R-domain interactions with distal regions of CFTR lead to phosphorylation and activation.
Cystic fibrosis is caused by the aberrant function of the cystic fibrosis transmembrane conductance regulator (CFTR) protein. We examined whether intramolecular binding interactions involving the regulatory (R) domain contributed to CFTR regulation and function. When the R-domain (amino acids 596-836) was coexpressed with Delta1-836 CFTR (a carboxyl hemi-CFTR beginning immediately after the R-domain), strong binding between the two polypeptides was exhibited. The R-domain that co-immunoprecipitated with Delta1-836 exhibited a slower mobility on SDS-PAGE that resulted from phosphorylation of the protein. A larger CFTR polypeptide that included the R-domain (M837X) also exhibited a phosphorylation-dependent mobility shift when coexpressed with Delta1-836. Moreover, coexpression of M837X and Delta1-836 led to enhanced halide permeability in living cells. The activity, unlike in full-length CFTR, was present without forskolin activation, but still sensitive to the PKA inhibitor, Rp-8-CPT-cAMPS. This PKA inhibition of activity was found to be dependent on the carboxy region of the R-domain, amino acids 723-836. Our results indicate that the R-domain binds CFTR residues after amino acid 836 and that this binding facilitates phosphorylation and CFTR activation. We have also characterized a subdomain within CFTR (residues 723-837) that is necessary for PKA-dependent constitutive activation. Finally, these experiments demonstrate that constitutive CFTR activity can be accomplished by at least two mechanisms: (1) direct modulation of the R-domain to abrogate PKA regulation and (2) modifications that increase R-domain susceptibility to steady-state phosphorylation through PKA.
Department of Physiology and Biophysics, Department of Medicine, University of Alabama at Birmingham, Birmingham, Alabama 35294-0005, USA.
S A
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King
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Sorscher
D000838:Anions / Q000378:metabolism; D001965:Bromides / Q000378:metabolism; D002463:Cell Membrane Permeability; D002712:Chlorides / Q000378:metabolism; D005576:Colforsin / Q000494:pharmacology; D000242:Cyclic AMP / Q000031:analogs & derivatives / Q000494:pharmacology; D017868:Cyclic AMP-Dependent Protein Kinases / Q000037:antagonists & inhibitors; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D007454:Iodides / Q000378:metabolism; D015640:Ion Channel Gating / Q000187:drug effects; D010446:Peptide Fragments / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000378:metabolism; D013873:Thionucleotides / Q000494:pharmacology
D000838:Anions; D001965:Bromides; D002712:Chlorides; D007454:Iodides; D010446:Peptide Fragments; D011994:Recombinant Proteins; D013873:Thionucleotides; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D005576:Colforsin; C015929:8-((4-chlorophenyl)thio)cyclic-3',5'-AMP; D000242:Cyclic AMP; D017868:Cyclic AMP-Dependent Protein Kinases