Fig. 5. COMP selectively inhibits the β-arrestin, but not G protein, in AT1 signaling.

a–d The effects of COMP on AngII-induced Gq and Gi activation through AT1. HEK293T cells were co-transfected with Gq BRET probes (Gq-RLucII, Gβ1, Gγ1-GFP10) (a, b) and Gi BRET probes (Gi1-RLuc8, Gβ3, Gγ9-GFP10) (c, d), respectively. The transfected cells were pre-incubated with purified COMP (100 nM) or losartan (1 μM), followed by stimulation with an increasing amount of AngII for 2 min (a, c), or with various concentrations of COMP or losartan, followed by stimulation with AngII (0.1 μM) for 2 min (b, d). The BRET signal was measured and presented as the ratio of the emission of GFP10 to RLucII (a, b) or RLuc8 (c, d). The data are presented as the means ± SEM from 3 independent experiments. One-way ANOVA followed by the Bonferroni test, *P < 0.05 vs vehicle. e–h The effects of COMP on AngII-induced β-arrestin-1 and β-arrestin-2 recruitment through AT1. HEK293T cells were co-transfected with the AT1-YFP and β-arrestin-1-RLuc plasmids (e, f) or the AT1-YFP and β-arrestin-2-RLuc plasmids (g, h), respectively. The transfected cells were pre-incubated with purified COMP (100 nM) or losartan (1 μM) for 30 min and then stimulated with an increasing amount of AngII for 5 min (e, g), or with various concentrations of COMP or losartan for 30 min and then stimulated with the AngII (0.1 μM) for 5 min (f, h). The BRET signal was measured and presented as the ratio of the emission of YFP to RLuc. The data are presented as the means ± SEM from 3 independent experiments performed in triplicate. One-way ANOVA followed by the Bonferroni test, *P < 0.05 vs vehicle. i, j The specific binding of [¹²⁵I]-AngII to WT AT1 receptor (i) or AT1–β-arrestin-2 fusion (j) with an increasing amount of unlabeled AngII in the absence or presence of purified COMP (5 μg/mL). k Schematics illustrating the AT1 receptor with a RLuc moiety fused to the C-terminus as well as a TC-tag (CCPGCC) inserted between K224 and A225 or Q229 and K230 in the ICL3 as a Gq- or β-arrestin-specific conformational sensor, respectively. l, m The effects of COMP on AngII-induced conformational change of AT1 Gq-specific sensor. HEK293T cells transfected with the Gq-specific conformational sensor of AT1 receptor were preincubated with purified COMP (100 nM) or losartan (1 μM) for 30 min followed by stimulation with an increasing amount of AngII for 2 min (l), or preincubated with various concentrations of COMP or losartan for 30 min followed by AngII (0.1 μM) stimulation for 2 min (m). n = 6; One-way ANOVA followed by the Bonferroni test. n, o The effects of COMP on AngII-induced conformational change of AT1 β-arrestin-specific sensor. HEK293T cells transfected with the β-arrestin-specific conformational sensor of AT1 receptor were preincubated with purified COMP (100 nM) or losartan (1 μM) for 30 min followed by stimulation with an increasing amount of AngII for 5 min (n), or preincubated with various concentrations of COMP or losartan for 30 min followed by AngII (0.1 μM) stimulation for 5 min (o). n = 6; One-way ANOVA followed by the Bonferroni test, *P < 0.05 vs vehicle.