Agonist-Dependent Coupling of the Promiscuous Adenosine A2B Receptor to Gα Protein Subunits

The adenosine A(2B) receptor (A(2B)AR) 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 A(2B)AR to different G proteins, mainly G, but in some cases G(q/11) or G(i), depending on the cell type. We have now utilized novel technologies, (i) heterologous expression of individual members of the G alpha(q/11 )protein family (G alpha(q), G alpha(11), G alpha(14), and G alpha(15)) in G alpha(q/11) knockout cells, and (ii) the TRUPATH platform, allowing the direct observation of G alpha protein activation for each of the G alpha subunits by bioluminescence resonance energy transfer (BRET) measurements. Three structurally diverse A(2B)AR 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 G alpha protein families (G alpha(s), G alpha(q/11), G alpha(i) and G alpha(12/13)). Significant differences in potencies and efficacies were observed; the highest efficacies were determined at the G alpha(15), G alpha(s), and G alpha(12) proteins, and for NECA additionally at the G alpha(i2) protein. In contrast, the partial agonist BAY 60-6583 only activated G alpha(15), G alpha(s) , and G alpha(12) proteins. Adenosine deaminase, an allosteric modulator of ARs, selectively increased the potency and efficacy of NECA and BAY 60-6583 at the G alpha(i2) protein, while it had no effect or decreased efficacy at the other G alpha proteins. We conclude that the A(2B)AR is preferably coupled to the G alpha(15), G alpha(s), and G alpha(12) proteins. Upon upregulation of receptor or G alpha protein expression, coupling to further G alpha proteins likely occurs. Importantly, different agonists can display different activation profiles.