Polarization-dependent apical membrane CFTR targeting underlies cAMP-stimulated Cl- secretion in epithelial cells.
The relationship between adenosine 3',5'-cyclic monophosphate (cAMP)-mediated Cl- secretion and the cellular location of the cystic fibrosis transmembrane conductance regulator (CFTR) was determined in both polarized (Cl.19A) and unpolarized (parental) HT-29 colonocytes expressing similar levels of CFTR mRNA and protein. CFTR immunolocalized to the apical membrane domain of polarized colonocytes exhibiting cAMP-responsive Cl- secretion. In contrast, CFTR staining was perinuclear in unpolarized colonocytes, which gave little or no cAMP-stimulated Cl- conductance responses. Thus cAMP-stimulated Cl- secretion coincided with an apical localization of CFTR. Brefeldin A (BFA) was used to perturb glycoprotein targeting in these cells. In polarized colonocytes, BFA caused a reversible, time-dependent decrease in the Cl-conductance response to cAMP but not Ca2+. Apical CFTR redistributed into large coalesced intracellular vesicles, located within the same plane as the microtubule organizing center, a marker for the trans-Golgi network (TGN). In preconfluent monolayers or unpolarized HT-29 cells, BFA had no effect on CFTR staining, which remained perinuclear. Mature, Golgi-processed CFTR protein was isolated from both polarized and unpolarized colonocytes. Thus the mechanism for polarization-dependent apical membrane CFTR targeting and the acquisition of cAMP-dependent Cl- secretion lies at or beyond the late Golgi-TGN in epithelial cells.
Morris
A P
AP
Department of Physiology and Biophysics, University of Alabama at Birmingham 35294-0005.
Cunningham
S A
SA
Tousson
A
A
Benos
D J
DJ
Frizzell
R A
RA
D020126:Brefeldin A; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D016764:Cell Polarity; D002712:Chlorides / Q000037:antagonists & inhibitors / Q000378:metabolism* / Q000502:physiology; D003106:Colon / Q000166:cytology / Q000378:metabolism*; D000242:Cyclic AMP / Q000494:pharmacology*; D003517:Cyclopentanes / Q000494:pharmacology; D003550:Cystic Fibrosis / Q000378:metabolism; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D004553:Electric Conductivity; D005455:Fluorescent Antibody Technique; D006031:Glycosylation; D006801:Humans; D007413:Intestinal Mucosa / Q000166:cytology / Q000378:metabolism*; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D011233:Precipitin Tests; D012333:RNA, Messenger / Q000378:metabolism
C505032:CFTR protein, human; D002712:Chlorides; D003517:Cyclopentanes; D008565:Membrane Proteins; D012333:RNA, Messenger; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D020126:Brefeldin A; D000242:Cyclic AMP