Figure 3.
Membrane preparation results in a loss of Ang II-induced AT1R to FP conformational cross-talk although the receptors still form a complex. A, ΔGαq/11/12/13 HEK 293 cells were transfected with FP-ICL3 P4-RLucII, the indicated wild-type receptor, and pcDNA3.1, or the indicated Gα subunit. Cells were split into intact cell or membrane preparation groups. Each group was treated as described under “Experimental procedures.” Basal BRET was recorded in the absence of receptor ligands. B, Western blot analysis demonstrating that Gαq is still present in the sample after membrane preparation. C and D, the same preparations used in A and B were stimulated with 1 μm PGF2α (C) or Ang II (D) and the change in BRET (ΔBRET) due to ligand stimulation is reported. E, Western blots demonstrating that AT1R (FLAG) can co-immunoprecipitate FP-ICL3 P4-RLucII in lysates from prepared membrane or intact ΔGαq/11/12/13 HEK 293 cell samples, independent of the expression of Gαq. A, C, and D, bars represent the mean of 3 independent biological replicates and error bars represent S.E. Tukey's test was used to compare all groups in graphs where: * = p < 0.5; ** = p < 0.01. B and E are representative blots of n = 2. E, expression of FP-ICL3 P4-RLucII in all conditions has been demonstrated based on quantification of luminescence from RLucII (data not shown).