Fig. 6: Pre-coupling of β-arrestin2 mediated by overexpression of GRK6 results in desensitisation and constitutive internalisation of the AT1R.

a, b ΔQ-GRK cells were transfected with hsp-AT1R-CFP (green), Rab5-mCherry (magenta), βarr2-YFP (not shown) and either EV, GRK2 or GRK6, as noted. Images were taken before (basal) and after 15 min of 100 nM AngII stimulation. Representative images are shown in (a) and Supplementary Fig. 12. The co-localisation of AT1R and βarr2 or Rab5 was quantified using Squassh and SquasshAnalyst (number of images per respective condition; ΔQ-GRK + EV (31), ΔQ-GRK + GRK2 (31), ΔQ-GRK + GRK6 (33)) (b). Data are presented as mean fold change in co-localisation signal + SEM normalised to unstimulated ΔQ-GRK + EV. Co-localisation prior to stimulation was compared using ANOVA and two-sided Dunnett’s test (*p < 0.05; **p < 0.01; ***p < 0.001; ns not significant; basal AT1R co-localisation with βarr2: ΔQ-GRK + GRK2 p = 0.812, ΔQ-GRK + GRK6 p = 0.027; basal AT1R co-localisation with Rab5: ΔQ-GRK + GRK2 p = 0.7649, ΔQ-GRK + GRK6 p = 0.0008). c ΔQ-GRK cells transiently transfected with hsp-AT1R-CFP, with or without co-transfection of either βarr2 alone or in combination with GRK2 or GRK6, were stimulated with AngII and real-time dynamic mass redistribution (DMR) responses were recorded as a measure of AT1R activity. DMR recordings are shown as mean + SEM of three technical replicates from a single experiment, representative of n = 3 independent experiments. d Concentration-effect curves derived from n = 3 independent experiments (representatively shown in (c)) ± SEM are plotted from the area under the curve (AUC) within 0 and 1800 s. Data are normalised to the picometre wavelength shifts evoked with 100 nM AngII in cells expressing hsp-AT1R-CFP alone. Statistical significance was calculated using a two-way ANOVA and two-sided Tukey’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.