Fig. 2.

Selectivity of G protein binding to M₃–R. (A) Agonist-dependent association and dissociation of M₃–R with G_o, G_q, G_s, or G₁₃ proteins were measured after nucleotide depletion by means of FRET similar to that described in Fig. 1F. Average traces of YFP/CFP emission ratio (normalized to initial values) reflecting FRET between YFP-labeled M₃–R and CFP-labeled Gγ₂ subunit are illustrated when Gα_o-WT (pink curve), Gα_q-WT (black), G_s (purple), G₁₃ (red), or empty pcDNA3 (dark green) was cotransfected. A much faster decay of the FRET signal after withdrawal of agonist was detected for G_o proteins compared with G_q. (B) The constants of dissociation (k_off; s⁻¹) for G_o and G_q proteins from M₃–R are shown (magenta, n = 14; black, n = 30, respectively). (C) Observed association kinetics (k_obs; s⁻¹) of G_q protein with M₃–R under GTP-depleted conditions were plotted over different CCh concentrations and fitted by a hyperbolic function. (D) Measured k_on value of M₃–R–G_q association kinetics (light gray, G_q meas.) obtained from fitted hyperbolic function using Eq. 2 compared with ${\text{k}}_{\text{on}}^{\text{C}}$ of G_q (black, G_q calc.) and G_o (magenta, G_o calc.), which were calculated based on steady-state experiments data as shown in Eq. 3. All data are plotted as means ± SEM for each condition; n of each experiment is shown in parentheses if not indicated. Statistical anlysis was performed using one-way ANOVA followed by Student’s t test (***P <0.001).