Agonist efficacy at the β 2 AR is driven by agonist-induced differences in receptor affinity for the G s protein, not ligand binding kinetics
Abstract
Introduction
The β 2 -adrenoceptor (β 2 AR) is a class A G protein-coupled receptor (GPCR). It is therapeutically relevant in asthma, whereby 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 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 β 2 AR agonists and examined the correlation between ligand and mini-G s binding kinetics and efficacy.
Methods
We 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 β 2 AR agonists binding to the human β 2 AR. TR-FRET between Lumi4-Tb 3+ on the N terminus of the β 2 AR and fluorescent CA200693 (S)-propranolol-green was measured using a PHERAstar FSX. The ability of these β 2 AR agonists to activate the heterotrimeric G s protein was measured using the CASE G s protein 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 β 2 AR-nLuc and purified Venus-mini-G s . A large excess of unlabelled mini-G s was used to dissociate the β 2 AR-nLuc: Venus-mini-G s complex.
Results
Characterisation of the molecular pharmacology of seven β 2 AR agonists showed a broad range of ligand binding affinities (p K i = 4.4 ± 0.09 to 9.2 ± 0.08) and kinetics parameters. There was no correlation between ligand residence times and their ability (log τ ) to activate the G s protein (R 2 =0.26, p=0.29). However, there were statistically significant differences in the association rate ( k on (fast) ) (3.36±0.64×10 5 to 9.19± 0.42×10 5 ) and affinity ( K d ) values of mini-G s binding to the agonist-β 2 AR complex (p K d =6.0 to 6.7). Both an increase in ligand driven mini-G s k on(fast) rate and associated increase in mini-G s p K d for the receptor, were moderately correlated with efficacy (log τ ) (R 2 =0.58 and R 2 =0.50 respectively).
Conclusions
These data support a model in which agonists of increased efficacy cause the β 2 AR 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.
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