FIGURE 5.

The K685R mutation impairs the induction of some angiotensin II-dependent genes in A4 cells. A, A4-V, -WT, -K685R, -Y705F, and -K685R/Y705F cells were infected with lentiviral constructs expressing hAT1R, and stable pools of cells were selected with puromycin. Western analysis of total cell lysates confirms human AT1R expression in each pool. IB, immunoblot. B, the hAT1R-expressing A4 cells were stimulated with 1 μm angiotensin II, and their response to angiotensin II-induced calcium mobilization was determined by measuring their respective calcium flux. C, the heat map of microarray done to compare genes induced in A4-Y705F STAT3 and A4-K685R/Y705F STAT3 cells expressing hAT1R upon angiotensin II (Ang II) treatment shows the increased expression of 73 genes. We include only genes whose expression changed by 2-fold, with differential p < 0.05 in Y705F STAT3/hAT1R cells when compared with vector control cells, and with average signals greater than 30 in WT cells, following treatment with 1 μm angiotensin II for 4 h. D, A4-Y705F and A4-K685R/Y705F cells were treated with angiotensin II (1 μm for 4 h), and the relative expression of the SOS2, LTA, and PLA2G10 genes was determined by qPCR. Values are the means, with standard deviations, from triplicate experiments. *, p ≤ 0.05; **, p ≤ 0.005.