Publications

@article{Harwood2025-cz,

title = {Agonist efficacy at the β2AR is driven by the faster  association rate of the Gs protein},

author = {Clare R Harwood and David A Sykes and Theo Redfern-Nichols and Owen Underwood and Colin Nicholson and Armin N Khoshgrudi and Eline J Koers and Graham Ladds and Stephen J Briddon and Dmitry B Veprintsev},

doi = {10.3389/fphar.2025.1367991},

year  = {2025},

date = {2025-03-01},

urldate = {2025-03-01},

journal = {Front. Pharmacol.},

volume = {16},

pages = {1367991},

publisher = {Frontiers Media SA},

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-Gs 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-Gs 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 Gs protein, measured using the Gs-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 Gs protein activation. Results: The 12 β2AR agonists 

 under study revealed a broad range of ligand potency and 

 efficacy values in the cellular Gs-CASE assays. Kinetic 

 characterisation of mini-Gs binding to the agonist β2AR 

 complex revealed a strong correlation between ligand efficacy 

 values (Emax) and mini-Gs 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 = {association   rate kon, dissociation rate koff, efficacy, G protein-coupled receptor, kinetics, β2-adrenoceptor},

pubstate = {published},

tppubtype = {article}

}