Agonist-induced phosphorylation of the endogenous AT1 angiotensin receptor in bovine adrenal glomerulosa cells.
A polyclonal antibody was raised in rabbits against a fusion protein immunogen consisting of bacterial maltose-binding protein coupled to a 92-amino acid C-terminal fragment of the rat AT1b angiotensin II (Ang II) receptor. The antibody immunoprecipitated the photoaffinity-labeled bovine AT1 receptor (AT1-R), but not the rat AT2 receptor, and specifically stained bovine adrenal glomerulosa cells and AT1a receptor-expressing Cos-7 cells, as well as the rat adrenal zona glomerulosa and renal glomeruli. The antibody was employed to analyze Ang II-induced phosphorylation of the endogenous AT1-R immunoprecipitated from cultured bovine adrenal glomerulosa cells. Receptor phosphorylation was rapid, sustained for up to 60 min, and enhanced by pretreatment of the cells with okadaic acid. Its magnitude was correlated with the degree of ligand occupancy of the receptor. Activation of protein kinase A and protein kinase C (PKC) also caused phosphorylation of the receptor, but to a lesser extent than Ang II. Inhibition of PKC by staurosporine augmented Ang II-stimulated AT1-R phosphorylation, suggesting a negative regulatory role of PKC on the putative G protein-coupled receptor kinase(s) that mediates the majority of AT1-R phosphorylation. The antibody should permit further analysis of endogenous AT1-R phosphorylation in Ang II target cells.
Smith
R D
RD
Endocrinology and Reproduction Research Branch, National Institute of Child Health and Human Development, Bethesda, Maryland 20892-4510, USA.
Baukal
A J
AJ
Zolyomi
A
A
Gaborik
Z
Z
Hunyady
L
L
Sun
L
L
Zhang
M
M
Chen
H C
HC
Catt
K J
KJ
D000804:Angiotensin II / Q000494:pharmacology; D000818:Animals; D019556:COS Cells; D002417:Cattle; D002478:Cells, Cultured; D015151:Immunoblotting; D007150:Immunohistochemistry; D010766:Phosphorylation; D011233:Precipitin Tests; D011817:Rabbits; D051381:Rats; D044140:Receptor, Angiotensin, Type 1; D044139:Receptor, Angiotensin, Type 2; D011945:Receptors, Angiotensin / Q000819:agonists* / Q000302:isolation & purification / Q000378:metabolism*; D015384:Zona Glomerulosa / Q000166:cytology / Q000378:metabolism*
D044140:Receptor, Angiotensin, Type 1; D044139:Receptor, Angiotensin, Type 2; D011945:Receptors, Angiotensin; D000804:Angiotensin II