The allosteric enhancer PD81,723 increases chimaeric A1/A2A adenosine receptor coupling with Gs

PD81,723 {(2-amino-4,5-dimethyl-3-thienyl)-[3-(trifluromethyl)-phenyl]methanone} is a selective allosteric enhancer of the G i -coupled A 1 AR (adenosine receptor) that is without effect on G s -coupled A 2A ARs. PD81,723 elicits a decrease in the dissociation kinetics of A 1 AR agonist radioligands and an increase in functional agonist potency. In the present study, we sought to determine whether enhancer sensitivity is dependent on coupling domains or G-protein specificity of the A 1 AR. Using six chimaeric A 1 /A 2A ARs, we show that the allosteric effect of PD81,723 is maintained in a chimaera in which the predominant G-protein-coupling domain of the A 1 receptor, the 3ICL (third intracellular loop), is replaced with A 2A sequence. These chimaeric receptors are dually coupled with G s and G i , and PD81,723 increases the potency of N 6 -cyclopentyladenosine to augment cAMP accumulation with or without pretreatment of cells with pertussis toxin. Thus PD81,723 has similar functional effects on chimaeric receptors with A 1 transmembrane sequences that couple with G i or G s . This is the first demonstration that an allosteric regulator can function in the context of a switch in G-protein-coupling specificity. There is no enhancement by PD81,723 of G i -coupled A 2A chimaeric receptors with A 1 sequence replacing A 2A sequence in the 3ICL. The results suggest that the recognition site for PD81,723 is on the A 1 receptor and that the enhancer acts to directly stabilize the receptor to a conformational state capable of coupling with G i or G s .