A2A and A2B adenosine receptors: The extracellular loop 2 determines high (A2A) or low affinity (A2B) 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(2A)ARs are activated by low (nanomolar) adenosine concentrations, while A(2B)ARs require micromolar concentrations. In the present study, we exchanged the extracellular loop 2 (ECL2) of the human A(2A)AR for that of the A(2B)AR. The resulting chimeric A(2A)(ECL2-A(2B))AR was investigated in radioligand binding and cAMP accumulation assays in comparison to the wildtype A(2A)AR. 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(2B)AR. 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.