A1 adenosine receptors of bovine brain couple to guanine nucleotide-binding proteins Gi1, Gi2, and Go.
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. Gi alpha 1, Gi alpha 2, Go alpha, G beta 35, and G beta 36 were detected. Of the total [35S]guanosine 5'-O-(3-thio)triphosphate [( 35S]GTP gamma S) binding sites, Gi alpha 1 and Go alpha each accounted for greater than 37% whereas Gi alpha 2 comprised less than 13%. G beta 35 was found in excess over G beta 36. 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 alpha-subunits restored GTP gamma S-sensitive high affinity binding of the agonist 125I-aminobenzyladenosine to a fraction (25%) of reconstituted receptors with a selectivity order of Gi2 greater than Go greater than or equal to Gi1 (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]GTP gamma S 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.
Department of Internal Medicine (Cardiology), University of Virginia, Charlottesville 22908.
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Munshi
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Pang
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Sternweis
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Linden
D000818:Animals; D002417:Cattle; D002462:Cell Membrane / Q000378:metabolism; D002540:Cerebral Cortex / Q000378:metabolism*; D002846:Chromatography, Affinity / Q000379:methods; D019204:GTP-Binding Proteins / Q000302:isolation & purification / Q000378:metabolism*; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism / Q000494:pharmacology; D015151:Immunoblotting; D007700:Kinetics; D008081:Liposomes; D046911:Macromolecular Substances; D010660:Phenylisopropyladenosine / Q000494:pharmacology; D011983:Receptors, Purinergic / Q000302:isolation & purification / Q000378:metabolism*
D008081:Liposomes; D046911:Macromolecular Substances; D011983:Receptors, Purinergic; D010660:Phenylisopropyladenosine; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D019204:GTP-Binding Proteins