Novel signaling pathways in Nephrogenic Syndrome of Inappropriate Antidiuresis: Functional implication of site-specific AQP2 phosphorylation.

Nephrogenic Syndrome of Inappropriate Antidiuresis (NSIAD) is a rare X-linked disease caused by gain-of-function mutations of the V2 vasopressin receptor (V2R). NSIAD patients are characterized by the inability to excrete a free water load and inappropriately increased urinary osmolality despite very low levels of plasma vasopressin resulting in euvolemic hyponatremia. To dissect the signaling downstream V2R constitutively active variants, Flp-In T-REx Madin-Darby canine kidney (FTM) cells, stably transfected with V2R mutants (R137L, R137C, and F229V) and AQP2-wt or non-phosphorylatable AQP2-S269A/AQP2-S256A, were used as cellular models. All three activating V2R mutations presented constitutive plasma membrane expression of AQP2-wt and significantly higher basal water permeability. In addition, V2R-R137L/C showed significantly higher activity of ROCK, a serine/threonine kinase previously suggested to be involved in S269-AQP2 phosphorylation downstream of these V2R mutants. Interestingly, FTM cells expressing V2R-R137L/C mutants and AQP2-S269A showed a significant reduction in AQP2 membrane abundance and a significant reduction in ROCK activity pointing to the crucial importance of S269-AQP2 phosphorylation in the gain-of-function phenotype. Conversely, V2R-R137L/C mutants retained the gain-of-function phenotype when AQP2-S256A was co-expressed. In contrast, cells expressing the F229V mutant and the non-phosphorylatable AQP2-S256A had a significant reduction in AQP2 membrane abundance along with a significant reduction in basal osmotic water permeability pointing to a crucial role of the ser-256 for this mutant. These data indicate that the constitutive AQP2 trafficking associated with the gain of function V2R-R137L/C mutants causing NSIAD is PKA-independent and requires an intact ser-269 in the AQP2 protein under the control of ROCK phosphorylation. KEY POINTS: NSIAD is caused by two constitutively active variant phenotypes of AVPR2, one sensitive to vaptans (V2R-F229V) and the other, vaptan resistant (V2R-R137C/L). In renal cells, all three activating V2R variants display constitutive AQP2 plasma membrane expression and high basal water permeability. In cells expressing V2R-R137L/C mutants, disruption of the AQP2-S269 phosphorylation site caused the loss of the gain-of-function phenotype, which in contrast, was retained in V2R-F229V expressing cells. Cells expressing the V2R-F229V mutant were instead sensitive to disruption of the AQP2-S256 phosphorylation site. The serine/threonine kinase ROCK was found involved in AQP2-S269 phosphorylation downstream of the V2R-R137L/C mutants. These findings may have clinical relevance for NSIAD patients. Abstract figure legend: Under physiological conditions, vasopressin binds to the type-2 receptor (V2R-WT) in principal cells of the collecting duct. Binding to the receptor activates the Gs protein leading to production of intracellular cyclic adenosine monophosphate (cAMP) as a consequence of activation of the adenylate cyclase. This pathway causes phosphorylation of the aquaporin-2 (AQP2) at S256 resulting in AQP2 insertion on the luminal plasma membrane. In cells expressing V2R-F229, AQP2 is constitutively expressed in the luminal membrane in the absence of vasopressin stimulation. Cells expressing the constitutively active AQP2-F229V mutant and the non-phosphorylatable AQP2-S256A, lost their gain-of-function phenotype indicating this mutant requires an intact S256 phosphorylation site for translocation. In cells expressing V2R-R137L/C the receptor is instead coupled to Gα12/13 causing activation of the serine/threonine kinase ROCK which is involved in S269-AQP2 phosphorylation. Disruption of the S269 phosphorylation site prevented the constitutive trafficking of AQP2 on the luminal plasma membrane, indicating that the gain-of-function phenotype depends on AQP2 phosphorylation at S269. This article is protected by copyright. All rights reserved.