Basal and agonist-induced binding of Lys139 mutants to GPCRs. A. Basal (agonist-independent) association of the indicated luciferase-tagged receptor and wild type or mutant Venus-Arrestin-3 in COS-7 cells at 10 min of vehicle treatment. Nonspecific (bystander) BRET was measured using non-receptor-binding arrestin-3 mutant (Arrestin3-KNC [23,30] ). Net BRET ratio was calculated by subtracting non-specific BRET from the raw BRET data in each experiment. B. The agonist-induced arrestin-3 recruitment to indicated receptors. The BRET change (ΔBRET) was determined by the difference between the BRET ratio between ligand and vehicle treated cells (nonspecific BRET was subtracted from both). Means ± S.E. of at least three independent experiments are shown. Each experiment was performed in quadruplicate. Statistical significance was determined by one-way ANOVA, followed by Dunnett’s post-hoc test: *, p < 0.05; **, p < 0.01; ***, p < 0.001, as compared with A87V base mutant binding to each receptor.