Phosphorylation of CFTR by PKA promotes binding of the regulatory domain.
The unphosphorylated regulatory (R) domain of the Cystic Fibrosis Transmembrane conductance Regulator (CFTR) is often viewed as an inhibitor that is released by phosphorylation. To test this notion, we studied domain interactions using CFTR channels assembled from three polypeptides. Nucleotides encoding the R domain (aa 635-836) were replaced with an internal ribosome entry sequence so that amino- and carboxyl-terminal half-molecules would be translated from the same mRNA transcript. Although only core glycosylation was detected on SplitDeltaR, biotinylation, immunostaining, and functional studies clearly demonstrated its trafficking to the plasma membrane. SplitDeltaR generated a constitutive halide permeability, which became responsive to cAMP when the missing R domain was coexpressed. Each half-molecule was co-precipitated by antibody against the other half. Contrary to expectations, GST-R domain was pulled down only if prephosphorylated by protein kinase A, and coexpressed R domain was precipitated with SplitDeltaR much more efficiently when cells were stimulated with cAMP. These results indicate that phosphorylation regulates CFTR by promoting association of the R domain with other domains rather than by causing its dissociation from an inhibitory site.
Chappe
Valerie
V
Department of Physiology, McGill University, Montréal, QC, Canada.
Irvine
Thomas
T
Liao
Jie
J
Evagelidis
Alexandra
A
Hanrahan
John W
JW
D000818:Animals; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D006224:Cricetinae; D000242:Cyclic AMP / Q000378:metabolism; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism*; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000378:metabolism*; D006801:Humans; D010766:Phosphorylation; D011485:Protein Binding; D017434:Protein Structure, Tertiary
C505032:CFTR protein, human; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D000242:Cyclic AMP; D017868:Cyclic AMP-Dependent Protein Kinases