DIRECT EVIDENCE FOR THE COUPLING OF ADENOSINE-A2 RECEPTOR TO STIMULATORY GUANINE NUCLEOTIDE-BINDING-PROTEIN IN BOVINE BRAIN STRIATUM

This study was designed to provide evidence for the coupling of A2-ade nosine receptor with stimulatory guanine nucleotide-binding protein (G (s)) protein and adenylate cyclase in bovine brain striatum. Binding s tudies were conducted in striatal membranes using rboxy-ethyl)phenethy lamino]-5'-N-ethylcarbox-amido adenosine (CGS-21680) as radioligand. C ompetition profiles of adenosine agonists and antagonists for the bind ing of [H-3]CGS-21680 were typical for A2-adenosine receptor subtype. [H-3]CGS-21680 binding in striatal membranes was saturable and recogni zed a single class of binding sites with a K(d) of 9.08 +/- 1 nM and a n apparent B(max) of 378 +/- 34.6 fmol/mg protein. CGS-21680 and 2-chl oroadenosine (CAD) stimulated adenylate cyclase in a concentration-dep endent fashion which was blocked significantly by G(salpha) antibody. To evaluate coupling of A2 receptor with G(s) protein, [H-3]GDP releas e assays were performed. The membranes were labeled with [alphaP-32]GT P in the presence of alpha or beta adrenergic agonists to label specif ically inhibitory G protein (G(i)) or G(s) pools. When membranes were labeled in the presence of isoproterenol, [P-32]GDP release was stimul ated by A2 receptor agonist but not by A1 receptor agonist, and vice v ersa when the membranes were labeled in the presence of alpha agonist. Thus, the adenylate cyclase activation by A2 agonist, its blockage by G(s) antibody and the [P-32]GDP release studies show that the A2-aden osine receptors are coupled to G(s) protein.