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Pharmacol Res Perspect. 2016 Aug 8;4(5):e00250. eCollection 2016 Oct.

Use of a new proximity assay (NanoBRET) to investigate the ligand-binding characteristics of three fluorescent ligands to the human β1-adrenoceptor expressed in HEK-293 cells.

Author information

1
Cell Signalling and Pharmacology Research Group School of Life Sciences University of Nottingham Nottingham NG7 2UH United Kingdom.
2
Heptares Therapeutics Ltd. Bio Park Welwyn Garden City AL7 3AX United Kingdom.

Abstract

Previous research has indicated that allosteric interactions across the dimer interface of β1-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 pKi values at the human β1-adenoceptor, which may result from such allosterism interactions. Three fluorescent β1-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 β1-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 yielding KD 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 3H-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 and β1-adrenoceptor competing antagonists, there were significant differences (P < 0.001) in the pKi values obtained for CGP20712a and CGP 12177 with the different fluorescent ligands and 3H-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 β1-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.

KEYWORDS:

Bioluminescence energy transfer; ligand binding; probe dependence; β‐adrenoceptors

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