Adenosine A2A receptor ligand recognition and signaling is blocked by A2B receptors.

The adenosine receptor (AR) subtypes A2A and A2B are rhodopsin-like Gs 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 A2A-A2BAR heteromeric complex formation. Moreover we observed a dramatically altered pharmacology of the A2AAR when co-expressed with the A2BAR (A2B ≥ A2A) in recombinant as well as in native cells. In the presence of A2BARs, A2A-selective ligands lost high affinity binding to A2AARs and displayed strongly reduced potency in cAMP accumulation and dynamic mass redistribution (DMR) assays. These results have major implications for the use of A2AAR ligands as drugs as they will fail to modulate the receptor in an A2A-A2B heteromer context. Accordingly, A2A-A2BAR heteromers represent novel pharmacological targets.

BAY60-6583 acts as a partial agonist at adenosine A2B receptors.

BAY60-6583 [2-({6-amino-3,5-dicyano-4-[4-(cyclopropylmethoxy)phenyl]pyridin-2-yl}sulfanyl)acetamide] is the most potent and selective adenosine A2B receptor (A2B AR) agonist known to date. Therefore, it has been widely used for in vitro and in vivo experiments. In the present study, we investigated the binding and functional properties of BAY60-6583 in various native and recombinant cell lines with different A2B AR expression levels. In cAMP accumulation and calcium mobilization assays, BAY60-6583 was found to be significantly less efficacious than adenosine or the adenosine derivative NECA. When it was tested in human embryonic kidney (HEK)293 cells, its efficacy correlated with the A2B expression level of the cells. In Jurkat T cells, BAY60-6583 antagonized the agonistic effect of NECA and adenosine as determined in cAMP accumulation assays. On the basis of these results, we conclude that BAY60-6583 acts as a partial agonist at adenosine A2B receptors. At high levels of the physiologic agonist adenosine, BAY60-6583 may act as an antagonist and block the effects of adenosine at A2B receptors. This has to be considered when applying the A2B-selective "agonist" BAY60-6583 in pharmacological studies, and previous research results may have to be reinterpreted.

The second extracellular loop of GPCRs determines subtype-selectivity and controls efficacy as evidenced by loop exchange study at A2 adenosine receptors.

The second extracellular loop (EL2) of G protein-coupled receptors (GPCRs), which represent important drug targets, may be involved in ligand recognition and receptor activation. We studied the closely related adenosine receptor (AR) subtypes A2A and A2B by exchanging the complete EL2 of the human A2BAR for the EL2 of the A2AAR. Furthermore, single amino acid residues (Asp148(45.27), Ser149(45.28), Thr151(45.30), Glu164(45.43), Ser165(45.44), and Val169(45.48)) in the EL2 of the A2BAR were exchanged for alanine. The single mutations did not lead to any major effects, except for the T151A mutant, at which NECA showed considerably increased efficacy. The loop exchange entailed significant effects: The A2A-selective agonist CGS21680, while being completely inactive at A2BARs, showed high affinity for the mutant A2B(EL2-A2A)AR, and was able to fully activate the receptor. Most strikingly, all agonists investigated (adenosine, NECA, BAY60-6583, CGS21680) showed strongly increased efficacies at the mutant A2B(EL2-A2A) as compared to the wt AR. Thus, the EL2 of the A2BAR appears to have multiple functions: besides its involvement in ligand binding and subtype selectivity it modulates agonist-bound receptor conformations thereby controlling signalling efficacy. This role of the EL2 is likely to extend to other members of the GPCR family, and the EL2 of GPCRs appears to be an attractive target structure for drugs.