Agonist-induced phosphorylation of the vascular type 1 angiotensin II receptor.
Agonist-induced receptor phosphorylation plays a role in transmembrane signal transduction systems. Although the cDNA for the rat vascular type 1 angiotensin II receptor (AT1AR) encodes a G protein-coupled receptor with several potential phosphorylation sites for serine/threonine and tyrosine kinases, little is known about the phosphorylation of this receptor. The aim of this study was to determine the effects of angiotensin II (Ang II) on phosphorylation of the AT1AR in rat aortic vascular smooth muscle cells. Using [32P]orthophosphate-labeled cells, immunoprecipitates with anti-AT1AR antibody revealed a labeled band of molecular weight 52 kD, corresponding to the Ang II receptor. Ang II induced a rapid and significant increase in phosphorylation of the Ang II receptor, with a peak at 20 minutes. Phosphoamino acid analysis showed that the major phosphoamino acid is serine, in both the basal and Ang II-stimulated states. Constitutive and agonist-stimulated tyrosine phosphorylation is also observed to a lesser extent. Immunoblotting of anti-phosphotyrosine immunoprecipitates with anti-AT1AR antibody showed that Ang II caused a delayed tyrosine phosphorylation of the receptor with a peak at 20 minutes in a dose-dependent manner. Forskolin increased total phosphorylation of AT1AR but had no effect on tyrosine phosphorylation. Neither phorbol 12-myristate-13-acetate nor ionomycin altered receptor phosphorylation. These findings suggest that Ang II induces the phosphorylation of its own G protein-coupled receptor through both serine and tyrosine kinases and raise the possibility that phosphorylation of the AT1AR is an important regulator of receptor function.
Kai
H
H
Department of Medicine, Emory University, Atlanta, Ga.
Griendling
K K
KK
Lassègue
B
B
Ollerenshaw
J D
JD
Runge
M S
MS
Alexander
R W
RW
D000804:Angiotensin II / Q000502:physiology*; D000818:Animals; D001345:Autoradiography; D002478:Cells, Cultured; D005576:Colforsin / Q000494:pharmacology; D015151:Immunoblotting; D015759:Ionomycin / Q000494:pharmacology; D008297:Male; D009131:Muscle, Smooth, Vascular / Q000378:metabolism*; D010766:Phosphorylation / Q000187:drug effects; D011233:Precipitin Tests; D051381:Rats; D017207:Rats, Sprague-Dawley; D011945:Receptors, Angiotensin / Q000187:drug effects / Q000378:metabolism*; D012694:Serine / Q000378:metabolism; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014443:Tyrosine / Q000378:metabolism
D011945:Receptors, Angiotensin; D000804:Angiotensin II; D005576:Colforsin; D014443:Tyrosine; D012694:Serine; D015759:Ionomycin; D013755:Tetradecanoylphorbol Acetate