title: Phosphorylation by cAMP-dependent protein kinase causes a conformational change in the R domain of the cystic fibrosis transmembrane conductance regulator.
abstract: Individuals with cystic fibrosis have a defect in the CFTR protein, a chloride channel regulated by cAMP-dependent protein kinase (PKA). The majority of the phosphorylation sites of PKA are located in the R domain of CFTR. It has been postulated that this domain may act as a gate for the chloride channel. Of the many possible mechanisms whereby the R domain could gate the channel, including interdomain interactions, charge distribution, or conformational change, we investigated the possibility that phosphorylation leads to conformational changes in the R domain. To test this hypothesis, a protocol for purification of human R domain peptide synthesized in a bacterial expression system was developed. Purified R domain was phosphorylated by PKA, and CD spectra were obtained. As a result of phosphorylation by PKA, a significant spectral change, indicative of a reduction in the alpha-helical content, was found. CD spectra of the R domain of a shark homologue of CFTR indicated similar changes in conformation as a result of phosphorylation by PKA. In contrast, phosphorylation of the human R domain by PKC, which has only a small influence on CFTR channel activity, failed to elicit CD spectral changes, indicating no conformational change comparable to those induced by PKA phosphorylation. These observations provide the first structural characterization of the R domain and suggest that the gating of the CFTR chloride channel by PKA may involve a conformational change in the R domain.
authors.affiliation: Research Institute and Biochemistry Department, Hospital for Sick Children, Toronto, Ontario, Canada.
authors.forename: A M
authors.initials: AM
authors.lastname: Dulhanty
authors.forename: J R
authors.initials: JR
authors.lastname: Riordan
mesh_terms: D000818:Animals; D018118:Chloride Channels / Q000378:metabolism* / Q000648:ultrastructure; D002942:Circular Dichroism; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism*; D003550:Cystic Fibrosis; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D004284:Dogfish; D006801:Humans; D008565:Membrane Proteins / Q000378:metabolism* / Q000648:ultrastructure; D010766:Phosphorylation; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry
chemical_list: C505032:CFTR protein, human; D018118:Chloride Channels; D008565:Membrane Proteins; D011994:Recombinant Proteins; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D017868:Cyclic AMP-Dependent Protein Kinases