title: Membrane reconstitution of high-affinity alpha 2 adrenergic agonist binding with guanine nucleotide regulatory proteins.
abstract: Regulation of adenylate cyclase by alpha 2 adrenergic receptors requires the inhibitory guanine nucleotide binding protein Ni. A role for this protein has also been suggested in the high-affinity binding of agonists to the alpha 2 receptor. We recently reported that alkaline treatment can selectively inactivate alpha 2 agonist binding and Ni in human platelet plasma membranes [Kim, M.H. & Neubig, R.R. (1985) FEBS Lett. 192, 321-325]. Binding of the full alpha 2 agonists epinephrine and 5-bromo-6-[N-(4,5-dihydroimidazol-2-yl)amino]quinoxaline (UK 14,304) to these membranes was determined by competition and direct radioligand binding, respectively. The high-affinity GTP-sensitive binding of the agonists is lost after alkaline treatment. Binding of [3H]UK 14,304 was reconstituted by poly(ethylene glycol)-induced fusion of alkaline-treated platelet membranes with cell membranes containing Ni but no alpha 2 receptor or with lipid vesicles containing purified guanine nucleotide binding proteins (N-proteins) from bovine brain. The reconstituted binding was of high affinity (Kd = 0.4 +/- 0.1 nM), accounted for a substantial fraction of the total alpha 2 receptors (Bmax for [3H]UK 14,304 was 78 +/- 23% of the Bmax for [3H]yohimbine), and was abolished in the presence of guanosine 5'-(beta, gamma-imidotriphosphate) (GppNHp). The brain-specific protein No (predominant guanine nucleotide regulatory protein from bovine brain) was also effective in reconstituting high-affinity alpha 2 agonist binding. The results presented here show that a guanine nucleotide regulatory protein of the No or Ni type is necessary for high-affinity alpha 2 agonist binding. These methods should also prove useful for future studies of receptor N-protein interactions.
mesh_terms: D037361:Adenylate Cyclase Toxin; D000316:Adrenergic alpha-Agonists / Q000494:pharmacology*; D000818:Animals; D001667:Binding, Competitive; D001792:Blood Platelets / Q000378:metabolism*; D001921:Brain / Q000378:metabolism*; D002417:Cattle; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000378:metabolism*; D005910:Glioma; D006801:Humans; D007700:Kinetics; D011942:Receptors, Adrenergic, alpha / Q000302:isolation & purification / Q000378:metabolism*; D010566:Virulence Factors, Bordetella / Q000494:pharmacology
chemical_list: D037361:Adenylate Cyclase Toxin; D000316:Adrenergic alpha-Agonists; D011942:Receptors, Adrenergic, alpha; D010566:Virulence Factors, Bordetella; D019204:GTP-Binding Proteins