RECONSTITUTION OF BOVINE A(1) ADENOSINE RECEPTORS AND G-PROTEINS IN PHOSPHOLIPID-VESICLES - BETA-GAMMA-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 recep tors were expressed in Sf9 cells and purified to homogeneity by sequen tial chromatography over heparin-sepharose, xanthine amino congener-ag arose, and nickel-nitrilotriacetic acid columns. These receptors were reconstituted with purl recombinant G proteins of defined subunit comp osition. Receptor-G protein complexes containing alpha(i2) and beta(1) gamma(2) or beta(1) gamma(3) and stimulated with the agonist, (R)-phe nylisopropyladenosine, exchange guanine nucleotide 2-3 times more rapi dly than do complexes containing beta(1) gamma(1). This difference is not overcome by increasing the concentration of beta gamma subunits. R eceptor-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 comp lexes containing beta(1) gamma(1) than from complexes containing the o ther 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. Take n together, these data implicate the gamma-subunit in influencing the interaction between the A(1) adenosine receptor and G proteins.