Direct evidence for the coupling of A2-adenosine receptor to stimulatory guanine nucleotide-binding-protein in bovine brain striatum.
This study was designed to provide evidence for the coupling of A2-adenosine receptor with stimulatory guanine nucleotide-binding protein (Gs) protein and adenylate cyclase in bovine brain striatum. Binding studies were conducted in striatal membranes using [3H]-2(-)[4-(2-carboxy-ethyl)phenethylamino]-5'-N-ethylcarbox++ +- amido adenosine (CGS-21680) as radioligand. Competition profiles of adenosine agonists and antagonists for the binding of [3H]CGS-21680 were typical for A2-adenosine receptor subtype. [3H]CGS-21680 binding in striatal membranes was saturable and recognized a single class of binding sites with a Kd of 9.08 +/- 1 nM and an apparent Bmax of 378 +/- 34.6 fmol/mg protein. CGS-21680 and 2-chloroadenosine (CAD) stimulated adenylate cyclase in a concentration-dependent fashion which was blocked significantly by Gs alpha antibody. To evaluate coupling of A2 receptor with Gs protein, [3H]GDP release assays were performed. The membranes were labeled with [alpha 32P]GTP in the presence of alpha or beta adrenergic agonists to label specifically inhibitory G protein (Gi) or Gs pools. When membranes were labeled in the presence of isoproterenol, [32P]GDP release was stimulated by A2 receptor agonist but not by A1 receptor agonist, and vice versa when the membranes were labeled in the presence of alpha agonist. Thus, the adenylate cyclase activation by A2 agonist, its blockage by Gs alpha antibody and the [32P]GDP release studies show that the A2-adenosine receptors are coupled to Gs protein.
Marala
R B
RB
Department of Pharmacology, School of Medicine, East Carolina University, Greenville, North Carolina.
Mustafa
S J
SJ
D000262:Adenylyl Cyclases / Q000378:metabolism; D000818:Animals; D002417:Cattle; D003342:Corpus Striatum / Q000378:metabolism*; D004789:Enzyme Activation; D019204:GTP-Binding Proteins / Q000378:metabolism*; D006153:Guanosine Diphosphate / Q000032:analysis / Q000378:metabolism; D010761:Phosphorus Radioisotopes; D011983:Receptors, Purinergic / Q000378:metabolism*
D010761:Phosphorus Radioisotopes; D011983:Receptors, Purinergic; D006153:Guanosine Diphosphate; D019204:GTP-Binding Proteins; D000262:Adenylyl Cyclases