Fig. 1. The β2AR CT autoinhibits agonist-stimulated receptor activation.

a Cell surface expression measured by the flow cytometry analysis. Note that amount of plasmid transfection of WT β₂AR was titrated down to 40 ng and 80 ng to adjust the expression levels equivalent to the ∆CT β₂AR. Data are mean±s.e.m. of 4 independent experiments. b Concentration-response curves of epinephrine-induced Gs heterotrimer dissociation in cells. The β₂AR-mediated Gs dissociation was measured by the NanoBiT-G-protein dissociation assay, stimulated by full agonist epinephrine in WT and the three ∆CT receptors. Data are mean±s.e.m. of 3 independent experiments. Νote that in many data points error bars are smaller than the size of symbols and thus are not visible. c The relative E_max and ∆pEC₅₀ parameters of the Gs dissociation responses stimulated by epinephrine in (b). d Partial agonist clenbuterol stimulated Gs dissociation in WT and three ∆CT receptors. Data are mean±s.e.m. of 3 independent experiments. e The relative E_max and ∆pEC₅₀ parameters of the Gs dissociation responses stimulated by clenbuterol in (d). f Removal of the β₂AR CT, by pretreating the β₂AR-s-CT with/without the SrtA, enhances the GTP turnover in the apo receptor and epinephrine-occupied receptor. Bars and error bars represent the mean±s.e.m. of 4 independent experiments. Statistical significances between ∆CT and expression-matched WT (∆C346 vs 80 ng; ∆C356 vs 80 ng; ∆C378 vs 40 ng) in c and e were performed by one-way ANOVA with the Sidak’s multiple comparison test. Statistical significances between Gs and the receptor-Gs mixture in f were performed by one-way ANOVA with Dunnett’s multiple comparison test. ^***P < 0.001; ns, P > 0.05.