Fig 1.
The OPC5 analogues activate Gs-cAMP pathway but not β-arrestin pathway.
(A) Structure of OPC5 and common skeleton of OPC5 analogues. (B) Concentration-response curve for cAMP accumulation upon the OPC analogues stimulation. For each experiment, ligand-induced cAMP responses were normalized to the response induced by 100 nM AVP. Symbols and error bars are mean and SEM, respectively, of three independent experiments with each performed in duplicate. AVP, OPC5, OPC16b, OPC16g, OPC16j, OPC19a, OPC19b, OPC23b, OPC23d, OPC23h and OPC23i are colored in black, red, magenta, light green, blue, orange, purple, cyan, yellow, green and brown, respectively. (C and D) Concentration-response curve for β-arrestin1 (C) and β-arrestin2 (D) recruitment upon the OPC5 analogues stimulation. For each experiment, ligand-induced β-arrestin recruitment was normalized to the response induced by 1 μM AVP. Symbols and error bars are mean and SEM, respectively, of three independent experiments with each performed in duplicate. (E) Heatmap representation of cAMP response and β-arrestin1/2 recruitment induced by AVP and the OPC analogues. Data are presented as Emax of each experiment and ligand-induced cAMP responses and β-arrestin1/2 recruitment were normalized to the response induced by 100 nM or 1 μM AVP, respectively.
Fig 2.
OPC5 analogues have less V2R internalizing activity than AVP.
(A) Schematic representation of the HiBiT-based luciferase-fragment complementation assay. The amount of plasma membrane receptor was assessed by reconstituting the luciferase with membrane-impermeable LgBiT. Agonist-induced internalization of the receptor results in a decline of luminescence signals. (B) Measurement of cell-surface expression levels with a titrated amount of wild-type V2R (0.04–25 ng). (C) Measurement of cell-surface expression levels of wild-type V2R. For each experiment, cell-surface expression levels were normalized to the control receptor. Bars and error bars are mean and SEM, respectively, of three independent experiments with each performed in duplicate. For the statistical analyses, data were analyzed by paired t-test. *P < 0.05, **P < 0.01, ***P < 0.001. (D) Measurement of cell-surface expression levels of wild-type V2R upon AVP or the OPC5 analogues stimulation for 20 hours. For each experiment, cell-surface expression levels were normalized to the expression levels upon vehicle stimulation. Bars and error bars are mean and SEM, respectively, of three independent experiments with each performed in duplicate. For the statistical analyses, data were analyzed by one-way ANOVA followed by the Dunnett’s test for multiple comparison analysis. *P < 0.05, **P < 0.01, ***P < 0.001 vs. AVP; # P < 0.05, ##P < 0.01, ###P < 0.001 vs. vehicle.
Fig 3.
V2R mutants impair prolonged increase of cAMP levels.
(A) Concentration-response curve for cAMP accumulation induced by wild-type and the six mutant V2Rs activation upon AVP stimulation. For each experiment, ligand-induced cAMP responses were normalized to the response induced by 1 μM AVP at wild-type V2R. WT, V882.53M, Y1283.41S, L1614.47P, T2736.37M, S3298.47R and S3338.51del are colored in red, blue, orange, purple, green and brown, respectively. Symbols and error bars are mean and SEM, respectively, of three independent experiments with each performed in duplicate. (B) pEC50 values for wild-type and six mutant V2Rs for AVP. Symbols and error bars are mean and SEM, respectively, of three independent experiments with each performed in duplicate. ***P < 0.001 vs. WT. (C-D) Relative cAMP levels for wild-type and the six mutant V2Rs after 15 min (C) and 20 hours (D) stimulation of AVP. Bars and error bars are mean and SEM, respectively, of three independent experiments with each performed in duplicate. ***P < 0.001 vs. WT.
Fig 4.
OPC5 analogues cause sustained cAMP signaling in V2R mutants with low expression levels.
Measurement of cAMP levels of wild-type and the six mutant V2Rs upon AVP and OPC5 analogues stimulation for 20 hours. For each experiment, ligand-induced cAMP responses were normalized to the response induced by AVP. Bars and error bars are mean and SEM, respectively, of six independent experiments with each performed duplicate. Data were analyzed by one-way ANOVA followed by the Dunnett’s test for multiple comparison analysis. *P < 0.05, **P < 0.01, ***P < 0.001 vs. AVP.
Fig 5.
Structural comparison of the AVP-bound V2R and OPC16g-docked V2R structure.
(A) Overall structure of the AVP-bound V2R-Gs structure (PDB ID: 7DW9) (left panel) and OPC16g-docked V2R structure (right panel). V2R, AVP and OPC16g are colored in gray, cyan and green, respectively. V882.53, Y1283.41, L1614.47, T2736.37, S3298.47 and S3338.51 are colored in red, blue, orange, purple, green and brown, respectively. (B and C) Conformational comparison of ligand binding pocket of AVP-bound V2R and OPC16g-docked V2R. Residues consisting of a hydrophobic cleft (B) and the hydrogen bond with L3127.40 of V2R (C) were shown as sticks.