AGONIST AND ANTAGONIST BINDING TO ALPHA-2-ADRENERGIC RECEPTORS IN PURIFIED MEMBRANES FROM HUMAN-PLATELETS - IMPLICATIONS OF RECEPTOR-INHIBITORY NUCLEOTIDE-BINDING PROTEIN STOICHIOMETRY

  • 1 January 1985
    • journal article
    • research article
    • Vol. 28  (5) , 475-486
Abstract
The agonist- and antagonist-binding properties of the .alpha.2-adrenergic receptor in a purified plasma membrane preparation from human platelets were determined both by direct binding of radiolabeled ligands and by competition with the labeled .alpha.2-antagonist, [3H]yohimbine. Binding of [3H]yohimbine was characterized by a single high affinity binding site (Kd = 6.2 .+-. 1.4 nM, Bmax = 507 .+-. 53 fmol/mg). In direct binding studies, the imidazoline full .alpha.2-agonist, [3H]-5-bromo-6-N(2-4,5-dihydroimidazolyl)quinoxaline ([3H] UK 14,304), bound to only one quantifiable high affinity site (Kd = 0.88 .+-. 0.17 nM), representing 65 .+-. 6% of the number of [3H]yohimbine sites. Binding of the partial agonist [3H]-p-aminoclonidine (PAC) showed nonlinear Scatchard plots. Analysis according to a model of multiple independent binding sites showed the data to be consistent with two sites (Kd1 = 0.62 .+-. 0.18 nM and Kd2 = 7.9 .+-. 1.4 nM). The high affinity site corresponded to 15 .+-. 6% and the low affinity site corresponded to 39 .+-. 6% of the number of [3H]yohimbine sites. Competition for binding of the .alpha.2-antagonist, [3H]yohimbine, with nonradiolabeled ligands revealed a single affinity for yohimbine. In contrast, competition for [3H]yohimbine binding by the full agonist UK 14,304 and epinephrine is best fit by a model with two independent binding sites. The partial agonist PAC was best characterized by a model with three distinct binding sites. The full agonists UK 14,304 and epinephrine inhibited adenylate cyclase approximately 30%, whereas PAC produced only 12% inhibition. The inhibitory guanine nucleotide-binding protein (Ni) with Mr 40,700 was the sole pertussis toxin substrate in the purified membranes. It was quantitated by pertussis toxin-catalyzed [32P]ADP ribosylation in cholate extracts. There is a 20- to 100-fold excess of Ni over .alpha.2-adrenergic receptors. Comparisons made between the experimental data for agonist binding and theoretical predictions of the simple ternary complex model suggest that there is compartmentalziation of Ni and/or that the .alpha.2 receptors are heterogeneous.