Fig. 4: GRK2, 3, 5 or 6 can individually mediate a ligand-independent interaction of the V2R and β-arrestin1.

a–d ΔQ-GRK or Control cells were transfected with V2R-Halo-Tag and one of the following β-arrestin1 (βarr1)-NanoLuc fusion constructs: wild type (a, b) or βarr1 lacking the finger loop region (dFLR; c, d). Additionally, either GRK2, 3, 5, 6 or the empty vector (EV) were transfected as indicated. The dynamic BRET changes are shown as ligand concentration-response curves normalised to baseline values and vehicle control. All data points are calculated as Δ net BRET fold change, mean of n = 3 independent experiments ± SEM. The same dataset is presented as bar graphs, displaying the mean BRET values + SEM before (baseline) and after stimulation with 10 µM [Arg⁸]-vasopressin (AVP; stimulated), normalised to the basal BRET ratio derived from the ΔQ-GRK + EV condition (dashed line). To test whether the baseline BRET ratios were significantly elevated compared to the respective ΔQ-GRK + EV baseline, an ANOVA and one-sided Dunnett’s test was performed (*p < 0.05; **p < 0.01; ns not significant; a, b ΔQ-GRK + GRK2 p = 0.0113, ΔQ-GRK + GRK3 p = 0.0142, ΔQ-GRK + GRK5 p = 0.0188, ΔQ-GRK + GRK6 p = 0.0036, Control + EV p = 0.6186; c, d: ΔQ-GRK + GRK2 p = 0.0073, ΔQ-GRK + GRK3 p = 0.0033, ΔQ-GRK + GRK5 p = 0.0024, ΔQ-GRK + GRK6 p = 0.0013, Control + EV p = 0.1054). e, f ΔQ-GRK or Control cells were transfected with V2R-CFP (green), Rab5-mCherry (magenta), βarr1-YFP (not shown) and either EV, GRK2 or GRK6 as indicated. Images were taken before (basal) and after 15 min of 100 nM AVP stimulation. Representative images are shown in (e) and Supplementary Fig. 8e–h. The co-localisation of V2R and βarr1 or Rab5 was quantified using Squassh and SquasshAnalyst (number of images per respective condition; ΔQ-GRK + EV (35), ΔQ-GRK + GRK2 (35), ΔQ-GRK + GRK6 (33), Control + EV (30)). f Data are presented as mean fold change in co-localisation signal + SEM normalised to unstimulated (baseline) ΔQ-GRK + EV condition. Co-localisation prior to stimulation was compared using ANOVA and two-sided Dunnett’s test (*p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001; ns not significant). All exact p values, test statistics, effect sizes, confidence intervals and degrees of freedom are provided in the Source Data files. g Schematic depiction of βarr1 interactions with the V2R in the absence and presence of ligand, facilitated by high expression levels of GRKs.