Fig. 4: Structure of the arrestin2-V2Rpp-3 complex and the conformational change related to MEK1 interaction.

a The loss of pS357 of V2Rpp-3 caused rotamer alterations in the side chains of R165, K138, and K160 of the V3 binding pocket. 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-3 complex (forest) with the arrestin2-V2Rpp-FP complex (PDB: 4JQI, light cyan) and inactive arrestin2 (PDB:1G4M, gray). F244TMSiPhe and K357TMSiPhe were marked as pink ball and blue ball, respectively. c Conformational changes occurred at the C loop (F243-T246), which is known to be involved in receptor binding. d Conformational changes in the region involved in the MEK1 interaction (K355-P361). e 1D ¹HNMR spectra of arrestin2-F244TMSiPhe in response to incubation with different phosphopeptides. Corresponding NMR shifts at 0.091 ppm, 0.026 ppm, and 0.019 ppm were assigned to the R0, R1, and R2 states, respectively. f 1D ¹HNMR spectra of arrestin2-K357TMSiPhe in response to incubation with different phosphopeptides. Corresponding NMR shifts at 0.055 ppm, 0.170 ppm, 0.180 ppm, and 0.026 ppm were designated M0, M1, M2, and M3, respectively. g Schematic representation of the experimental design used to monitor vasopressin-induced MEK1 recruitment to arrestin2 downstream of V2R by FlAsH-BRET between arrestin2-Rluc and MEK1-FlAsH. The blue ball: K357 site in arrestin2; Yellow pentagram: the position of FlAsH labeled in MEK1. The yellow circles: phosphorylation. h Vasopressin-induced recruitment of MEK1 to arrestin2 in HEK293 cells overexpressing wild-type V2R or different mutants. Data are expressed as ΔBRET ratio relative values and represent 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-3, V2Rpp-4, V2Rpp-6-7 mutants; #P < 0.05; ##P < 0.01; ###P < 0.001; ns no significant difference. Source data are provided in the Source Data file.