Use of a new proximity assay (NanoBRET) to investigate the ligand‐binding characteristics of three fluorescent ligands to the human β (1)‐adrenoceptor expressed in HEK‐293 cells

Previous research has indicated that allosteric interactions across the dimer interface of β (1)‐adrenoceptors may be responsible for a secondary low affinity binding conformation. Here we have investigated the potential for probe dependence, in the determination of antagonist pK(i) values at the human β (1)‐adenoceptor, which may result from such allosterism interactions. Three fluorescent β (1)‐adrenoceptor ligands were used to investigate this using bioluminescence energy transfer (BRET) between the receptor‐bound fluorescent ligand and the N‐terminal NanoLuc tag of a human β (1)‐adrenoceptor expressed in HEK 293 cells (NanoBRET). This proximity assay showed high‐affinity‐specific binding to the NanoLuc‐ β (1)‐adrenoceptor with each of the three fluorescent ligands yielding K (D) values of 87.1 ± 10 nmol/L (n = 8), 38.1 ± 12 nmol/L (n = 7), 13.4 ± 2 nmol/L (n = 14) for propranolol‐Peg8‐BY630, propranolol‐ β(Ala‐Ala)‐BY630 and CGP‐12177‐TMR, respectively. Parallel radioligand‐binding studies with (3)H‐CGP12177 and TIRF microscopy, to monitor NanoLuc bioluminescence, confirmed a high cell surface expression of the NanoLuc‐ β (1)‐adrenoceptor in HEK 293 cells (circa 1500 fmol.mg protein(−1)). Following a 1 h incubation with fluorescent ligands and β (1)‐adrenoceptor competing antagonists, there were significant differences (P < 0.001) in the pK(i) values obtained for CGP20712a and CGP 12177 with the different fluorescent ligands and (3)H‐CGP 12177. However, increasing the incubation time to 2 h removed these significant differences. The data obtained show that the NanoBRET assay can be applied successfully to study ligand‐receptor interactions at the human β (1)‐adrenoceptor. However, the study also emphasizes the importance of ensuring that both the fluorescent and competing ligands are in true equilibrium before interpretations regarding probe dependence can be made.