Phosphorylation of CFTR by PKA promotes binding of the regulatory domain

Valerie Chappe, Thomas Irvine, Jie Liao, Alexandra Evagelidis, John W. Hanrahan*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

62 Scopus citations

Abstract

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 SplitAR, biotinylation, immunostaining, and functional studies clearly demonstrated its trafficking to the plasma membrane. SplitΔR generated a constitutive halide permeability, which became responsive to cAMP when the missing R domain was coexpressed. Each half-molecule was coprecipitated 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 SplitΔR 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.

Original languageEnglish (US)
Pages (from-to)2730-2740
Number of pages11
JournalEMBO Journal
Volume24
Issue number15
DOIs
StatePublished - Aug 3 2005

Keywords

  • Chloride channel
  • Cystic fibrosis
  • Domain-domain interactions
  • Ion channel regulation
  • Protein kinase

ASJC Scopus subject areas

  • Neuroscience(all)
  • Molecular Biology
  • Biochemistry, Genetics and Molecular Biology(all)
  • Immunology and Microbiology(all)

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