RECONSTITUTION OF RECOMBINANT BOVINE A(1) ADENOSINE RECEPTORS IN SF9 CELL-MEMBRANES WITH RECOMBINANT G-PROTEINS OF DEFINED COMPOSITION

We investigated the coupling of A(1) adenosine receptors to recombinan t G proteins. Recombinant baculoviruses were used to express bovine A( 1) adenosine receptors in Sf9 insect cells that lack endogenous adenos ine receptors. Binding parameters for recombinant receptors expressed in Sf9 cell membranes using the antagonist radioligand [(125)l]BW-A844 U cyclopentyl-3-iodoaminophenethyl-1-propylxanthine} are B-max = 2-5 p mol/mg of protein and K-D = 0.53 +/- 0.12 nM. In competition assays, t he potency order of agonists is (R)-phenylisopropyladenosine > (S)-phe nylisopropyladenosine > 5'-N-ethylcarboxamidoadenosine, properties cha racteristic of native bovine A(1) adenosine receptors. The agonist rad ioligand (125)l-N-6-4-aminobenzyladenosine binds to two affinity state s of the recombinant A(1) adenosine receptors with K-D values of 0.09 and 10.4 nM. The high affinity binding site represents <10% of total s ites and is increased 7-fold on reconstitution with both or and beta g amma G protein subunits but not with either subunit alone; thus, exoge nous alpha and beta gamma subunits do not functionally interact with e ndogenous Sf9 beta gamma and alpha subunits, respectively. Four differ ent alpha subunits (alpha i1, alpha i2, alpha i3, and alpha o) and six different beta gamma subunits (beta 1 gamma 1, beta 1 gamma 2, beta 1 gamma 3, beta 2 gamma 2, beta 2 gamma 3, and bovine brain beta gamma) ) increased GTP-sensitive, high affinity agonist binding. The results indicate that bovine A(1) adenosine receptors couple equally well to G protein alpha i and alpha o subunits in combination with beta gamma s ubunits containing the beta 1 or beta 2 subunits and gamma 2 or gamma 3 subunits. G protein heterotrimers that contain the beta 1 gamma 1 di mer couple with similar potency but reduced efficacy to A(1) adenosine receptors.