Agonist and antagonist binding to alpha 2-adrenergic receptors in purified membranes from human platelets. Implications of receptor-inhibitory nucleotide-binding protein stoichiometry.
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 compartmentalization of Ni and/or that the alpha 2 receptors are heterogeneous.
Neubig
R R
RR
Gantzos
R D
RD
Brasier
R S
RS
D000246:Adenosine Diphosphate Ribose / Q000378:metabolism; D000067956:Adenylyl Cyclase Inhibitors; D000316:Adrenergic alpha-Agonists / Q000378:metabolism*; D000317:Adrenergic alpha-Antagonists / Q000378:metabolism*; D001792:Blood Platelets / Q000378:metabolism*; D000068438:Brimonidine Tartrate; D003000:Clonidine / Q000031:analogs & derivatives / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000032:analysis*; D006165:Guanylyl Imidodiphosphate / Q000494:pharmacology; D006801:Humans; D066298:In Vitro Techniques; D009243:NAD / Q000378:metabolism; D011810:Quinoxalines / Q000378:metabolism; D011942:Receptors, Adrenergic, alpha / Q000378:metabolism*; D015016:Yohimbine / Q000378:metabolism
D000067956:Adenylyl Cyclase Inhibitors; D000316:Adrenergic alpha-Agonists; D000317:Adrenergic alpha-Antagonists; D011810:Quinoxalines; D011942:Receptors, Adrenergic, alpha; D009243:NAD; D000246:Adenosine Diphosphate Ribose; D015016:Yohimbine; D006165:Guanylyl Imidodiphosphate; D000068438:Brimonidine Tartrate; C016986:apraclonidine; D019204:GTP-Binding Proteins; D003000:Clonidine