AGONIST-INDUCED PHOSPHORYLATION OF THE ENDOGENOUS AT(1) ANGIOTENSIN RECEPTOR IN BOVINE ADRENAL GLOMERULOSA CELLS

A polyclonal antibody was raised in rabbits against a fusion protein i mmunogen consisting of bacterial maltose-binding protein coupled to a 92-amino acid C-terminal fragment of the rat AT(1b) angiotensin II (An g II) receptor. The antibody immunoprecipitated the photoaffinity-labe led bovine AT(1) receptor (AT(1)-R), but not the rat AT(2) receptor, a nd specifically stained bovine adrenal glomerulosa cells and AT(1a) re ceptor-expressing Cos-7 cells, as well as the rat adrenal zona glomeru losa and renal glomeruli. The antibody was employed to analyze Ang II- induced phosphorylation of the endogenous AT(1)-R immunoprecipitated f rom cultured bovine adrenal glomerulosa cells. Receptor phosphorylatio n 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 kina se A and protein kinase C (PKC) also caused phosphorylation of the rec eptor, but to a lesser extent than Ang II. Inhibition of PKC by stauro sporine augmented Ang II-stimulated AT(1)-R phosphorylation, suggestin g a negative regulatory role of PKC on the putative G protein-coupled receptor kinase(s) that mediates the majority of AT(1)-R phosphorylati on. The antibody should permit further analysis of endogenous AT(1)-R phosphorylation in Ang II target cells.