Agonist efficacy at the β2AR is driven by agonist-induced differences in receptor affinity for the Gsprotein, not ligand binding kinetics

Abstract

AbstractIntroductionThe β2-adrenoceptor (β2AR) is a class A G protein-coupled receptor (GPCR). It is therapeutically relevant in asthma, 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 ligand binding and GPCR-G protein interactions could play a role in ligand efficacy. We characterised the molecular pharmacology of a range of β2AR agonists and examined the correlation between ligand and mini-Gsbinding 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 β2AR agonists binding to the human β2AR. TR-FRET between Lumi4-Tb3+on the N terminus of the β2AR and fluorescent CA200693 (S)-propranolol-green was measured using a PHERAstar FSX. The ability of these β2AR agonists to activate the heterotrimeric Gsprotein was measured using the CASE Gsprotein biosensor. This assay senses a reduction in NanoBRET between the nano-luciferase (nLuc) donor on the Gα subunit and Venus acceptor on the Gψ, on receptor activation, quantified using the operational model of agonism. NanoBRET was also used to measure interactions between DDM solubilised β2AR-nLuc and purified Venus-mini-Gs. A large excess of unlabelled mini-Gswas used to dissociate the β2AR-nLuc: Venus-mini-Gscomplex.ResultsCharacterisation of the molecular pharmacology of seven β2AR 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 (logτ) to activate the Gsprotein (R2=0.26, p=0.29). However, there were statistically significant differences in the association rate (kon (fast)) (3.36±0.64×105to 9.19± 0.42×105) and affinity (Kd) values of mini-Gsbinding to the agonist-β2AR complex (pKd=6.0 to 6.7). Both an increase in ligand driven mini-Gskon(fast)rate and associated increase in mini-GspKdfor the receptor, were moderately correlated with efficacy (logτ) (R2=0.58 and R2=0.50 respectively).ConclusionsThese 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.