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

Previous research has indicated that allosteric interactions across the dimer interface of -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 values at the human -adenoceptor, which may result from such allosterism interactions. Three fluorescent -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 -adrenoceptor expressed in HEK 293 cells (NanoBRET). This proximity assay showed high-affinity-specific binding to the NanoLuc- -adrenoceptor with each of the three fluorescent ligands yielding values of 87.1 ± 10 nmol/L ( = 8), 38.1 ± 12 nmol/L ( = 7), 13.4 ± 2 nmol/L ( = 14) for propranolol-Peg8-BY630, propranolol- (Ala-Ala)-BY630 and CGP-12177-TMR, respectively. Parallel radioligand-binding studies with H-CGP12177 and TIRF microscopy, to monitor NanoLuc bioluminescence, confirmed a high cell surface expression of the NanoLuc- -adrenoceptor in HEK 293 cells (circa 1500 fmol.mg protein). Following a 1 h incubation with fluorescent ligands and -adrenoceptor competing antagonists, there were significant differences ( < 0.001) in the pK values obtained for CGP20712a and CGP 12177 with the different fluorescent ligands and 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 -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.