Figure 7. G_s signalling of dualsteric probes is augmented in the W422^7.35A mutant.

(a,c) Maximum G_i- and G_s-DMR effects of the indicated compounds in wild-type CHO-hM₂ cells (a) or CHO-hM₂ W422^7.35A cells (c). Data represent means±s.e.m. from three independent experiments in a and c. Cells were pretreated with PTX 100 ng ml⁻¹ for 16–24 h to disclose G_s signalling. All concentrations used provide maximum effects for both the G_i and the G_s pathway according to GTPγS binding and cAMP accumulation assays, respectively. Data were normalized on the respective effect of iperoxo 10 μM, which was set 100% for each condition. Dashed lines represent balanced agonism. Continuous lines define a binodal curve resulting from a best fit to the indicated data points in a. Concentrations of ligands: iperoxo 10 μM, iper-6-phth (i-6-p) 100 μM, iper-8-phth (i-8-p) 10 μM, iper-6-naph (i-6-n) 100 μM, iper-8-naph (i-8-n) 10 μM, pilocarpine 100 μM and McN-A-343 100 μM. (b) Iper-6-phth and iper-6-naph induce significant G_s signalling at the hM₂ W422^7.35A mutant in contrast to wild-type receptors. Shown are maximal DMR responses normalized to the DMR of iperoxo 10 μM (set 100%) in CHO-hM₂ cells (wild type, open bars) and CHO-hM₂ W422^7.35A cells (filled bars). Data are means±s.e.m of at least three independent experiments. *P<0.05,**P<0.01, ***P<0.001, significant to vehicle according to a two-way ANOVA with Bonferroni's multiple comparison test.