title: Evidence for agonist-induced interaction of angiotensin receptor with a guanine nucleotide-binding protein in bovine adrenal zona glomerulosa.
abstract: Previous studies have documented that high affinity binding of [125I]angiotensin II to adrenal cortex receptors was modulated by guanine nucleotides. Since in other receptor systems, similar properties of hormone-receptor interactions were shown to be specific for agonists, we studied the differential binding characteristics of agonists and antagonists to this receptor using a new radiolabeled antagonist [125I] [Sar1,Ile8] angiotensin II. Receptor saturation studies indicate that the antagonist is binding to a homogeneous population of sites with a Kd of 0.6-2.0 nM and with a receptor density around 1 pmol/mg of protein. Competition curves using unlabeled antagonists are characterized by a slope factor of 1 and a single Kd of 1-3 nM. Addition of guanylylimidodiphosphate to the assay is absolutely without effect on radiolabeled antagonist binding. In contrast, competition curves using the full agonists angiotensin II, [Sar1]angiotensin II, angiotensin III, and [des-Arg]angiotensin III display slope factors of 0.79, 0.87, 0.70, and 0.84, respectively. These curves can be explained by two apparent forms of the receptor having high and low affinity for the agonist. The higher affinity form associated with these four agonists is characterized by a Kd of 1.2 nM, 0.25 nM, 0.8 nM, and 3 microM, and corresponds to 60, 56, 42, and 25% of angiotensin II-binding sites, respectively. The other form displays 13- to 33-fold lower affinity. Addition of guanine nucleotide to the assay results in a 2-4-fold shift to the right and a steepening (slope factor 0.9-1.0) of agonist competition curves. Angiotensin II receptors, occupied by the full agonist [131I] [Sar1] angiotensin II or by the antagonist [125I] [Sar1, Ile8]angiotensin II, were then solubilized with the nonionic detergent octylglucoside. Dissociation of the agonist [131I] [Sar1] angiotensin II from solubilized receptors is enhanced by guanylylimidodiphosphate or sodium acetate, while dissociation of the antagonist [125I] [Sar1, Ile8]angiotensin II displays little sensitivity towards guanine nucleotides or increased ionic strength. Inclusion of bile salts in the solubilization medium preferentially destabilizes receptor-bound agonist, presumably by interfering with protein-protein interactions required for high affinity agonist binding. Separation of radiolabeled agonist and antagonist-occupied solubilized receptor complexes by steric exclusion high performance liquid chromatography reveals that the agonist-occupied receptor complex behaves as a larger protein than the antagonist-occupied receptor complex.(ABSTRACT TRUNCATED AT 400 WORDS)
mesh_terms: D000302:Adrenal Cortex / Q000378:metabolism*; D000804:Angiotensin II / Q000031:analogs & derivatives / Q000037:antagonists & inhibitors / Q000378:metabolism; D057911:Angiotensin Receptor Antagonists; D000818:Animals; D001667:Binding, Competitive; D002417:Cattle; D002462:Cell Membrane / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000378:metabolism*; D006160:Guanosine Triphosphate / Q000031:analogs & derivatives*; D006165:Guanylyl Imidodiphosphate / Q000494:pharmacology*; D007700:Kinetics; D008954:Models, Biological; D011945:Receptors, Angiotensin / Q000187:drug effects / Q000302:isolation & purification / Q000378:metabolism*; D011956:Receptors, Cell Surface / Q000378:metabolism*
chemical_list: D057911:Angiotensin Receptor Antagonists; D011945:Receptors, Angiotensin; D011956:Receptors, Cell Surface; D000804:Angiotensin II; D006165:Guanylyl Imidodiphosphate; C009210:sarleucin; D006160:Guanosine Triphosphate; D019204:GTP-Binding Proteins