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Results: 8

1.
Fig. 6.

Fig. 6. From: Functional Characterization of Vasopressin Type 2 Receptor Substitutions (R137H/C/L) Leading to Nephrogenic Diabetes Insipidus and Nephrogenic Syndrome of Inappropriate Antidiuresis: Implications for Treatments.

Effects of the R137C-V2R and R137L-V2R substitutions on receptor maturation profile. HEK293 cells transfected with the indicated receptors were incubated in the absence (top) or presence (bottom) of SR121463 (10 μM) for 16 h. Cells extracts were then resolved by SDS-PAGE, and receptors were visualized by Western blotting using an anti-FLAG antibody. Arrowheads indicate the fully glycosylated mature form of V2R. Data shown are representative of three to four experiments.

Moulay D. Rochdi, et al. Mol Pharmacol. 2010 May;77(5):836-845.
2.
Fig. 7.

Fig. 7. From: Functional Characterization of Vasopressin Type 2 Receptor Substitutions (R137H/C/L) Leading to Nephrogenic Diabetes Insipidus and Nephrogenic Syndrome of Inappropriate Antidiuresis: Implications for Treatments.

Effect of V2R inverse agonist SR121463 on the constitutive cAMP production and constitutive endocytosis promoted by R137C-V2R and R137L-V2R. A, HEK293 cells were transfected with the indicated constructs. Forty-eight hours later, cells were incubated for another 16 h in the absence or presence of SR121463 (10 μM), and basal cAMP production was measured by using a HTRF-based technology as described under Materials and Methods. B, HEK293 cells stably expressing β2-adaptin-EYFP were transfected with the β-arrestin2-Rluc and the indicated receptor constructs. Cells were subsequently incubated for 16 h in the presence or absence of SR121463 (10 μM), and BRET1 was measured as described under Materials and Methods.

Moulay D. Rochdi, et al. Mol Pharmacol. 2010 May;77(5):836-845.
3.
Fig. 5.

Fig. 5. From: Functional Characterization of Vasopressin Type 2 Receptor Substitutions (R137H/C/L) Leading to Nephrogenic Diabetes Insipidus and Nephrogenic Syndrome of Inappropriate Antidiuresis: Implications for Treatments.

V2R-promoted MAPK activation. AVP-induced MAPK activation was assessed in HEK293 cells transfected with the indicated V2R constructs. After 5-min incubation in the presence (+) or absence (−) of AVP (1 μM), cells were lysed in Laemmli sample buffer and extracts were resolved by SDS-PAGE. MAPK activity was detected by Western blot using a phosphospecific anti-ERK1/2 antibody (P-ERK1/2). Expression level of ERK1/2 was controlled by using an antibody directed against the total kinase population (ERK1/2). Data are expressed as the percentage of P-ERK/ERK of the level observed for AVP-stimulated WT-V2R conditions. Data represent the mean ± S.E.M. of at least three independent experiments. The Western blots shown are representative of three independent experiments.

Moulay D. Rochdi, et al. Mol Pharmacol. 2010 May;77(5):836-845.
4.
Fig. 1.

Fig. 1. From: Functional Characterization of Vasopressin Type 2 Receptor Substitutions (R137H/C/L) Leading to Nephrogenic Diabetes Insipidus and Nephrogenic Syndrome of Inappropriate Antidiuresis: Implications for Treatments.

Cell surface expression of WT-V2R, R137H-V2R, R137C-V2R, and R137L-V2R and their constitutive internalization. A, HEK293 cells, transiently expressing the indicated V2R constructs along with the overexpression of DynK44A or an empty vector (pcDNA3), were assessed for receptor surface expression by ELISA using an anti-FLAG antibody as described under Materials and Methods. B, HEK293 cells stably expressing the β2-adaptin-EYFP subunit of the AP-2 complex were transfected with the indicated V2R constructs and β-arrestin2-Rluc, with or without DynK44A overexpression. β-Arrestin2 and AP-2 interaction was monitored by BRET. Data shown are the mean ± S.E.M. of three to five independent experiments. Statistical significance of the difference was assessed by paired Student's t test. ***, p < 0.001; **, p < 0.01.

Moulay D. Rochdi, et al. Mol Pharmacol. 2010 May;77(5):836-845.
5.
Fig. 8.

Fig. 8. From: Functional Characterization of Vasopressin Type 2 Receptor Substitutions (R137H/C/L) Leading to Nephrogenic Diabetes Insipidus and Nephrogenic Syndrome of Inappropriate Antidiuresis: Implications for Treatments.

Combined effects of misfolding and constitutive internalization on the constitutive activity of R137C-V2R and R137L-V2R. HEK293 cells were transfected with the indicated V2R construct and either DynK44A or a control plasmid. Cells were then incubated in the presence or absence of SR121463 (10 μM) for 16 h, and basal cAMP production was measured by using HTRF-based technology. Data shown are the mean ± S.E.M. of three to five independent experiments. Statistical significance of the difference was assessed by using paired Student's t test. ***, p < 0.001; **, p < 0.01. One-way ANOVA combined with the Tukey's multiple comparison test was also performed. ¤¤¤, p < 0.001; ¤¤, p < 0.01.

Moulay D. Rochdi, et al. Mol Pharmacol. 2010 May;77(5):836-845.
6.
Fig. 3.

Fig. 3. From: Functional Characterization of Vasopressin Type 2 Receptor Substitutions (R137H/C/L) Leading to Nephrogenic Diabetes Insipidus and Nephrogenic Syndrome of Inappropriate Antidiuresis: Implications for Treatments.

Constitutive cAMP signaling of the mutant V2Rs. A, HEK293 cells were cotransfected with the indicated V2R and the CRE-luciferase reporter constructs, and lysates were assayed for luciferase 24 h later. In each experiment, reporter gene activity was measured in three replicate wells, and the average value from cells expressing R137L-V2R, R137C-V2R, or R137H-V2R mutant was normalized to the average value from cells expressing WT-V2R. The bars indicate the mean of normalized luciferase activity across three independent experiments. B, HEK293 cells were cotransfected with the indicated receptors and either the DynK44A construct or a control plasmid (pcDNA3). cAMP production was measured by using a HTRF-based technology as described under Materials and Methods. C, stably transfected HEK293 cell clones expressing FLAG-tagged WT-V2R or R137L-V2R, which were matched for receptor expression (see Supplementary Fig. 2), were transfected with the CRE-luciferase reporter construct, and lysates were assayed for luciferase activity 24 h later. In each experiment, reporter gene activity was measured in three replicate wells, and the average value from cells expressing R137L-V2R mutant was normalized to the average value from cells expressing WT-V2R. Data shown are the mean ± S.E.M. of three to five independent experiments. Statistical significance of the difference was assessed by using paired Student's t test. ***, p < 0.001; *, p < 0.05. One-way ANOVA combined with Tukey's multiple comparison test was also performed. ¤¤, p < 0.01; ¤, p < 0.05.

Moulay D. Rochdi, et al. Mol Pharmacol. 2010 May;77(5):836-845.
7.
Fig. 2.

Fig. 2. From: Functional Characterization of Vasopressin Type 2 Receptor Substitutions (R137H/C/L) Leading to Nephrogenic Diabetes Insipidus and Nephrogenic Syndrome of Inappropriate Antidiuresis: Implications for Treatments.

Agonist-induced internalization of wild-type and mutant V2R assessed by fluorescence microscopy, cell surface ELISA, and BRET. A, HEK293 cells transiently expressing the FLAG-tagged WT-V2R (a and b), R137L-V2R (c and d), R137C-V2R (e and f), or R137H-V2R (g and h) were surface-labeled with anti-FLAG monoclonal antibody as described under Materials and Methods and incubated in the absence (a, c, e, and g) or presence (b, d, f, and h) of 10 μM AVP for 30 min before fixation. Representative epifluorescence images are shown. B, HEK293 cells were transfected with the indicated constructs, and receptor internalization was determined by measuring cell surface receptor expression after the incubation of the transfected cells in the absence or presence of AVP (1 μM) using the cell surface ELISA and anti-FLAG antibody. C, HEK293 cells stably expressing the EYFP-fused β2-adaptin subunit of the AP-2 complex were transfected with the indicated V2R constructs and β-arrestin2-Rluc. BRET1 measurements of the β-arrestin2/AP-2 biosensor were done after 20-min incubation with or without AVP (1 μM). Data shown are the mean ± S.E.M. of three to five independent experiments. Statistical significance of the difference was assessed by paired Student's t test. ***, p < 0.001. One-way ANOVA combined with Tukey's multiple comparison test was also performed. ¤¤, p < 0.01.

Moulay D. Rochdi, et al. Mol Pharmacol. 2010 May;77(5):836-845.
8.
Fig. 4.

Fig. 4. From: Functional Characterization of Vasopressin Type 2 Receptor Substitutions (R137H/C/L) Leading to Nephrogenic Diabetes Insipidus and Nephrogenic Syndrome of Inappropriate Antidiuresis: Implications for Treatments.

AVP-dependent signaling and G protein coupling estimated by cAMP production measurement and potassium channel currents in oocytes. A, HEK293 cells were transfected with WT or mutant V2R constructs, as indicated. Agonist-induced cAMP production was assessed after 15-min incubation in the presence or absence of AVP (1 μM) using a HTRF-based method as described under Materials and Methods. B, HEK293 cells stably expressing the indicated V2R were transfected with the CRE-luciferase reporter construct, incubated in the absence or presence of 1 μM AVP for 24 h, and lysed for luciferase activity measurements. In each experiment, triplicate wells were assayed for each condition and the luminescence units measured from AVP-exposed cells were normalized to the WT-V2R expressing cells not exposed to AVP. Data shown are the mean ± S.E.M. from three independent experiments. C, oocytes were injected with 4 ng of cRNA of either WT-V2R or the R137L-V2R mutant along with 1 ng each of Gαs, GIRK1, and GIRK4 cRNA. Current amplitude produced by the application of AVP was then measured. The inset shows a representative trace of AVP evoked current in an oocyte injected with either WT-V2R cRNA (thick gray trace) or mutant R137L-V2R cRNA (thin black trace). The horizontal bar above the traces indicates AVP application. Data shown are the mean ± S.E.M. of three to five independent experiments. Statistical significance of the difference was assessed by using one-way ANOVA combined with Tukey's multiple comparison test. ¤¤, p < 0.01.

Moulay D. Rochdi, et al. Mol Pharmacol. 2010 May;77(5):836-845.

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