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., and 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 14304 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.