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

Clare Harwood ¹ David A Sykes ¹ Theo Redfern-Nichols ² Graham Ladds ² Stephen J Briddon ^1* Dmitry B Veprintsev ^1*

• ¹ University of Nottingham, Nottingham, United Kingdom
• ² University of Cambridge, Cambridge, England, United Kingdom

The β2-adrenoceptor (β2AR) is a class A G protein-coupled receptor (GPCR). It is therapeutically relevant in asthma and chronic obstructive pulmonary disease, whereby 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 hypothesized that the kinetics of GPCR-G protein interactions could play a role in determining ligand efficacy. 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.We used NanoBRET technology to measure ligand-induced binding of purified Venus-mini-Gs to β2AR-nLuc in membrane preparations, in equilibrium and kinetic modes. 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 senses a reduction in NanoBRET between the nano-luciferase (nLuc) donor on the Ga subunit and Venus acceptor on the Gg, upon Gs protein activation.The twelve β2AR agonists under study revealed a broad range of ligand potency and efficacy values in both the cellular Gs-CASE and membrane-based mGs recruitment assays. Kinetic characterisation of mGs binding to the agonist-β2AR complex revealed a strong correlation between ligand efficacy values (Emax) and mini-Gs affinity (Kd) and its association rate (kon). In contrast, there was no correlation between ligand efficacy and reported ligand dissociation rates (or residence times).The association rate (kon) of the G protein to the agonist-β2AR complex is directly correlated with ligand efficacy. These data support a model in which agonists of increased efficacy cause the β2AR to adopt a conformation that is more likely to recruit G protein. Conversely, these data did not support a role for agonist binding kinetics in the molecular basis of efficacy.