THE ALLOSTERIC ENHANCER, PD-81,723, STABILIZES HUMAN A(1)-ADENOSINE RECEPTOR COUPLING TO G-PROTEINS

2-Amino-3-benzoylthiophenes such as PD 81,273 (PD) have been shown to increase agonist, but not antagonist, radioligand binding and to enhance functional effects of A(1) adenosine receptor (A(1)AR) activation in tissues derived from rats and guinea-pigs. The mechanism by which PD produces this allosteric enhancement, and its effect on human A(1)ARs was not known. In this study, we demonstrate that PD modifies recombinant human A(1)AR binding and function in stably transfected CHO cells. In membranes, PD (20 mu M) causes: (i) a 3-fold increase in the fraction of receptors found in a high affinity G-protein coupled conformation as assessed by the binding of [I-125]N-6-(3-iodo-4-aminobenzyladenosine (I-125-ABA), an A(1)AR agonist; (ii) a 2.44-fold increase in the potency of the agonist R-N-6-phenylisopropyladenosine (R-PIA) to compete far binding with the antagonist radioligand, [H-3]8-cyclopentyl-1,3-dipropylxanthine ([H-3]CPX); (iii) a 1.5-fold increase in the t(1/2) of I-125-ABA to dissociate from A(1)AR; and (iv) a 2.2-fold increase in the concentration of guanosine-5'-3-O-(thio)triphosphate (GTP gamma S) required to half-maximally uncouple receptor-G-protein complexes. In intact CHO cells expressing A,AR, PD increases the potency of R-PIA to decrease forskolin-stimulated cAMP accumulation by 3.3-fold. We speculate that PD binds to A(1)AR at a site distinct from the agonist binding site and stabilizes agonist-R-G complexes.