Fig. 1: GRK knockout cells enable the characterisation of GRK-specific β-arrestin recruitment.

a Single (ΔGRK2, 3, 5 and 6), double (ΔGRK2/3 or 5/6), triple (ΔGRK3/5/6, 2/5/6, 2/3/6 and 2/3/5) and quadruple (ΔQ-GRK) GRK knockout cells were generated using the CRISPR/Cas9 technology and established as single-cell clones. The absence of GRK2, 3, 5 or 6 was confirmed by Western blot analysis. A representative blot of n = 4 independent experiments is shown (for quantification, see Supplementary Fig. 1d). b Schematic depiction of the performed NanoBRET β-arrestin (βarr) recruitment assay and colour-coding for GRK-specific conditions used throughout the paper. The Halo-Tag-βarr fusion protein is recruited to a NanoLuciferase (NanoLuc)-tagged GPCR upon agonist activation and subsequent receptor phosphorylation. The resulting change in proximity of the Halo-Tag and the NanoLuc increases measured BRET ratios, enabling the agonist concentration-dependent analysis of βarr recruitment. c Halo-Tag-βarr2 recruitment to the b2AR-NanoLuc upon stimulation with isoprenaline (Iso) in cells expressing all endogenous GRKs (Control + empty vector (EV)) or only one remaining endogenous GRK (triple knockout cells ΔGRK3/5/6, ΔGRK2/5/6, ΔGRK2/3/6 and ΔGRK2/3/5). d The relative GRK protein expression in Control cells determined by Western blot of n = 3 independent lysates as described in Reichel et al.²⁷. Data are depicted as mean ± SEM of n = 3 independent blots. GRK expression levels were compared using ANOVA and two-sided Tukey’s test (*p < 0.05; **p < 0.01; ***p < 0.001). e βarr2 recruitment to the b2AR in quadruple GRK knockout cells (ΔQ-GRK), overexpressing a single GRK (ΔQ-GRK + GRK) or expressing all endogenous GRKs (Control + EV). BRET data in (c) and (e) are presented as Δ net BRET fold change, mean of n = 3 independent experiments ± SEM. For better comparison, the Control and ΔQ-GRK curves are shown multiple times. f GRK–YFP fusion proteins were transfected in ΔQ-GRK and YFP fluorescence was measured to confirm similar expression levels of all transfected GRKs. YFP fluorescence was compared using ANOVA and two-sided Tukey’s test (ns not significant). Corresponding experiments, confirming the catalytic activity of GRK–YFP fusion constructs are shown in Supplementary Fig. 3a. Measured fluorescence is depicted as a mean of n = 3 independent experiments + SEM as normalised fluorescence. All exact p values, test statistics, effect sizes, confidence intervals and degrees of freedom are provided in the Source Data files.