Fig. 1. Gαi protein recruitment, inhibition of cAMP synthesis, and β-arrestin recruitment by biased agonists of CXCR3.
(A) Arrangement of the nanoBiT luciferase fragments used to assess G protein recruitment. (B) HEK293T transiently expressing CXCR3-smBiT and Gαi-LgBiT were treated with 100 nM CXCL9, 100 nM CXCL10, 100 nM CXCL11, 1 μM VUF10661, 1 μM VUF11418, or vehicle, and the resulting luminescence was analyzed for 750 s after an initial preread. (C) Gαi recruitment to CXCR3 after treatment with the indicated concentrations of agonists 1 min after treatment. (D) Scheme illustrating the Gαi-regulated cAMP assay. Before the cells were treated with biased agonists of CXCR3, the cellular cAMP concentration was increased by treatment with 10 μM forskolin. (E) HEK293T cells transiently expressing cAMP-activated modified firefly luciferase and CXCR3 were treated with vehicle, 100 nM CXCL9, 100 nM CXCL10, 100 nM CXCL11, 1 μM VUF10661, or 1 μM VUF11418. The inhibition of cAMP synthesis is displayed as a percentage of that in VUF10661-treated cells. (F) Arrangement of the luciferase fragments CXCR3-LgBiT and smBiT–β-arrestin2 to assess β-arrestin2 recruitment to CXCR3. (G) HEK293T transiently expressing CXCR3-LgBiT and smBiT–β-arrestin2 were treated with 100 nM CXCL9, 100 nM CXCL10, 100 nM CXCL11, 1 μM VUF10661, 1 μM VUF11418, or vehicle and then were analyzed for 750 s after an initial preread. (H) β-arrestin2 recruitment after treatment with the indicated concentrations of agonist 6 min after treatment. Data are means ± SEM of three or four independent experiments. *P < 0.05 by two-way ANOVA; Dunnett’s post hoc analysis showed a significant difference relative to pretreatment.