Fig. 5: Activation trajectories and conformational equilibria define ligand efficacy in living cells.
From: Ligand-specific activation trajectories dictate GPCR signalling in cells
a, TRUPATH assay principle. The BRET-based Gαβγ biosensors sense the conformational rearrangement during receptor-promoted G-protein activation as a decrease in BRET due to the increased distance between Gα and Gγ subunits53. Created in BioRender. Thomas, R. (2025) https://BioRender.com/loxqlqf. b–e, Representative traces of agonist-promoted changes in normalized ΔBRET (%) for all G-protein biosensors after activation of WT M2R (SP-M2R-WT) with the indicated agonists. Agonists were applied (black arrows) at saturating concentrations of ACh (b), iperoxo (c), arecoline (d) and pilocarpine (e). Number of independent experiments for ACh, iperoxo, arecoline and pilocarpine, respectively: 4, 6, 4 and 6 (Gi1); 4, 7, 4 and 6 (Gi2); 4, 6, 4 and 5 (Gi3); 6, 5, 3 and 6 (GoA); 5, 8, 3 and 5 (GoB); 5, 6, 4 and 4 (Gz); and 6, 6, 4 and 5 (G15). f, G protein-coupling selectivity profile of the M2R. Overview of agonist-promoted ΔBRET normalized to ACh (set to 1) obtained after agonist-promoted, M2R-mediated stimulation of BRET-based G-protein biosensors (TRUPATH). The coloured boxes (using the colour code from the planes in Fig. 4) on top of each column represent the equilibrium of receptor–G-protein complexes stabilized by the indicated ligand (Fig. 4). Heat-map values represent the mean ± s.e.m. (Supplementary Table 4). Statistical analysis was performed using one-way analysis of variance (ANOVA) with Dunnett’s post-hoc test for multiple comparisons (ACh as reference); ****P < 0.0001, ***P < 0.001, **P < 0.01, *P < 0.05; NS, not significant.
