Agonist-Dependent Coupling of the Promiscuous Adenosine A Receptor to Gα Protein Subunits.

The adenosine A receptor (AAR) belongs to the rhodopsin-like G protein-coupled receptor (GPCR) family. It is upregulated under hypoxic conditions, in inflammation and cancer. Previous studies indicated the coupling of the AAR to different G proteins, mainly G, but in some cases G or G, depending on the cell type. We have now utilized novel technologies, (i) heterologous expression of individual members of the Gα protein family (Gα, Gα, Gα, and Gα) in Gα knockout cells, and (ii) the TRUPATH platform, allowing the direct observation of Gα protein activation for each of the Gα subunits by bioluminescence resonance energy transfer (BRET) measurements. Three structurally diverse AAR agonists were studied: the cognate agonist adenosine, its metabolically stable analog NECA, and the non-nucleosidic partial agonist BAY 60-6583. Adenosine and NECA activated most members of all four Gα protein families (Gα, Gα, Gα, and Gα). Significant differences in potencies and efficacies were observed; the highest efficacies were determined at the Gα, Gα, and Gα proteins, and for NECA additionally at the Gα protein. In contrast, the partial agonist BAY 60-6583 only activated Gα, Gα, and Gα proteins. Adenosine deaminase, an allosteric modulator of ARs, selectively increased the potency and efficacy of NECA and BAY 60-6583 at the Gα protein, while it had no effect or decreased efficacy at the other Gα proteins. We conclude that the AAR is preferably coupled to the Gα, Gα, and Gα proteins. Upon upregulation of receptor or Gα protein expression, coupling to further Gα proteins likely occurs. Importantly, different agonists can display different activation profiles.