Journal Article

Agonist efficacy at the β 2 AR is driven by the faster association rate of the G s protein

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

2025

Overview

The β -adrenoceptor (β AR) is a class A G protein-coupled receptor (GPCR). It is therapeutically relevant in asthma and chronic obstructive pulmonary disease (COPD), where β AR agonists relieve bronchoconstriction. The β AR is a prototypical GPCR for structural and biophysical studies. However, the molecular basis of agonist efficacy at the β AR 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 binding to the β AR and ligand efficacy, along with the ability of individual ligands to activate the G protein in cells. We used NanoBRET technology to measure ligand-induced binding of purified Venus-mini-G to β AR-nLuc in membrane preparations under both equilibrium and kinetic conditions. In addition, we examined the ability of these β AR agonists to activate the heterotrimeric G protein, measured using the G -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 protein activation. The 12 β AR agonists under study revealed a broad range of ligand potency and efficacy values in the cellular G -CASE assays. Kinetic characterisation of mini-G binding to the agonist β AR complex revealed a strong correlation between ligand efficacy values (E ) and mini-G affinity ( ) and its association rate ( ). In contrast, there was no correlation between ligand efficacy and reported ligand dissociation rates (or residence times). The association rate ( ) of the G protein to the agonist β AR complex is directly correlated with ligand efficacy. These data support a model in which higher-efficacy agonists induce the β AR 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.