Fig. 3.

Comparison of receptor–G-protein binding and activation of G proteins. (A) Concentration–response curves of G_q (black) and G_o (pink) binding to M₃–R under GTP-free conditions. FRET imaging in single permeabilized cells was performed as described in Fig. 1F. Increasing concentrations of CCh were applied as shown in a representative trace of a G_q-expressing cell (B). The change of YFP/CFP emission ratio was normalized to the amplitude observed on application of saturating CCh concentration (1 mM; maximum response) and the YFP/CFP ratio measured under the application of GTPγS (minimum response). To achieve steady-state conditions at low CCh concentrations (under 100 nM), cells were incubated for 10 min. (C and D) Concentration–response curves of G_q and G_o activation by M₃–R were determined in intact cells using established G-protein FRET assays (61). (D) Representative traces of FRET between YFP-labeled Gα subunit and CFP-labeled Gγ₂ subunit were measured in duplicates (overflow technique) simultaneously in 12 individual wells derived from a 96-well plate and normalized to the maximal response (1 mM CCh). All concentration–response data are represented as means ± SEM for each condition; n of each experiment is shown in parentheses. Statistical analysis was performed by one-way ANOVA with Bonferroni post hoc test (P < 0.05).