biostudies-literatureUnknown650(8103)Thomas RG-protein-coupled receptors (GPCRs) are key mediators of cell communication and represent the most important class of drug targets^1,2. Biophysical studies with purified GPCRs in vitro have suggested that they exist in an equilibrium of distinct inactive and active states, which is modulated by ligands in an efficacy-dependent manner^3-11. However, how efficacy is encoded and whether multiple receptor states occur in living cells remain unclear. Here we use genetic code expansion¹² and bioorthogonal labelling^13-16 to generate a panel of fluorescence-based biosensors for a prototypical GPCR, the M₂ muscarinic acetylcholine receptor (M₂R). These biosensors enable real-time monitoring of agonist-promoted conformational changes acNature1053-1062https://www.ebi.ac.uk/biostudies/studies/S-EPMC12935549biostudies-literatureLigand-specific activation trajectories dictate GPCR signalling in cells.PMC12935549Jacoby PSStaubert CLohse MJThomas RDe Faveri CCoin ILiebing ADMelkes BBermudez MBock AMartini HJDerieux CfalseLigand-specific activation trajectories dictate GPCR signalling in cells.G-protein-coupled receptors (GPCRs) are key mediators of cell communication and represent the most important class of drug targets^1,2. Biophysical studies with purified GPCRs in vitro have suggested that they exist in an equilibrium of distinct inactive and active states, which is modulated by ligands in an efficacy-dependent manner^3-11. However, how efficacy is encoded and whether multiple receptor states occur in living cells remain unclear. Here we use genetic code expansion¹² and bioorthogonal labelling^13-16 to generate a panel of fluorescence-based biosensors for a prototypical GPCR, the M₂ muscarinic acetylcholine receptor (M₂R). These biosensors enable real-time monitoring of agonist-promoted conformational changes ac2026-01-01T00:00:00Z2026 Feb2026-07-16T21:58:54.314Z2026-07-11T03:09:04.628ZS-EPMC129355494153547210.1038/s41586-025-09963-3