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

Introduction The β 2 -adrenoceptor (β 2 AR) is a class A G protein-coupled receptor (GPCR). It is therapeutically relevant in asthma and chronic obstructive pulmonary disease (COPD), where β 2 AR agonists relieve bronchoconstriction. The β 2 AR is a prototypical GPCR for structural and biophysical studies. However, the molecular basis of agonist efficacy at the β 2 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 s binding to the β 2 AR 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 β 2 AR-nLuc in membrane preparations under both equilibrium and kinetic conditions. In addition, we examined the ability of these β 2 AR 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 β 2 AR 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 β 2 AR 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 β 2 AR complex is directly correlated with ligand efficacy. These data support a model in which higher-efficacy agonists induce the β 2 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.