Assay-Dependent Inverse Agonism at the A₃ Adenosine Receptor: When Neutral Is Not Neutral

The A₃ adenosine receptor (A₃AR) is implicated in a variety of (patho)physiological conditions. While most research has focused on agonists and antagonists, inverse agonism at A₃AR has been scarcely studied. Therefore, this study aimed at exploring inverse agonism, using two previously engineered cell lines (hA₃ARLgBiT-SmBiTβarr2 and hA₃ARLgBiT-SmBiTminiGα_i), both employing the NanoBiT technology. The previously established inverse agonist PSB-10 showed a decrease in basal signal in the β-arrestin 2 (βarr2) but not the miniGα_i recruitment assay, indicative of inverse agonism in the former assay. Control experiments confirmed the specificity and reversibility of this observation. Evaluation of a set of presumed neutral antagonists (MRS7907, MRS7799, XAC, and MRS1220) revealed that all displayed concentration-dependent signal decreases when tested in the A₃AR-βarr2 recruitment assay, yielding EC₅₀ and E_max values for inverse agonism. Conversely, in the miniGα_i recruitment assay, no signal decreases were observed. To assess whether this observation was caused by the inability of the ligands to induce inverse agonism in the G protein pathway, or rather by a limitation inherent to the employed A₃AR-miniGα_i recruitment assay, a GloSensor cAMP assay was performed. The outcome of the latter also suggests inverse agonism by the presumed neutral antagonists in this latter assay. These findings emphasize the importance of prior characterization of ligands in the relevant test system. Moreover, it showed the suitability of the NanoBiT βarr2 recruitment and the GloSensor cAMP assays to capture inverse agonism at the A₃AR, as opposed to the NanoBiT miniGα_i recruitment assay.