Supplementary Figure 9: Agonist-stimulated BRET signals and Phos-tag analysis of carboxyl-terminal V1bR mutants.

(A) Agonist increased BRET signal between μ-receptor-Luc and β-arrestin 2-Venus. In one-way ANOVA, drug treatments significantly increased the BRET signal in V1aR-transfected cells (F_2,15 = 6.356, P = 0.01) and in μ-receptor-transfected cells (F_2,15 = 30.65, P < 0.0001). Multiple comparisons with Tukey’s method detected significant difference; V1a-transfected cells, control vs. 1 μM DAMGO, P = 0.0222, control vs. 1 μM endomorphin, P = 0.0169; μ-receptor-transfected cells, control vs. DAMGO, P < 0.0001, control vs. endomorphin, P = 0.0002. *P < 0.05, ***P < 0.001. (n = 6 independent experiments). (B) The phosphorylated form was not detected in the deletion mutant V1bRs lacking the entire C-terminal region. Phos-tag SDS-PAGE and western blot detection of wild-type V1bR showed two bands, which correspond to the phosphorylated and unphosphorylated forms. Two consecutive cysteine residues, Cys³⁵² and Cys³⁵³, in the V1bR C-terminus correspond to putative palmitoylation sites ‡. Residues were removed from the C-terminus up to Cys³⁵² or Cys³⁵³, and termed V1bR-Ala³⁵¹-stop or V1bR-Cys³⁵²-stop, respectively. Both of the mutant V1bRs showed a single population of unphosphorylated forms in Phos-tag analysis. The data are representative from three experiments. V1b-p indicates the phosphorylated form of V1bR. Graphs represent the mean ± S.E.M. Dots represent data from individual data points. Immunoblot in B are cropped; full image is shown in Supplementary Fig. 14. ‡ Qanbar, R. & Bouvier, M. Role of palmitoylation/depalmitoylation reactions in G-protein-coupled receptor function. Pharmacol. Ther. 97, 1-33 (2003).