cAMP-regulated trafficking of epitope-tagged CFTR.
The cystic fibrosis transmembrane conductance regulator (CFTR) is a phosphorylation-activated chloride channel responsible for cAMP-induced Cl secretion across the apical membranes of epithelial cells. To optimize its detection in membrane localization studies, we tagged CFTR with epitope sequences at the carboxy terminus or in the fourth external loop. The function of six different tagged-CFTRs was tested in two different physiological assays. CFTRs containing the M2 epitope responded to cAMP, whereas cells expressing CFTR with the hemagglutinin HA tag showed little or no cAMP response. Using CFTR tagged in the fourth external loop, we demonstrate that cAMP activation using forskolin results in an increase in CFTR in the plasma membrane of HeLa cells. Forskolin inhibited CFTR endocytosis, and this contributes to the increase in cell surface CFTR expression. Our results indicate that regulation of cell surface CFTR contributes to the increase in plasma membrane Cl conductance evoked by cAMP stimulation.
Howard
M
M
Department of Physiology and Biophysics, University of Alabama at Birmingham, USA.
Jilling
T
T
DuVall
M
M
Frizzell
R A
RA
D000595:Amino Acid Sequence; D000818:Animals; D001692:Biological Transport / Q000502:physiology; D002460:Cell Line / Q000737:chemistry / Q000378:metabolism; D002462:Cell Membrane / Q000737:chemistry / Q000378:metabolism; D005576:Colforsin / Q000494:pharmacology; D000242:Cyclic AMP / Q000502:physiology*; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D004285:Dogs; D004705:Endocytosis / Q000502:physiology; D000939:Epitopes / Q000378:metabolism*; D005260:Female; D005455:Fluorescent Antibody Technique; D015870:Gene Expression / Q000187:drug effects; D006031:Glycosylation; D006367:HeLa Cells / Q000378:metabolism; D006801:Humans; D007686:Kidney Tubules, Distal / Q000166:cytology; D008565:Membrane Proteins / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation / Q000502:physiology; D009865:Oocytes / Q000378:metabolism; D017434:Protein Structure, Tertiary; D012333:RNA, Messenger / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics; D014616:Vaccinia virus; D014982:Xenopus laevis
C505032:CFTR protein, human; D000939:Epitopes; D008565:Membrane Proteins; D012333:RNA, Messenger; D011994:Recombinant Proteins; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D005576:Colforsin; D000242:Cyclic AMP