Ligand-mediated activation of G-protein-coupled receptors (GPCRs) requires their coupling to transducers such as G proteins to initiate downstream signaling. Prior biophysical studies using fluorescent, NMR and double electron–electron resonance spectroscopy revealed that GPCRs exist in an equilibrium between inactive and active states that is dictated by ligand efficacy, which translates into diverse GPCR–G protein affinities. To determine if such distinct states were present in living cells, Thomas et al. devised a genetic code expansion strategy using unnatural amino acids to attach a cyanine fluorophore at particular amino acid residues on the extracellular surface of the M2 muscarinic acetylcholine receptor (M2R). This way, they engineered seven biosensors that exhibited fluorescent changes after M2R activation with preserved G-protein coupling. They tested these biosensors with a variety of agonists: the endogenous agonist acetylcholine, the super-agonist iperoxo, and the partial agonists arecoline and pilocarpine. For each agonist, they observed diverse effects with each biosensor in terms of activation or inhibition, with distinct effects on amplitude and kinetics, producing a unique conformational fingerprint. Analysis of the conformational fingerprint from each agonist argued for a range of distinct M2R active states in intact cells. Acetylcholine and iperoxo produced a high efficacy GPCR complex called C1 that was associated with efficacious G-protein activation. At later stages, a low-efficacy GPCR signaling complex called C2 was identified that correlated strongly with the activity of weaker agonists. The biosensor strategy developed by Thomas et al. could be extended to other GPCRs and may reveal unique conformational complexes in a more physiologically relevant environment that may inform drug discovery campaigns.
Original reference: Nature https://doi.org/10.1038/s41586-025-09963-3 (2026)
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