A₁ adenosine receptors of bovine brain couple to guanine nucleotide-binding proteins G_i1, G_i2, and G_o

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 pertussis toxin-sensitive G proteins.