Adenosine is an important physiological regulator and interacts with four G protein-coupled receptor (GPCR) subtypes, the adenosine A₁, A_2A, A_2B and A₃ receptors(1). The adenosine A_2B receptor (A_2BAR) has a low affinity for the endogenous agonist, adenosine, and is expressed throughout the cardiovascular system(2). It is implicated in diseases including inflammation, angiogenesis and ischaemia(2). Recently, it has been shown that the adenosine A₁AR partial agonist capadenoson and the A₁AR hybrid molecule VCP746 can bind and activate the human A_2BAR(3,4). These ligands are currently being investigated in an in vivo rat haemodynamics model, however the complexity of a whole animal system necessitates further studies into the effects of capadenoson and VCP746 at the rat adenosine receptors. Therefore, we investigated ligand binding at the rat A_2BAR using the NanoBRET proximity assay(5).

Full length cDNA encoding the rat A_2BAR was amplified and fused in frame to a vector containing NanoLuc to generate the rat NanoLuc-A_2BAR (NL-A_2BAR). This construct was stably transfected into whole HEK 293 cells. These cells were used in saturation, competition and kinetic NanoBRET experiments as previously described(5,6). All data are mean ± SEM representative of n experiments.

Specific binding of the fluorescent adenosine antagonist CA200645(5) was detected at the rat NL-A_2BAR in whole living cells after two hours incubation using endpoint saturation assays (K_D 89.9 ± 14.3 nM, n=6), and via a continuously monitoring kinetic assay (K_D 35.4 ± 6.4 nM, n=6). From the kinetic assay, CA200645 was observed to be a slowly dissociating ligand (K_off 0.05 ± 0.01 min^-1). The specific CA200645 binding enabled it to be used as a probe to measure the affinities of a panel of unlabelled ligands at the rat NL-A_2BAR (Table 1). 

These data represent the first time ligand binding kinetics have been monitored in real time at the rat A_2BAR. The data presented here will be a useful resource for future experiments in a whole animal system. 

(1) Alexander, S.P.H. et al., G protein-coupled receptors, in The Concise Guide to Pharmacology 2015/2016, 5904-5941 (2015). 

(2) Burnstock, G. Purinergic Signalling: Therapeutic Developments. Front. Pharmacol. 8, 1–55 (2017). 

(3) Baltos, J., Vecchio, E. A., Harris, M. A., Xue, C., Ritchie, R. H., Christopoulos, A. May, L. T. Capadenoson, a clinically trialed partial adenosine A1 receptor agonist, can stimulate adenosine A2B receptor biased agonism. Biochem, Pharmacol., 135, 79–89 (2017). 

(4) Vecchio, E. A. et al. The hybrid molecule, VCP746, is a potent adenosine A2B receptor agonist that stimulates anti-fibrotic signalling. Biochem. Pharmacol. 117, 46–56 (2016). 

(5) Stoddart, L. A. et al. Application of BRET to monitor ligand binding to GPCRs. Nat. Methods 3, 661–663 (2015).  

(6) Stoddart, L.A. et al., (2018). Development of novel fluorescent histamine H1-receptor antagonists to study ligand-binding kinetics in living cells. Scientific Reports, 8(1), 1572.