title: Use of a new proximity assay (NanoBRET) to investigate the ligand-binding characteristics of three fluorescent ligands to the humanβ1-adrenoceptor expressed in HEK-293 cells.
abstract: Previous research has indicated that allosteric interactions across the dimer interface ofβ1-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 pKivalues at the humanβ1-adenoceptor, which may result from such allosterism interactions. Three fluorescentβ1-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 humanβ1-adrenoceptor expressed in HEK 293 cells (NanoBRET). This proximity assay showed high-affinity-specific binding to the NanoLuc-β1-adrenoceptor with each of the three fluorescent ligands yieldingKDvalues 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 with3H-CGP12177 and TIRF microscopy, to monitor NanoLuc bioluminescence, confirmed a high cell surface expression of the NanoLuc-β1-adrenoceptor in HEK 293 cells (circa 1500 fmol.mg protein-1). Following a 1 h incubation with fluorescent ligands andβ1-adrenoceptor competing antagonists, there were significant differences (P< 0.001) in the pKivalues obtained for CGP20712a and CGP 12177 with the different fluorescent ligands and3H-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 humanβ1-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.
authors: Cell Signalling and Pharmacology Research Group School of Life Sciences University of Nottingham Nottingham NG7 2UH United Kingdom.
authors: Mark
authors: M
authors: Soave
authors: Cell Signalling and Pharmacology Research Group School of Life Sciences University of Nottingham Nottingham NG7 2UH United Kingdom.
authors: Leigh A
authors: LA
authors: Stoddart
authors: Heptares Therapeutics Ltd. Bio Park Welwyn Garden City AL7 3AX United Kingdom.
authors: Alastair
authors: A
authors: Brown
authors: Cell Signalling and Pharmacology Research Group School of Life Sciences University of Nottingham Nottingham NG7 2UH United Kingdom.
authors: Jeanette
authors: J
authors: Woolard
authors: Cell Signalling and Pharmacology Research Group School of Life Sciences University of Nottingham Nottingham NG7 2UH United Kingdom.
authors: Stephen J
authors: SJ
authors: Hill
keywords: Bioluminescence energy transfer; ligand binding; probe dependence; β‐adrenoceptors