title: Failure of cAMP agonists to activate rescued deltaF508 CFTR in CFBE41o- airway epithelial monolayers.
abstract: The cystic fibrosis transmembrane conductance regulator (CFTR) is a cyclic AMP-regulated chloride channel. Mutations in the CFTR gene result in cystic fibrosis (CF). The most common mutation, deltaF508, results in endoplasmic reticulum-associated degradation (ERAD) of CFTR. DeltaF508 CFTR has been described as a temperature-sensitive mutation that can be rescued following growth at 27 degrees C. In order to study the processing and function of wild-type and rescued deltaF508 CFTR at the cell surface under non-polarized and polarized conditions, we developed stable cell lines expressing deltaF508 or wild-type CFTR. CFBE41o- is a human airway epithelial cell line capable of forming high resistance, polarized monolayers when cultured on permeable supports, while HeLa cells are normally grown under non-polarizing conditions. Immunoprecipitation, cell surface biotinylation, immunofluorescence, and functional assays confirmed the presence of deltaF508 CFTR at the cell surface in both cell lines after incubating the cells for 48 h at 27 degrees C. However, stimulators of wild-type CFTR such as forskolin, beta2-adrenergic or A2B-adenosine receptor agonists failed to activate rescued deltaF508 CFTR in CFBE41o- monolayers. Rescued deltaF508 CFTR could be stimulated with genistein independent of pretreatment with cAMP signalling agonists. Interestingly, rescued deltaF508 CFTR in HeLa cells could be efficiently stimulated with either forskolin or genistein to promote Cl- transport. These results indicate that deltaF508 CFTR, when rescued in CFBE41o- human airway epithelial cells, is poorly responsive to signalling pathways known to regulate wild-type CFTR. Furthermore, the differences in rescue and activation of deltaF508 CFTR in the two cell lines suggest that cell-type specific differences in deltaF508 CFTR processing are likely to complicate efforts to identify potentiators and/or correctors of the deltaF508 defect.
authors.affiliation: Gregory Fleming James Cystic Fibrosis Research Center, University of Alabama at Birmingham, Birmingham, AL 35233, USA.
authors.forename: Zsuzsa
authors.initials: Z
authors.lastname: Bebok
authors.forename: James F
authors.initials: JF
authors.lastname: Collawn
authors.forename: John
authors.initials: J
authors.lastname: Wakefield
authors.forename: William
authors.initials: W
authors.lastname: Parker
authors.forename: Yao
authors.initials: Y
authors.lastname: Li
authors.forename: Karoly
authors.initials: K
authors.lastname: Varga
authors.forename: Eric J
authors.initials: EJ
authors.lastname: Sorscher
authors.forename: J P
authors.initials: JP
authors.lastname: Clancy
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D002460:Cell Line; D002462:Cell Membrane / Q000737:chemistry / Q000187:drug effects; D005576:Colforsin / Q000494:pharmacology*; D000242:Cyclic AMP / Q000819:agonists* / Q000032:analysis / Q000378:metabolism*; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000235:genetics / Q000378:metabolism*; D005260:Female; D019833:Genistein / Q000494:pharmacology; D006367:HeLa Cells; D006801:Humans; D007150:Immunohistochemistry; D047468:Immunoprecipitation; D009154:Mutation; D043562:Receptors, G-Protein-Coupled / Q000502:physiology*; D020545:Respiratory Mucosa / Q000737:chemistry / Q000166:cytology / Q000502:physiology*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology
chemical_list: D043562:Receptors, G-Protein-Coupled; C093349:cystic fibrosis transmembrane conductance regulator delta F508; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D005576:Colforsin; D000255:Adenosine Triphosphate; D019833:Genistein; D000242:Cyclic AMP