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

Armstrong SP, Seeber RM, Ayoub MA, Feldman BJ, Pfleger KD - PLoS ONE (2013)

Bottom Line: 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.Hence, the M311V V2R retains greater activity than the R181C mutant, consistent with the milder phenotype of NDI associated with this mutant.

View Article: PubMed Central - PubMed

Affiliation: Laboratory for Molecular Endocrinology-G Protein-Coupled Receptors, Western Australian Institute for Medical Research and Centre for Medical Research, The University of Western Australia, Nedlands, Perth, Western Australia, Australia.

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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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.
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pone-0065885-g002: 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.

Mentions: Transfected HEK293FT or COS7 cells were treated as per Figure 1 or 2. Data shown are the pEC50 values for AVP treatment (30 min) and are the mean ± SEM of 3 or 5 independent experiments for cAMP and IP1 respectively. Fold change from the wild-type receptor EC50 data are given in adjacent columns.


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

Armstrong SP, Seeber RM, Ayoub MA, Feldman BJ, Pfleger KD - PLoS ONE (2013)

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.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3675069&req=5

pone-0065885-g002: 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.
Mentions: Transfected HEK293FT or COS7 cells were treated as per Figure 1 or 2. Data shown are the pEC50 values for AVP treatment (30 min) and are the mean ± SEM of 3 or 5 independent experiments for cAMP and IP1 respectively. Fold change from the wild-type receptor EC50 data are given in adjacent columns.

Bottom Line: 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.Hence, the M311V V2R retains greater activity than the R181C mutant, consistent with the milder phenotype of NDI associated with this mutant.

View Article: PubMed Central - PubMed

Affiliation: Laboratory for Molecular Endocrinology-G Protein-Coupled Receptors, Western Australian Institute for Medical Research and Centre for Medical Research, The University of Western Australia, Nedlands, Perth, Western Australia, Australia.

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.

Show MeSH
Related in: MedlinePlus