Glycosylation status of endogenous CFTR does not affect cAMP-stimulated Cl- secretion in epithelial cells.
Cystic fibrosis (CF) impairs Cl- secretion across epithelial tissues and is caused by mutations in an N-linked glycoprotein, the cystic fibrosis transmembrane conductance regulator (CFTR). We modified the glycosylation pattern of CFTR using inhibitors of oligosaccharide processing and determined their effects on both agonist-induced Cl- secretion and CFTR location in human colon (HT-29) cell lines. In both polarized and unpolarized HT-29 cells, immunoprecipitation of cell extracts using a monoclonal antibody against CFTR gave a single band at 170 kDa. Inhibitors of N-linked glycosylation reduced the molecular mass of this band: swainsonine by 10 kDa, deoxymannojirimycin by 30 kDa, and deoxynojirimycin by 10-20 kDa. However, the transepithelial Cl- current and conductance stimulated by adenosine 3',5'-cyclic monophosphate (cAMP)- or Ca(2+)-dependent secretagogues was not affected. In the polarized cells, CFTR was localized in the apical membrane domain. Treatment of the monolayers with glycoprocessing inhibitors did not affect CFTR's location. Thus, in human colonocytes that endogenously express CFTR, the extent of CFTR glycosylation does not influence the targeting of CFTR to the apical membrane domain or its function as an agonist-stimulated Cl- channel.
Morris
A P
AP
Department of Physiology and Biophysics, University of Alabama at Birmingham 35294-0005.
Cunningham
S A
SA
Benos
D J
DJ
Frizzell
R A
RA
D002460:Cell Line; D016764:Cell Polarity; D002712:Chlorides / Q000378:metabolism*; D005576:Colforsin / Q000494:pharmacology; D003106:Colon / Q000166:cytology / Q000378:metabolism*; D000242:Cyclic AMP / Q000494:pharmacology*; D003550:Cystic Fibrosis / Q000378:metabolism; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D005455:Fluorescent Antibody Technique; D006031:Glycosylation; D006801:Humans; D007413:Intestinal Mucosa / Q000166:cytology / Q000378:metabolism*; D008565:Membrane Proteins / Q000378:metabolism*; D009496:Neurotensin / Q000494:pharmacology; D009844:Oligosaccharides / Q000037:antagonists & inhibitors / Q000378:metabolism; D011499:Protein Processing, Post-Translational
C505032:CFTR protein, human; D002712:Chlorides; D008565:Membrane Proteins; D009844:Oligosaccharides; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D005576:Colforsin; D009496:Neurotensin; D000242:Cyclic AMP