Fig. 3.
Effect of different β2AR ligands with varying efficacies on the conformational changes in β-arrestin. HEK-293 cells overexpressing β2AR (A) or β2ARTYY (B) mutant were transiently transfected with β-arrestin 2 double brilliance biosensor (Luc–β-arr–YFP). Forty-eight hours after transfection, cells were stimulated with receptor-saturating concentration of different ligands for 10 min, and changes in intramolecular BRET ratio were monitored. Data are mean ± SD of four independent experiments, each performed in triplicates. P < 0.05 between basal and stimulated condition as analyzed by one-way ANOVA with Bonferroni's post-test. (C) Conformational change in β-arrestin upon direct interaction with β2AR. Purified Luc–β-arr–YFP was incubated with β2AR reconstituted in lipid vesicles with isoproterenol (1 μM), with propranolol (10 μM), or preincubated with propranolol followed by addition of isoproterenol (n = 4, two independent purifications of Luc–β-arr–YFP).