Fig. 4: Differential GPCR phosphorylation by cytosolic and membrane-bound GRK isoforms induces distinct β-arrestin interactions with (chimeric) class A and B GPCRs.

a–h Measurements of proximal and distal C-terminal phosphorylation in ΔQ-GRK stably expressing b2AR (a, e), V2b2 (b, f), b2V2 (c, g) or V2R (d, h) and GRK2-YFP or GRK6-YFP, analogously to Fig. 2c–j. Data are shown as optical density (OD) at 405 nm ±SEM of n = 5 independent experiments, normalized to the maximum ligand concentration for each receptor in Control cells, respectively. Statistical differences between measurements in ΔQ-GRK + GRK2, ΔQ-GRK + GRK6 or Control cells were compared using a two-way analysis of variance (ANOVA), followed by a Tukey’s test for vehicle or highest stimulating concentrations (*p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001; ns not significant). Complete results of the statistical analysis can be accessed in Supplementary Table 2. i–l Fingerprint of β-arrestin2 conformational change sensors measured in ΔQ-GRK cells, individually overexpressing GRK2 or GRK6, in presence of untagged b2AR (i), V2b2 (j), b2V2 (k) or V2R (l). The Δ net BRET changes at 10 µM Iso (i, k) or 3 µM AVP (j, l) are shown as Δ net BRET change (%) in radar plots, analogously to Fig. 2s–v. Sensor conditions, which did not fulfill the set pharmacological parameters (Hill slope and EC₅₀ analysis as described in methods) were classified as non-responding conditions and assigned zero. m, n The Δ net BRET changes (%) at highest stimulating ligand concentrations for each FlAsH position were clustered for all receptor–β-arrestin pairs in presence of GRK2 (m) or GRK6 (n) according to Manhattan distance. GPCR transmembrane helix bundle or C-terminus identity is indicated by colored bar (b2AR in orange, V2R in blue). Source data are provided as a Source Data file.