Agonist efficacy at the β2AR is driven by the faster association rate of the Gs protein

Clare R Harwood; David A Sykes; Theo Redfern-Nichols; Owen Underwood; Colin Nicholson; Armin N Khoshgrudi; Eline J Koers; Graham Ladds; Stephen J Briddon; Dmitry B Veprintsev

doi:10.3389/fphar.2025.1367991

Agonist efficacy at the β₂AR is driven by the faster association rate of the G_s protein

Front Pharmacol. 2025 Mar 27:16:1367991. doi: 10.3389/fphar.2025.1367991. eCollection 2025.

Authors

Clare R Harwood^{1

2}, David A Sykes^{1

2

3}, Theo Redfern-Nichols⁴, Owen Underwood³, Colin Nicholson^{1

2}, Armin N Khoshgrudi^{1

2}, Eline J Koers^{1

2}, Graham Ladds^{2

4}, Stephen J Briddon^{1

2}, Dmitry B Veprintsev^{1

2

3}

Affiliations

¹ Division of Physiology, Pharmacology and Neuroscience, School of Life Sciences, University of Nottingham, Nottingham, United Kingdom.
² Centre of Membrane Proteins and Receptors (COMPARE), University of Birmingham and University of Nottingham, Nottingham, United Kingdom.
³ Z7 Biotech Ltd., Nottingham, United Kingdom.
⁴ Department of Pharmacology, University of Cambridge, Cambridge, United Kingdom.

Abstract

Introduction: The β₂-adrenoceptor (β₂AR) is a class A G protein-coupled receptor (GPCR). It is therapeutically relevant in asthma and chronic obstructive pulmonary disease (COPD), where β₂AR agonists relieve bronchoconstriction. The β₂AR is a prototypical GPCR for structural and biophysical studies. However, the molecular basis of agonist efficacy at the β₂AR is not understood. We hypothesised that the kinetics of GPCR-G protein interactions could play a role in determining ligand efficacy. By studying a range of agonists with varying efficacy, we examined the relationship between ligand-induced mini-G_s binding to the β₂AR and ligand efficacy, along with the ability of individual ligands to activate the G protein in cells.

Methods: We used NanoBRET technology to measure ligand-induced binding of purified Venus-mini-G_s to β₂AR-nLuc in membrane preparations under both equilibrium and kinetic conditions. In addition, we examined the ability of these β₂AR agonists to activate the heterotrimeric G_s protein, measured using the G_s-CASE protein biosensor in living cells. This assay detects a reduction in NanoBRET between the nano-luciferase (nLuc) donor on the Gα subunit and Venus acceptor on the Gγ upon G_s protein activation.

Results: The 12 β₂AR agonists under study revealed a broad range of ligand potency and efficacy values in the cellular G_s-CASE assays. Kinetic characterisation of mini-G_s binding to the agonist β₂AR complex revealed a strong correlation between ligand efficacy values (E_max) and mini-G_s affinity (K _d) and its association rate (k _on). In contrast, there was no correlation between ligand efficacy and reported ligand dissociation rates (or residence times).

Conclusion: The association rate (k _on) of the G protein to the agonist β₂AR complex is directly correlated with ligand efficacy. These data support a model in which higher-efficacy agonists induce the β₂AR to adopt a conformation that is more likely to recruit G protein. Conversely, these data did not support the role of agonist binding kinetics in determining the molecular basis of efficacy.

Keywords: dissociation rate koff; G protein-coupled receptor; association rate kon; efficacy; kinetics; β2-adrenoceptor.

Grants and funding

The author(s) declare that financial support was received for the research and/or publication of this article. CH was funded by a Medical Research Council (MRC) IMPACT PhD studentship. TR-N was funded by a UK Biotechnology and Biological Sciences Research Council iCase studentship (BB/V509334/1) co-funded with AstraZeneca. AK is funded by the BBSRC Doctorate Training Program at the University of Nottingham. GL is a Royal Society Industry Fellow (NF\R2\212001). EK, CN, DS and DV gratefully acknowledge funding by the Medical Research Council [grant number MR/Y003667/1] and Biotechnology and Biological Sciences Research Council [BB/Y51407X/1] and [BB/Z514500/1].