Reconstitution of bovine A1 adenosine receptors and G proteins in phospholipid vesicles:: βγ-subunit composition influences guanine nucleotide exchange and agonist binding

We have studied the interactions of purified A(1) adenosine receptors and G proteins reconstituted into phospholipid vesicles to investigate how the beta gamma composition of G protein heterotrimers influences coupling. Recombinant hexahistidine-tagged bovine A(1) adenosine receptors were expressed in Sf9 cells and purified to homogeneity by sequential chromatography over heparin-sepharose, xanthine amino congener-agarose, and nickel-nitrilotriacetic acid columns. These receptors were reconstituted with purl recombinant G proteins of defined subunit composition. Receptor-G protein complexes containing alpha(i2) and beta(1) gamma(2) or beta(1) gamma(3) and stimulated with the agonist, (R)-phenylisopropyladenosine, exchange guanine nucleotide 2-3 times more rapidly than do complexes containing beta(1) gamma(1). This difference is not overcome by increasing the concentration of beta gamma subunits. Receptor-G protein complexes containing beta(1) gamma(1) also bind less of the agonist, [I-125]-iodoaminobenzyladenosine (I-125-ABA), than do complexes containing beta(1) gamma(3). Kinetic experiments show that I-125-ABA dissociates 2-fold more rapidly from receptor-G protein complexes containing beta(1) gamma(1) than from complexes containing the other py subunits. The affinity of the interaction between immobilized G(alpha i2) subunits and beta(1) gamma(1) or beta(1) gamma(2) measured with an optical biosensor in the absence of receptor is similar. Taken together, these data implicate the gamma-subunit in influencing the interaction between the A(1) adenosine receptor and G proteins.