Open AccessUse 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
Author(s) -
Soave Mark,
Stoddart Leigh A.,
Brown Alastair,
Woolard Jeanette,
Hill Stephen J.
Publication year - 2016
Publication title -
pharmacology research and perspectives
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.975
H-Index - 27
ISSN - 2052-1707
DOI - 10.1002/prp2.250
Subject(s) - hek 293 cells , ligand (biochemistry) , allosteric regulation , fluorescence , chemistry , receptor , radioligand , biophysics , ligand binding assay , dimer , wild type , stereochemistry , biochemistry , biology , mutant , gene , physics , quantum mechanics , organic chemistry
Abstract 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.