title: Role of tyrosine phosphorylation in the muscarinic activation of the cystic fibrosis transmembrane conductance regulator (CFTR).
abstract: Cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride (Cl(-)) channel, which plays an important role in physiological anion and fluid secretion, and is defective in several diseases. Although its activation by PKA and PKC has been studied extensively, its regulation by receptors is less well understood. To study signaling involved in CFTR activation, we measured whole-cell Cl(-) currents in BHK cells cotransfected with GPCRs and CFTR. In cells expressing the M3 muscarinic acetylcholine receptor, the agonist carbachol (Cch) caused strong activation of CFTR through two pathways; the canonical PKA-dependent mechanism and a second mechanism that involves tyrosine phosphorylation. The role of PKA was suggested by partial inhibition of cholinergic stimulation by the specific PKA inhibitor Rp-cAMPS. The role of tyrosine kinases was suggested by Cch stimulation of 15SA-CFTR and 9CA-CFTR, mutants that lack 15 PKA or 9 PKC consensus sequences and are unresponsive to PKA or PKC stimulation, respectively. Moreover the residual Cch response was sensitive to inhibitors of the Pyk2 and Src tyrosine kinase family. Our results suggest that tyrosine phosphorylation acts on CFTR directly and through inhibition of the phosphatase PP2A. Results suggest that PKA and tyrosine kinases contribute to CFTR regulation by GPCRs that are expressed at the apical membrane of intestinal and airway epithelia.
authors.affiliation: Department of Physiology, McGill University and Research Institute of the McGill University Hospital Centre, Montréal, Quebec H3G 1Y6, Canada. arnaud.billet@mcgill.ca
authors.forename: Arnaud
authors.initials: A
authors.lastname: Billet
authors.forename: Yishan
authors.initials: Y
authors.lastname: Luo
authors.forename: Haouaria
authors.initials: H
authors.lastname: Balghi
authors.forename: John W
authors.initials: JW
authors.lastname: Hanrahan
mesh_terms: D000818:Animals; D001565:Benzoates / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism; D002217:Carbachol / Q000494:pharmacology; D002460:Cell Line; D018679:Cholinergic Agonists / Q000494:pharmacology; D000242:Cyclic AMP / Q000031:analogs & derivatives / Q000378:metabolism / Q000494:pharmacology; D017868:Cyclic AMP-Dependent Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D051418:Focal Adhesion Kinase 2 / Q000037:antagonists & inhibitors / Q000378:metabolism; D006801:Humans; D015151:Immunoblotting; D008564:Membrane Potentials / Q000187:drug effects; D009154:Mutation; D018408:Patch-Clamp Techniques; D010766:Phosphorylation / Q000187:drug effects; D054648:Protein Phosphatase 2 / Q000037:antagonists & inhibitors / Q000378:metabolism; D043587:Receptor, Muscarinic M3 / Q000819:agonists / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology*; D053778:Thiazolidines / Q000494:pharmacology; D013873:Thionucleotides / Q000494:pharmacology; D014443:Tyrosine / Q000235:genetics / Q000378:metabolism*; D019061:src-Family Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism
chemical_list: C482900:3-((3-trifluoromethyl)phenyl)-5-((3-carboxyphenyl)methylene)-2-thioxo-4-thiazolidinone; D001565:Benzoates; D018679:Cholinergic Agonists; D004791:Enzyme Inhibitors; D043587:Receptor, Muscarinic M3; D053778:Thiazolidines; D013873:Thionucleotides; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; C016957:adenosine-3',5'-cyclic phosphorothioate; D014443:Tyrosine; D002217:Carbachol; D000242:Cyclic AMP; D051418:Focal Adhesion Kinase 2; D019061:src-Family Kinases; D017868:Cyclic AMP-Dependent Protein Kinases; D054648:Protein Phosphatase 2; D002118:Calcium
keywords: CFTR; G Protein-coupled Receptors (GPCR); Muscarinic 3 Receptor; PP2A; Src; Tyrosine Protein Kinase (Tyrosine Kinase)