Fig. 3: ΔQ-GRK cells reveal GRK-specificity of β-arrestin1 and 2 recruitment to different GPCRs and allow assessment of GRK-dependent GPCR internalisation and β-arrestin2 translocation.

a–d GRK-specific β-arrestin (βarr)1 (a, c) or βarr2 (b, d) recruitment to the M5R upon acetylcholine (ACh) stimulation (a, b) or the PTH1R upon parathyroid hormone 1–34 (PTH(1-34)) stimulation (c, d). Shown are concentration-response curves depicted as Δ net BRET fold change, mean of n = 3 independent experiments ± SEM. The panels display the recruitment in presence of either GRK2 or 3 or GRK5 or 6. For better comparison, the Control and ΔQ-GRK curves are shown multiple times. e, f Control, ΔGRK2/3, ΔGRK5/6 and ΔQ-GRK cells were transfected with either M5R-CFP or PTH1R-CFP (blue), the early endosome marker Rab5-mCherry (red) and βarr2-YFP (green) expression constructs. The cells were grown on coverslips and subjected to confocal live-cell microscopy. Shown are representative images, taken before and after 15 min of stimulation with either 100 µM ACh or 100 nM PTH(1–34), respectively. The normalised co-localisation of M5R (g) or PTH1R (h) with βarr2 or Rab5 was quantified using Squassh and SquasshAnalyst (number of images per respective condition; Control: M5R (39), PTH1R (38); ΔGRK2/3: M5R (35), PTH1R (32); ΔGRK5/6: M5R (36), PTH1R (33); ΔQ-GRK: M5R (38), PTH1R (55)). Data are presented as mean fold change in co-localisation signal + SEM. Statistical analysis was performed using a two-way mixed model ANOVA followed by a two-sided paired t-test (*p < 0.05; **p < 0.01; ***p < 0.001; ns not significant). i Clustering heatmap representing the statistical multiple comparisons of βarr recruitment data for ten different GPCRs. Conditions with overexpressed GRKs were tested against ΔQ-GRK + empty vector (EV) or Control + EV, as indicated. Additionally, ΔQ-GRK + EV as compared to Control + EV. BRET fold changes at saturating ligand concentrations of at least n = 3 independent experiments were compared using ANOVA and two-sided Bonferroni’s test (Supplementary Table 3, data derived from Supplementary Fig. 5). Transformed unadjusted p values are plotted. GPCR–βarr pairs are clustered according to Canberra distance. j Overview of clustering from i in GPCR–βarr pairs regulated by any tested GRK (GRK2/3/5/6 regulated), by GRK2 or 3 only (GRK2/3 regulated) and a third group, which is comprised of GPCR–βarr pairs that do not consistently show significant differences between the tested conditions. All exact p values, test statistics, effect sizes, confidence intervals and degrees of freedom are provided in the Source Data files.