Fig. 2
Functional characterization in HEK-293T cells expressing the AT1R-AT2R heteromer. HEK-293T cells were pretreated with selective receptor antagonists (300 nM candesartan for AT1R or 1 μM PD123319 for AT2R) and subsequently treated with selective agonists (100 nM angiotensin II for AT1R and 300 nM CGP-42112A for AT2R) a–c Cytosolic calcium detection assay were performed in HEK-293T cells transfected with the cDNAs for an engineered calcium sensor, 6GCaMP (1 μg), AT1R (1 μg), and/or AT2R (1 μg). Values are the mean ± S.E.M. of 5 independent experiments performed in duplicates. d–f Intracellular cAMP levels were determined by TR-FRET as described in Methods. HEK-293T cells were transfected with cDNAs for AT1R (1 μg) and/or AT2R (1 μg). When Gi coupling was assessed, decreases in [cAMP] were determined using 0.5 μM forskolin added 15 min after the agonists stimulation. Values are the mean ± S.E.M. of 6 independent experiments performed in triplicates. In cAMP one-way ANOVA followed by Bonferroni’s multiple comparison post-hoc test were used for statistical analysis. Signaling output was the dependent variable and the different treatments were the independent variables. (*p < 0.05, **p < 0.01, ***p < 0.001 versus forskolin treatment; +++p < 0.001 versus Ang II treatment; &&&p < 0.001 versus CGP-42112A treatment)