Extracellular ATP and structurally related molecules potentiate adenosine A_2a receptor-stimulated cAMP production

Extracellular ATP has been reported to potentiate signalling by several Class B G protein-coupled receptors (GPCRs). The adenosine A_2a receptor (A_2aR) is a Class A GPCR that regulates many physiological processes, and a potential therapeutic target for many diseases. In vivo, A_2aR is exposed transiently to extracellular ATP within the cellular microenvironment under both physiological and pathological conditions. The modulating effects of extracellular ATP seen with Class B GPCRs have not previously been investigated in other classes of GPCRs. In the present study, we investigated the effects of extracellular ATP on A_2aR signalling. We also studied the actions of similar molecules to explore the structure-activity relationship. Cyclic 3',5'-adenosine monophosphate (cAMP) levels were monitored following agonist-induced receptor activation in cells co-transfected with plasmids encoding A_2aR and a luminescent cAMP biosensor. Extracellular ATP increased the potency of both adenosine and selective A_2aR agonists by approximately an order of magnitude. In the absence of agonist, ATP did not activate A_2aR, arguing against an effect due to ATP metabolism to adenosine. The potentiating effect of ATP was mimicked by other nucleotides and similarly by phosphorylated sugars. Non-phosphorylated sugars produced comparable effects, but higher concentrations were required to do so. This difference in potency implies that the phosphate group is important for modulating A_2aR activity. Here, we present the first evidence that A_2aR can be positively modulated by extracellular ATP, thus the effect of ATP is not limited to Class B GPCRs.