Membrane reconstitution of high-affinity alpha 2 adrenergic agonist binding with guanine nucleotide regulatory proteins

Biochemistry. 1987 Jun 16;26(12):3664-72. doi: 10.1021/bi00386a061.


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.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Adenylate Cyclase Toxin
  • Adrenergic alpha-Agonists / pharmacology*
  • Animals
  • Binding, Competitive
  • Blood Platelets / metabolism*
  • Brain / metabolism*
  • Cattle
  • Cell Line
  • Cell Membrane / metabolism
  • GTP-Binding Proteins / metabolism*
  • Glioma
  • Humans
  • Kinetics
  • Receptors, Adrenergic, alpha / isolation & purification
  • Receptors, Adrenergic, alpha / metabolism*
  • Virulence Factors, Bordetella / pharmacology


  • Adenylate Cyclase Toxin
  • Adrenergic alpha-Agonists
  • Receptors, Adrenergic, alpha
  • Virulence Factors, Bordetella
  • GTP-Binding Proteins