A2A and A2B adenosine receptors: the extracellular loop 2 determines high (A2A) or low affinity (A2B) for adenosine

Abstract A2A and A2B adenosine receptors (ARs) are closely related G protein-coupled receptor subtypes, which represent important (potential) drug targets. Despite their almost identical binding sites for adenosine, A2AARs are activated by low (nanomolar) adenosine concentrations, while A2BARs require micromolar concentrations. In the present study, we exchanged the extracellular loop 2 (ECL2) of the human A2AAR for that of the A2BAR. The resulting chimeric A2A(ECL2-A2B)AR was investigated in radioligand binding and cAMP accumulation assays in comparison to the wildtype A2AAR. While the ribose-modified adenosine analog N-ethylcarboxamidoadenosine (NECA) and its 2-substituted derivative CGS-21680 did not exhibit significant changes, adenosine showed dramatically reduced potency and affinity for the A2A(ECL2-A2B)AR mutant displaying similarly low potency as for the wt A2BAR. Supervised molecular dynamics simulation studies predicted a meta-binding site with high affinity for adenosine, but not for NECA, which may contribute to the observed effects.