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

Abstract

The adenosine A2B receptor (A2BAR) 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 A2BAR to different G proteins, mainly Gs, but in some cases Gq/11 or Gi, depending on the cell type. We have now utilized novel technologies, (i) heterologous expression of individual members of the Gαq/11 protein family (Gαq, Gα11, Gα14, and Gα15) in Gαq/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 A2BAR 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 (Gαs, Gαq/11, Gαi, and Gα12/13). Significant differences in potencies and efficacies were observed; the highest efficacies were determined at the Gα15, Gαs, and Gα12 proteins, and for NECA additionally at the Gαi2 protein. In contrast, the partial agonist BAY 60-6583 only activated Gα15, Gαs, 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 A2BAR is preferably coupled to the Gα15, Gαs, 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.

Irreversible Antagonists for the Adenosine A2B Receptor

“…The inhibitory potency of the compounds on A 2B AR activation was determined in G protein activation assays using N-ethylcarboxamidoadenosine (NECA), a stable analog of adenosine, as an agonist. In the assay, a bioluminescence resonance energy transfer (BRET) ratio between a renilla luciferase 8 (RLuc8)-tagged Gα 15 protein-a key effector protein of the A 2B AR [38]-and a green fluorescent protein 2 (GFP2)-tagged Gγ 13 protein is measured [39] (Figure 4a). Upon receptor activation by NECA, the BRET ratio decreases.…”

“…In washout experiments, the cells were washed twice with 100 µL of assay buffer after antagonist incubation. Subsequently, 10 µL of substrate solution (50 µM Deep Blue C, Biomol, Hamburg, Germany) was added to the cells and the mixture was incubated for 5 min before addition of agonist (10 µL of NECA at its EC 80 concentration, 100 nM final concentration, as previously determined [38]). RLuc8-luminescence (395 nm emission filter) and GFP2-fluorescence (515 nm emission filter) were measured 5 min after agonist addition using a Mithras LB940 plate reader.…”

Cancer-Associated Mutations Enhance The Sensitivity Of The Trupath GαQ/11 System

“…Optimized sensors consisting of Gα-Rluc8 and Gβɣ-GFP2 were developed for all non-visual G proteins, with the exception of G olf and G 14 , and included the first sensors for G 15 and G gust (Olsen et al, 2020). TRUPATH has since been employed to assess the G protein activation profile of a number of GPCRs (Knight et al, 2021; Lieb et al, 2021; Singh, Senatorov, Cheshmehkani, Karmokar, & Moniri, 2022; Voss, Mahardhika, Inoue, & Müller, 2022; Wall et al, 2022).…”

“…Pleiotropic signalling has been documented for numerous GPCRs (Avet et al, 2022; Hauser et al, 2022; Knight et al, 2021; Lieb et al, 2021; Olsen et al, 2020; Singh et al, 2022; Voss et al, 2022; Wall et al, 2022). Class B1 GPCRs, for example, are classically considered Gα s- coupled GPCRs but have now been demonstrated to consistently exhibit secondary G protein coupling (Wootten et al, 2017).…”

“…TRUPATH has since been employed to assess the G protein activation profile of a number of GPCRs (Knight et al, 2021;Lieb et al, 2021;Singh, Senatorov, Cheshmehkani, Karmokar, & Moniri, 2022;Voss, Mahardhika, Inoue, & Müller, 2022;Wall et al, 2022).…”