Fig. 3: Structure of the arrestin2-V2Rpp-1 complex and correlation of phospho-binding at the V1 site with the conformational change related to the c-Raf-1 interaction.

a Structural comparisons of residues near the V1 site in the structure of the arrestin2-V2Rpp-1 complex (salmon) and arrestin2-V2Rpp-FP (PDB: 4JQI, light cyan). Residues F61, Y63, and L243 underwent obvious structural rearrangement. The orange balls indicate that the phosphorylated peptide remains bound to arrestin, as observed in the previously solved arrestin2-V2Rpp-FP complex structure, whereas the green balls indicate loss of the original interaction of the phospho-residue. b Overall structural comparisons of the arrestin2-V2Rpp-1 complex (salmon), arrestin2-V2Rpp-FP complex (PDB: 4JQI, light cyan) and inactive arrestin2 (PDB:1G4M, gray). R307TMSiPhe was marked as red ball. c Comparisons of conformational changes at the back loop in the arrestin2-V2Rpp-1 complex with the inactive arrestin2 and arrestin2-V2Rpp-FP complex. The back loop experienced conformational rearrangement by approximately 10° rotation in the arrestin2-V2Rpp-1 complex compared to inactive arrestin2. d The loop at the region from S193 to K195 underwent significant structural rearrangement in arrestin2-V2Rpp-1 compared to that in the arrestin2-V2Rpp-FP complex structure. e 1D ¹H NMR spectra of arrestin2-R307TMSiPhe in response to incubation with different phosphopeptides or control vehicles. The corresponding NMR shifts at 0.116, 0.195, and 0.219 ppm are designated S0, S1, and S2, respectively. f Schematic representation of the experimental design used to monitor vasopressin-induced c-Raf-1 recruitment to arrestin2 downstream of V2R. The interaction between arrestin2 and c-Raf-1 was measured by the FlAsH-BRET method. The red ball: R307 site in arrestin2; Yellow pentagram: the position of FlAsH labeled in c-Raf-1. g Vasopressin-induced recruitment of c-Raf-1 to arrestin2 in HEK293 cells overexpressing wild-type V2R or different mutants. Data are expressed as relative values of ΔBRET ratio with the mean ± SEM of three independent experiments (n = 3). Statistical differences were determined by two-sided one-way ANOVA with Tukey’s test. * on behalf of differences between V2R-WT and mutants; *P < 0.05; **P < 0.01; ***P < 0.001; & on behalf of differences between V2R-1 and V2Rpp-4, V2Rpp-6-7 mutants; &P < 0.05; &&P < 0.01; &&&P < 0.001; # on behalf of differences between V2R-3 and V2Rpp-4, V2Rpp-6-7 mutants; #P < 0.05; ##P < 0.01; ###P < 0.001; ns no significant difference. Equal surface expression of all V2R constructs was achieved by adjusting the amounts of transfected plasmids and monitored by ELISA. Source data are provided in the Source. Data file.