The allosteric enhancer, PD 81,723, stabilizes human A 1 adenosine receptor coupling to G proteins

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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 1AR) activation in tissues derived from rats and guinea-pigs. The mechanism by which PD produces this allosteric enhancement, and its effect on human A 1ARs was not known. In this study, we demonstrate that PD modifies recombinant human A 1AR binding and function in stably transfected CHO cells. In membranes, PD (20 μM) 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 [ 125 I]N 6-(3- iodo-4- aminobenzyladenosine) ( 125I-ABA), an A 1AR agonist; (ii) a 2.44-fold increase in the potency of the agonist R- N 6-phenylisopropyladenosine ( R-PIA) to compete for binding with the antagonist radioligand, [ 3H]8-cyclopentyl-1,3-dipro-pylxanthine ([ 3H]CPX); (iii) a 1.5-fold increase in the t 1 2 of 125I-ABA to dissociate from A 1AR; and (iv) a 2.2-fold increase in the concentration of guanosine-5′-3-0-(thio)triphosphate (GTPγS) required to half-maximally uncouple receptor-G-protein complexes. In intact CHO cells expressing A 1AR, PD increases the potency of R-PIA to decrease forskolin-stimulated cAMP accumulation by 3.3-fold. We speculate that PD binds to A 1AR at a site distinct from the agonist binding site and stabilizes agonist-R-G complexes.