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. 2013 Jun 6;8(6):e65885.
doi: 10.1371/journal.pone.0065885. Print 2013.

Characterization of three vasopressin receptor 2 variants: an apparent polymorphism (V266A) and two loss-of-function mutations (R181C and M311V)

Stephen P Armstrong  1 , Ruth M Seeber, Mohammed Akli Ayoub, Brian J Feldman, Kevin D G Pfleger
Affiliations

Characterization of three vasopressin receptor 2 variants: an apparent polymorphism (V266A) and two loss-of-function mutations (R181C and M311V)

Stephen P Armstrong et al. PLoS One. .

Abstract

Arginine vasopressin (AVP) is released from the posterior pituitary and controls water homeostasis. AVP binding to vasopressin V2 receptors (V2Rs) located on kidney collecting duct epithelial cells triggers activation of Gs proteins, leading to increased cAMP levels, trafficking of aquaporin-2 water channels, and consequent increased water permeability and antidiuresis. Typically, loss-of-function V2R mutations cause nephrogenic diabetes insipidus (NDI), whereas gain-of-function mutations cause nephrogenic syndrome of inappropriate antidiuresis (NSIAD). Here we provide further characterization of two mutant V2Rs, R181C and M311V, reported to cause complete and partial NDI respectively, together with a V266A variant, in a patient diagnosed with NSIAD. Our data in HEK293FT cells revealed that for cAMP accumulation, AVP was about 500- or 30-fold less potent at the R181C and M311V mutants than at the wild-type receptor respectively (and about 4000- and 60-fold in COS7 cells respectively). However, in contrast to wild type V2R, the R181C mutant failed to increase inositol phosphate production, while with the M311V mutant, AVP exhibited only partial agonism in addition to a 37-fold potency decrease. Similar responses were detected in a BRET assay for β-arrestin recruitment, with the R181C receptor unresponsive to AVP, and partial agonism with a 23-fold decrease in potency observed with M311V in both HEK293FT and COS7 cells. Notably, the V266A V2R appeared functionally identical to the wild-type receptor in all assays tested, including cAMP and inositol phosphate accumulation, β-arrestin interaction, and in a BRET assay of receptor ubiquitination. Each receptor was expressed at comparable levels. Hence, the M311V V2R retains greater activity than the R181C mutant, consistent with the milder phenotype of NDI associated with this mutant. Notably, the R181C mutant appears to be a Gs protein-biased receptor incapable of signaling to inositol phosphate or recruiting β-arrestin. The etiology of NSIAD in the patient with V266A V2R remains unknown.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. AVP-induced cAMP accumulation.
Transfected HEK293FT cells (A, B) or COS7 cells (C) expressing either wild-type or mutant HA-tagged V2R (A, C) or Rluc8-tagged V2R (B), were treated for 30 minutes with stimulation buffer containing 0.5 mM IBMX together with the indicated concentrations of AVP or 50 mM forskolin. Following additions, cells were lysed and cAMP accumulation measured by HTRF. Data shown are HTRF signal in arbitrary units (AU) normalized to the forskolin response. Results shown are the mean ± SEM of 3 independent experiments.
Figure 2
Figure 2. AVP-induced IP1 accumulation.
Transfected HEK293FT cells expressing either wild-type or mutant HA-tagged V2R were treated for 30 minutes with the indicated concentrations of AVP. Following additions, cells were lysed and IP1 accumulation measured by HTRF. Data shown are HTRF signal in arbitrary units (AU) normalized to the wild-type receptor response. Results shown are the mean ± SEM of 5 independent experiments.
Figure 3
Figure 3. Effect of V2R mutations on the kinetics of AVP-induced β-arrestin2/Venus recruitment.
Extended BRET kinetic profiles were generated with HEK293FT cells (A, B, C, D) or COS7 cells (E, F, G, H) expressing β-arrestin2/Venus with wild-type, R181C, V266A, or M311V V2R/Rluc8, as indicated. Cells were treated with indicated concentrations of AVP and kinetic BRET responses recorded. Data shown are the mean ± SEM of 3 independent experiments.
Figure 4
Figure 4. Dose-response curves for the effect of V2R mutations on AVP-induced β-arrestin2/Venus recruitment.
BRET dose-response data were generated with HEK293FT cells (A) or COS7 cells (B) expressing β-arrestin2/Venus with wild-type, R181C, V266A, or M311V V2R/Rluc8. Data shown are the ligand-induced BRET response at 60 minutes post AVP treatment, and are the mean ± SEM of 3 independent experiments.
Figure 5
Figure 5. Quantification of HA-V2R cell surface expression by automated imaging.
COS7 cells expressing HA-V2R constructs were stained with anti-HA antibody, initially as live intact cells, prior to image acquisition and analysis using an IN Cell Analyzer 1000. Data shown are an Expression Index defined as the percentage of positive cells (>10% above background) multiplied by their mean fluorescence intensity (A), and are the mean ± SEM of 3 experiments. Representative images of nuclei stained with Hoechst 33258 (B), cell surface HA-V2R stain (C), and automated image segmentation (D) are shown. As illustrated in (D), IN Cell Analyzer software was used to define perimeters of nuclei (blue) and cells (green or red) with a filter to distinguish cells in which staining was >10% above background (green perimeters (1)) or <10% above background (red perimeters (0)). Each of the image panels corresponds to a width of 250 µm and represents approximately 1/10th of the area captured in each field of view.
Figure 6
Figure 6. Effect of V2R mutations on AVP-induced ubiquitin/Venus recruitment.
HEK293FT cells expressing ubiquitin/Venus and wild-type or mutant V2R/Rluc8 constructs were stimulated with the indicated concentrations of AVP for 120 min. Data shown are the mean ± SEM of 4 independent experiments.
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References

    1. Ball SG (2007) Vasopressin and disorders of water balance: the physiology and pathophysiology of vasopressin. Ann Clin Biochem 44: 417–431. - PubMed
    1. Morello J-P, Bichet DG (2001) Nephrogenic Diabetes Insipidus. Annu Rev Physiol 63: 607–630. - PubMed
    1. Sands JM, Bichet DG (2006) Nephrogenic Diabetes Insipidus. Ann Intern Med 144: 186–194. - PubMed
    1. Lolait SJ, O'Carroll A-M, McBride OW, Konig M, Morel A, et al. (1992) Cloning and characterization of a vasopressin V2 receptor and possible link to nephrogenic diabetes insipidus. Nature 357: 336–339. - PubMed
    1. Birnbaumer M, Seibold A, Gilbert S, Ishido M, Barberis C, et al. (1992) Molecular cloning of the receptor for human antidiuretic hormone. Nature 357: 333–335. - PubMed

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This research was funded by the Australian Research Council (www.arc.gov.au) Future Fellowship (FT100100271) and a Priming Grant from the Raine Medical Research Foundation (www.raine.uwa.edu.au), both awarded to KDGP. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.