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.