Skip to main content
. 2017 Jun 5;292(29):12139–12152. doi: 10.1074/jbc.M117.793877

Figure 2.

Figure 2.

Functional Gαq/11 is required for Ang II-induced conformational cross-talk between AT1R and FP. A, HEK 293 cells were transfected with the FP-ICL3 P4-RLucII biosensor and AT1R-WT. Cells were pre-treated with 100 nm of the Gαq inhibitor FR900359 for 1 h followed by buffer, 10 μm AS604872, or 10 μm losartan for 30 min prior to BRET recording. Cells were stimulated with 1 μm PGF2α or Ang II and the change in BRET (ΔBRET) due to ligand stimulation is reported. B, ΔGαq/11/12/13 HEK 293 cells were transfected with FP-ICL3 P4-RLucII, AT1R-WT, and pcDNA3.1 or the indicated Gα subunit. Cells were stimulated with 1 μm PGF2α or Ang II and the change in BRET (ΔBRET) due to ligand stimulation is reported. C, ΔGαq/11/12/13 HEK 293 cells were transfected with FP-ICL3 P4-RLucII, AT1R-WT, and pcDNA3.1 or the indicated Gα subunit. Cells were pre-treated with buffer or 100 ng/ml of pertussis toxin for 16 h before FlAsH labeling. Cells were stimulated with 1 μm PGF2α or Ang II and the change in BRET (ΔBRET) due to ligand stimulation is reported. Bars represent the mean of 3 independent biological replicates and error bars represent S.E. A and B, Dunnett's test was performed to compare conditions with buffer (A) or pcDNA3.1 (B), where ** = p < 0.01. C, a two-way analysis of variance was used to analyze the 2 graphs. For both graphs, the G protein factor and interaction were nonsignificant, whereas the PTX factor was significant (PGF2α = p < 0.01, Ang II = p < 0.001). Bonferroni corrected tests were used to make post hoc comparisons where: * = p < 0.05; ** = p < 0.01. D and E, kinetic traces of conformational biosensors responding to ligand in the presence and absence of G proteins. ΔGαq/11/12/13 cells were transfected with FP-ICL3 P4-RLucII and AT1R-WT (D) or FP-ICL3 P4-RLucII, AT1R-WT, and Gαq (E). The specified ligand was injected onto the cells at the 50th repeat measure as denoted by an arrow. Inset to D: offset basal traces for PGF2α and Ang II to better demonstrate differences in biosensor responses. Traces represent the mean of 3 independent experiments.