Adenosine A_2A receptor ligand recognition and signaling is blocked by A_2B receptors

The adenosine receptor (AR) subtypes A_2A and A_2B are rhodopsin-like G_s protein-coupled receptors whose expression is highly regulated under pathological, e.g. hypoxic, ischemic and inflammatory conditions. Both receptors play important roles in inflammatory and neurodegenerative diseases, are blocked by caffeine, and have now become major drug targets in immuno-oncology. By Förster resonance energy transfer (FRET), bioluminescence resonance energy transfer (BRET), bimolecular fluorescence complementation (BiFC) and proximity ligation assays (PLA) we demonstrated A_2A-A_2BAR heteromeric complex formation. Moreover we observed a dramatically altered pharmacology of the A_2AAR when co-expressed with the A_2BAR (A_2B ≥ A_2A) in recombinant as well as in native cells. In the presence of A_2BARs, A_2A-selective ligands lost high affinity binding to A_2AARs and displayed strongly reduced potency in cAMP accumulation and dynamic mass redistribution (DMR) assays. These results have major implications for the use of A_2AAR ligands as drugs as they will fail to modulate the receptor in an A_2A-A_2B heteromer context. Accordingly, A_2A-A_2BAR heteromers represent novel pharmacological targets.