Agonist efficacy at the B2AR is driven by agonist-induced differences in receptor affinity for the Gs protein, not ligand binding kinetics.

IntroductionThe {beta}2-adrenoceptor ({beta}2AR) is a class A G protein-coupled receptor (GPCR). It is therapeutically relevant in asthma, whereby {beta}2AR agonists relieve bronchoconstriction. The {beta}2AR is a prototypical GPCR for structural and biophysical studies. However, the molecular basis of agonist efficacy at the {beta}2AR is not understood. We hypothesized that the kinetics of ligand binding and GPCR-G protein interactions could play a role in ligand efficacy. We characterised the molecular pharmacology of a range of {beta}2AR agonists and examined the correlation between ligand and mini-Gs binding kinetics and efficacy. MethodsWe used a Time-resolved Fluorescence Resonance Energy Transfer (TR-FRET) based competition ligand binding assay to measure the affinity and residence times of a range of {beta}2AR agonists binding to the human {beta}2AR. TR-FRET between Lumi4-Tb3+ on the N terminus of the {beta}2AR and fluorescent CA200693 (S)-propranolol-green was measured using a PHERAstar FSX. The ability of these {beta}2AR agonists to activate the heterotrimeric Gs protein was measured using the CASE Gs protein biosensor. This assay senses a reduction in NanoBRET between the nano-luciferase (nLuc) donor on the G subunit and Venus acceptor on the G{psi}, on receptor activation, quantified using the operational model of agonism. NanoBRET was also used to measure interactions between DDM solubilised {beta}2AR-nLuc and purified Venus-mini-Gs. A large excess of unlabelled mini-Gs was used to dissociate the {beta}2AR-nLuc: Venus-mini-Gs complex. ResultsCharacterisation of the molecular pharmacology of seven {beta}2AR agonists showed a broad range of ligand binding affinities (pKi = 4.4 {+/-} 0.09 to 9.2 {+/-} 0.08) and kinetics parameters. There was no correlation between ligand residence times and their ability (log{tau} ) to activate the Gs protein (R2=0.26, p=0.29). However, there were statistically significant differences in the association rate (kon (fast)) (3.36{+/-}0.64x105 to 9.19{+/-} 0.42x105) and affinity (Kd) values of mini-Gs binding to the agonist-{beta}2AR complex (pKd =6.0 to 6.7). Both an increase in ligand driven mini-Gs kon(fast) rate and associated increase in mini-Gs pKd for the receptor, were moderately correlated with efficacy (log{tau} ) (R2 =0.58 and R2 =0.50 respectively). ConclusionsThese data support a model in which agonists of increased efficacy cause the {beta}2AR 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.