title: Phosphorylation of the type 1A angiotensin II receptor by G protein-coupled receptor kinases and protein kinase C.
abstract: The type 1A angiotensin II receptor (AT1A-R), which mediates cardiovascular effects of angiotensin II, has been shown to undergo rapid agonist-induced desensitization. We investigated the potential role of second messenger-activated kinases and G protein-coupled receptor kinases (GRKs) in the regulation of this receptor. In 293 cells transfected with the AT1A-R, a 3-min challenge with angiotensin II engendered a 46% decrease in subsequent angiotensin II-stimulated phosphoinositide hydrolysis in intact cells. This agonist-induced desensitization correlated temporally and dose-dependently with the phosphorylation of the receptor to a stoichiometry of 1 mol of phosphate/mol of receptor, as assessed by immunoprecipitation of receptors from cells metabolically labeled with 32Pi. Agonist-induced receptor phosphorylation was reduced by 40-50% by either overexpression of a dominant negative K220R mutant GRK2 or treatment of the cells with the protein kinase C (PKC) inhibitor staurosporine, in a virtually additive fashion. Cellular overexpression of GRK2K220R not only inhibited agonist-induced AT1A-R phosphorylation, but also prevented receptor desensitization, as assessed by angiotensin II-stimulated GTPase activity in membranes prepared from agonist-treated and control cells. In contrast, PKC inhibition by staurosporine did not affect homologous desensitization of the AT1A-R. Overexpression of GRKs 2, 3, or 5 significantly augmented the agonist-induced AT1A-R phosphorylation 1.5- to 1.7-fold (p < 0.001). These findings suggest a role for receptor phosphorylation by one or several GRKs in the rapid agonist-induced desensitization of the AT1A-R.
authors.affiliation: Howard Hughes Medical Institute, Department of Medicine, Duke University Medical Center, Durham, North Carolina 27710, USA.
authors.forename: M
authors.initials: M
authors.lastname: Oppermann
authors.forename: N J
authors.initials: NJ
authors.lastname: Freedman
authors.forename: R W
authors.initials: RW
authors.lastname: Alexander
authors.forename: R J
authors.initials: RJ
authors.lastname: Lefkowitz
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D017931:DNA Primers; D019204:GTP-Binding Proteins / Q000378:metabolism*; D006801:Humans; D008969:Molecular Sequence Data; D016296:Mutagenesis; D010766:Phosphorylation; D011493:Protein Kinase C / Q000378:metabolism*; D051381:Rats; D020794:Receptor Protein-Tyrosine Kinases / Q000235:genetics / Q000378:metabolism*; D011945:Receptors, Angiotensin / Q000378:metabolism*; D015290:Second Messenger Systems
chemical_list: D017931:DNA Primers; D011945:Receptors, Angiotensin; D020794:Receptor Protein-Tyrosine Kinases; D011493:Protein Kinase C; D019204:GTP-Binding Proteins