A1 adenosine receptors and associated guanine nucleotide-binding proteins (G proteins) were purified from bovine cerebral cortex by affinity chromatography (Munshi, R., and Linden, J. (1989) J. Biol. Chem. 264, 14853-14859). In this study we have identified the pertussis toxin-sensitive G protein subunits that co-purify with A1 adenosine receptors by immunoblotting with specific antipeptide antisera. G(iα1), G(iα2), G(oα), G(β35), and G(β36) were detected. Of the total [35S]guanosine 5'-O-(3-thio)triphosphate ([35S]GTPγS) binding sites, G(iα1) and G(oα) each accounted for >37% whereas G(iα2) comprised <13%. G(β35) was found in excess over G(β36). Low molecular mass (21-25 kDa) GTP-binding proteins were not detected. We also examined the characteristics of purified receptors and various purified bovine brain G proteins reconstituted into phospholipid vesicles. All three α-subunits restored GTPγS-sensitive high affinity binding of the agonist 125I-aminobenzyladenosine to a fraction (25%) of reconstituted receptors with a selectivity order of G(i2) > G(o) ≥ G(i1) (ED50 values of G proteins measured as fold excess over the receptor concentration were 4.7 ± 1.2, 24 ± 5, and 34 ± 7, respectively). Furthermore, receptors occupied with the agonist R-phenylisopropyladenosine catalytically increased the rate of binding of [35S]GTPγS to reconstituted G proteins by 6.5-8.5-fold. These results suggest that A1 adenosine receptors couple indiscriminately to pertusis toxin-sensitive G proteins.
|Number of pages||5|
|Journal||Journal of Biological Chemistry|
|Publication status||Published - 1 Jan 1991|