Fig. 2: GRK knockout cells are a viable cellular platform to assess various features of b2AR–β-arrestin complex formation.

a Analysis of β-arrestin2 (βarr2) conformational changes. ΔQ-GRK or Control cells were transfected with an untagged b2AR expression construct and the βarr2-F5-NanoLuc conformational change biosensor (more details on the intramolecular BRET sensor will be published elsewhere), in the absence or presence of GRKs as noted and stimulated with isoprenaline (Iso). Conformational change data are shown as Δ net BRET change in per cent, mean of n = 3 independent repetitions ± SEM. b–d The recruitment of βarr2 to the b2AR following stimulation with Iso (b), Epinephrine (c) or Norepinephrine (d) in presence of all endogenous GRKs or individually overexpressed GRK2 or GRK6 in ΔQ-GRK as indicated. Data are depicted as a mean of n = 3 independent experiments ± SEM and normalised to individual maxima. The tables below depict the EC₅₀ ± SEM of the corresponding concentration-response curves. The EC₅₀ of the indicated conditions were compared to the EC₅₀ in Control using ANOVA and two-sided Dunnett’s test (*p < 0.05; **p < 0.01; ns not significant). e, f Utilisation of the βarr recruitment assay for specificity determination of the GRK inhibitor cmpd101 in living cells. ΔQ-GRK or Control cells were transfected with b2AR-NanoLuc, Halo-Tag-βarr2 and either GRK2, 3, 5, 6 or EV as noted. The cells were incubated with different concentrations of cmpd101 (e) or the b2AR antagonist pindolol (f) for 10 min prior to stimulation with 1 µM Iso. The recruitment-induced BRET changes were measured and calculated as Δ net BRET change in per cent, represented as the mean of n = 4 independent experiments ± SEM. All exact p values, test statistics, effect sizes, confidence intervals and degrees of freedom are provided in the Source Data files.