CFTR in cells stained with monoclonal antibodies against different regions of the protein: the R (regulatory) domain (M13-1), the COOH terminus (M1-4), and a predicted extracellular domain (M6-4). All three antibodies immunoprecipitated a 155-170-kD polypeptide from cells expressing CFTR. Each antibody stained HeLa and 3T3 cells expressing recombinant CFTR, but not cells lacking endogenous CFTR: HeLa, NIH-3T3, and endothelial cells. For localization studies, we used epithelial cell lines that express endogenous CFTR and have a cAMP-activated apical Cl- permeability: T84, CaCo2, and HT29 clone 19A. Our results demonstrate that CFTR is an apical membrane protein in these epithelial cells because (a) staining for CFTR resembled staining for several apical membrane markers, but differed from staining for basolateral membrane proteins; (b) thin sections of cell monolayers show staining at the apical membrane; and (c) M6-4, an extracellular domain antibody, stained the apical surface of nonpermeabilized cells. Our results do not exclude the possibility that CFTR is also located beneath the apical membrane. Increasing intracellular cAMP levels did not change the apical membrane staining pattern for CFTR. Moreover, insertion of channels by vesicle fusion with the apical membrane was not required for cAMP-mediated increases in apical membrane Cl- conductance. These results indicate that CFTR is located in the apical plasma membrane of Cl(-)-secreting epithelia, a result consistent with the conclusion that Cl TR is an apical membrane chloride channel.
Denning
G M
GM
Howard Hughes Medical Institute, Department of Internal Medicine, University of Iowa College of Medicine, Iowa City 52242.
Ostedgaard
L S
LS
Cheng
S H
SH
Smith
A E
AE
Welsh
M J
MJ
D000911:Antibodies, Monoclonal; D000918:Antibody Specificity; D002460:Cell Line; D002462:Cell Membrane / Q000737:chemistry / Q000187:drug effects; D016764:Cell Polarity / Q000502:physiology; D018118:Chloride Channels; D002712:Chlorides / Q000378:metabolism*; D000242:Cyclic AMP / Q000494:pharmacology; D003550:Cystic Fibrosis / Q000378:metabolism*; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D004789:Enzyme Activation; D004847:Epithelial Cells; D004848:Epithelium / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism; D005455:Fluorescent Antibody Technique; D006801:Humans; D008565:Membrane Proteins / Q000032:analysis* / Q000737:chemistry; D008856:Microscopy, Fluorescence; D008956:Models, Chemical; D011487:Protein Conformation
D000911:Antibodies, Monoclonal; C505032:CFTR protein, human; D018118:Chloride Channels; D002712:Chlorides; D008565:Membrane Proteins; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D000242:Cyclic AMP