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
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 β...
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Wiley
2016
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| Online Access: | https://eprints.nottingham.ac.uk/34685/ |
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| author | Soave, Mark Stoddart, Leigh A. Brown, Alastair Woolard, Jeanette Hill, Stephen J. |
| author_facet | Soave, Mark Stoddart, Leigh A. Brown, Alastair Woolard, Jeanette Hill, Stephen J. |
| author_sort | Soave, Mark |
| building | Nottingham Research Data Repository |
| collection | Online Access |
| description | 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 ± 10nM (n=8), 38.1 ± 12nM (n=7), 13.4 ± 2nM (n=14) for propranolol-Peg8-BY630, propranolol-3(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- 31-adrenoceptor in HEK 293 cells (circa 1500 fmol.mg protein-1). Following a 1h 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 2h 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. |
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| institution | University of Nottingham Malaysia Campus |
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| publishDate | 2016 |
| publisher | Wiley |
| recordtype | eprints |
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| spelling | nottingham-346852020-05-04T18:07:39Z https://eprints.nottingham.ac.uk/34685/ 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 Soave, Mark Stoddart, Leigh A. Brown, Alastair Woolard, Jeanette Hill, Stephen J. 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 ± 10nM (n=8), 38.1 ± 12nM (n=7), 13.4 ± 2nM (n=14) for propranolol-Peg8-BY630, propranolol-3(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- 31-adrenoceptor in HEK 293 cells (circa 1500 fmol.mg protein-1). Following a 1h 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 2h 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. Wiley 2016-08-08 Article PeerReviewed Soave, Mark, Stoddart, Leigh A., Brown, Alastair, Woolard, Jeanette and Hill, Stephen J. (2016) 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. Pharmacology Research and Perspectives, 4 (5). e00250/1-e00250/13. ISSN 2052-1707 Bioluminescence energy transfer; Ligand-binding; 3-adrenoceptors; Probe dependence http://onlinelibrary.wiley.com/doi/10.1002/prp2.250/abstract doi:10.1002/prp2.250 doi:10.1002/prp2.250 |
| spellingShingle | Bioluminescence energy transfer; Ligand-binding; 3-adrenoceptors; Probe dependence Soave, Mark Stoddart, Leigh A. Brown, Alastair Woolard, Jeanette Hill, Stephen J. 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 |
| title | 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 |
| title_full | 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 |
| title_fullStr | 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 |
| title_full_unstemmed | 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 |
| title_short | 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 |
| title_sort | 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 |
| topic | Bioluminescence energy transfer; Ligand-binding; 3-adrenoceptors; Probe dependence |
| url | https://eprints.nottingham.ac.uk/34685/ https://eprints.nottingham.ac.uk/34685/ https://eprints.nottingham.ac.uk/34685/ |