title: Inhibition of adenylate cyclase is mediated by the high affinity conformation of the alpha 2-adrenergic receptor.
abstract: The functional significance of high affinity agonist binding to receptors that interact with guanine nucleotide regulatory proteins has remained controversial. Preincubation of human platelet membranes with the full alpha 2-agonist UK 14,304 in the absence of GTP increases the potency of the agonist to inhibit adenylate cyclase in a pre-steady state (15-sec) assay. The EC50 after preincubation (6 +/- 1 nM) is within a factor of 2 of the high affinity Kd for [3H]UK 14,304 binding determined under identical conditions (2.7 +/- 0.1 nM). In contrast, in the usual steady state measurements (15 min) or in pre-steady state measurements without agonist preincubation, the EC50 values (74 +/- 1 and 207 +/- 8 nM, respectively) are near the low affinity Kd for [3H]UK 14,304 binding. Reduction of the GTP concentration in steady state adenylate cyclase assays also decreases the EC50 for UK 14,304 from 40 +/- 5 nM at 10 microM GTP to 14 +/- 5 nM with no added GTP. Both sets of experimental observations are accommodated by a complete kinetic model of inhibition in which the high affinity ternary complex of drug, receptor, and G protein leads to the response. Explicit rate parameters are included for agonist binding, receptor-G protein interactions, GTP binding, and hydrolysis. Despite the functional role of the high affinity state of the alpha 2-receptor in this model, the steady state EC50 for agonist-mediated inhibition correlates best with the Kd of low affinity agonist binding in the presence of high levels of GTP. Under conditions in which formation of the high affinity ternary complex is favored, the EC50 for responses approaches the high affinity Kd.
authors.affiliation: Department of Pharmacology, University of Michigan, Ann Arbor 48109-0626.
authors.forename: W J
authors.initials: WJ
authors.lastname: Thomsen
authors.forename: J A
authors.initials: JA
authors.lastname: Jacquez
authors.forename: R R
authors.initials: RR
authors.lastname: Neubig
mesh_terms: D000067956:Adenylyl Cyclase Inhibitors; D000316:Adrenergic alpha-Agonists / Q000378:metabolism / Q000494:pharmacology; D001792:Blood Platelets / Q000378:metabolism; D000068438:Brimonidine Tartrate; D019204:GTP-Binding Proteins / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism; D006801:Humans; D066298:In Vitro Techniques; D007700:Kinetics; D008954:Models, Biological; D011487:Protein Conformation; D011810:Quinoxalines / Q000378:metabolism / Q000494:pharmacology; D011869:Radioligand Assay; D011942:Receptors, Adrenergic, alpha / Q000378:metabolism / Q000502:physiology*
chemical_list: D000067956:Adenylyl Cyclase Inhibitors; D000316:Adrenergic alpha-Agonists; D011810:Quinoxalines; D011942:Receptors, Adrenergic, alpha; D000068438:Brimonidine Tartrate; D006160:Guanosine Triphosphate; D019204:GTP-Binding Proteins