A_2A and A_2B adenosine receptors: The extracellular loop 2 determines high (A_2A) or low affinity (A_2B) for adenosine

A_2A and A_2B 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, A_2AARs are activated by low (nanomolar) adenosine concentrations, while A_2BARs require micromolar concentrations. In the present study, we exchanged the extracellular loop 2 (ECL2) of the human A_2AAR for that of the A_2BAR. The resulting chimeric A_2A(ECL2-A_2B)AR was investigated in radioligand binding and cAMP accumulation assays in comparison to the wildtype A_2AAR. 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 A_2A(ECL2-A_2B)AR mutant displaying similarly low potency as for the wt A_2BAR. 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.