title: Mechanism of agonist and antagonist binding to alpha 2 adrenergic receptors: evidence for a precoupled receptor-guanine nucleotide protein complex.
abstract: The alpha 2 adrenergic receptor (AR) inhibits adenylate cyclase via an interaction with Ni, a guanine nucleotide binding protein. The early steps involved in the activation of the alpha 2 AR by agonists and the subsequent interaction with Ni are poorly understood. In order to better characterize these processes, we have studied the kinetics of ligand binding to the alpha 2 AR in human platelet membranes on the second time scale. Binding of the alpha 2 antagonist [3H]yohimbine was formally consistent with a simple bimolecular reaction mechanism with an association rate constant of 2.5 X 10(5) M-1 s-1 and a dissociation rate constant of 1.11 X 10(-3) s-1. The low association rate constant suggests that this is not a diffusion-limited reaction. Equilibrium binding of the alpha 2 adrenergic full agonist [3H]UK 14,304 was characterized by two binding affinities: Kd1 = 0.3-0.6 nM and Kd2 = 10 nM. The high-affinity binding corresponds to approximately 65% and the low-affinity binding to 35% of the total binding. The kinetics of binding of [3H]UK 14,304 were complex and not consistent with a mass action interaction at one or more independent binding sites. The dependence of the kinetics on [3H]UK 14,304 concentration revealed a fast phase with an apparent bimolecular reaction constant kappa + of 5 X 10(6) M-1 s-1. The rate constants and amplitudes of the slow phase of agonist binding were relatively independent of ligand concentration. These results were analyzed quantitatively according to several variants of the "ternary complex" binding mechanism. In the model which best accounted for the data, (1) approximately one-third of the alpha 2 adrenergic receptor binds agonist with low affinity and is unable to couple with a guanine nucleotide binding protein (N protein), (2) approximately one-third is coupled to the N protein prior to agonist binding, and (3) the remainder interacts by a diffusional coupling of the alpha 2 AR with the N protein or a slow, ligand-independent conformational change of the alpha 2 AR-N protein complex. The rates of interaction of liganded and unliganded receptor with N protein are estimated.
authors.affiliation: Department of Pharmacology, University of Michigan, Ann Arbor 48109-0010.
authors.forename: R R
authors.initials: RR
authors.lastname: Neubig
authors.forename: R D
authors.initials: RD
authors.lastname: Gantzos
authors.forename: W J
authors.initials: WJ
authors.lastname: Thomsen
mesh_terms: D000959:Antihypertensive Agents / Q000097:blood*; D001792:Blood Platelets / Q000378:metabolism*; D000068438:Brimonidine Tartrate; D002462:Cell Membrane / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D008433:Mathematics; D008954:Models, Biological; D011810:Quinoxalines / Q000097:blood*; D011942:Receptors, Adrenergic, alpha / Q000378:metabolism*; D015016:Yohimbine / Q000097:blood*
chemical_list: D000959:Antihypertensive Agents; D011810:Quinoxalines; D011942:Receptors, Adrenergic, alpha; D015016:Yohimbine; D000068438:Brimonidine Tartrate; D019204:GTP-Binding Proteins