Receptor phosphorylation independent conformational changes in β-arrestin 2. (A) HEK-293 cells were cotransfected with AT1aRΔ324 and Luc–β-arr–YFP, and stimulated with Ang II (100 nM, 10 min) and SII (10 μM, 5 min), and changes in intramolecular BRET ratio were measured. (B) Cells expressing AT1aRΔ324 mutant and Luc–β-arr–YFP were pretreated with H-89 (10 μM, 10 min) or GFX (10 μM, 10 min), and stimulated with Ang II (100 nM, 10 min) and SII (10 μM, 5 min), and changes in intramolecular BRET ratio were measured. (C) HEK-293 cells cotransfected with β2ARGRK-/PKA- mutant and Luc–β-arr–YFP were stimulated by isoproterenol (1 μM, 10 min), and changes in the intramolecular BRET ratio were measured. (D) HEK-293 cells overexpressing AT1aR were transfected with Luc–β-arr (R169E)-YFP and stimulated with Ang II (100 nM, 10 min) and SII (10 μM, 5 min), and changes in intramolecular BRET ratio were measured. Data are mean ± SD of four to six independent experiments, each performed at least in triplicates. *, P < 0.05; **, P < 0.01—between basal and stimulated condition as analyzed by one-way ANOVA with Bonferroni's post-test (A, B, and D) or paired t test (C).