Agonist-Dependent Coupling of the Promiscuous Adenosine A_2B Receptor to Gα Protein Subunits

The adenosine A_2B receptor (A_2BAR) 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_2BAR to different G proteins, mainly G_s, 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 Gα_q/11 protein family (Gα_q, Gα₁₁, Gα₁₄, and Gα₁₅) in Gα_q/11 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 A_2BAR 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_s, Gα_q/11, Gα_i, and Gα_12/13). Significant differences in potencies and efficacies were observed; the highest efficacies were determined at the Gα₁₅, Gα_s, and Gα₁₂ proteins, and for NECA additionally at the Gα_i2 protein. In contrast, the partial agonist BAY 60-6583 only activated Gα₁₅, Gα_s, and Gα₁₂ proteins. Adenosine deaminase, an allosteric modulator of ARs, selectively increased the potency and efficacy of NECA and BAY 60-6583 at the Gα₁₅ protein, while it had no effect or decreased efficacy at the other Gα proteins. We conclude that the A_2BAR is preferably coupled to the Gα₁₅, Gα_s, and Gα₁₂ proteins. Upon upregulation of receptor or Gα protein expression, coupling to further Gα proteins likely occurs. Importantly, different agonists can display different activation profiles.