2004 Global Researcher Conference Proceeding

April 09 - 11, 2004

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Conference: 2004 Global Researcher Conference
Title: Lack of AVP-induced phosphorylation of the Aquaporin-2 mutants AQP2-R254L and AQP2-R254Q explains dominant Nephrogenic Diabetes Insipidus
Authors: de Mattia, Fabrizio; Savelkoul, Paul J.M.; Oksche, Alexander; Robertson, Gary; Deen, Peter M.T.
Institutions: University of Nijmegen, NHGRI, Institut Fuer Pharmakologie, Northwestern University Medical School
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deMattia Vasopressin regulates the human water homeostasis by translocating vesicles containing homotetrameric Aquaporin-2 (AQP2) water channels to and from the apical membrane of renal principal cells, a process in which phosphorylation of AQP2 at S256 by cAMP-dependent protein kinase A (PKA) is thought to be essential. Mutations in the AQP2 gene cause recessive and dominant Nephrogenic Diabetes Insipidus (NDI), a disease in which the kidney is unable to concentrate urine in response to vasopressin. Here, we identified two families, in which dominant NDI was caused by R254L and R254Q mutations in AQP2, which destroy the PKA consensus site. Expressed in oocytes, both mutants appeared functional water channels, but were impaired in their transport to the cell surface to the same degree as AQP2-S256A, which mimics non-phosphorylated AQP2. In polarized MDCK cells, they were retained in intracellular vesicles in a distribution pattern that was similar to that of unstimulated wt-AQP2 or AQP2-S256A. In contrast to wild-type AQP2, their level of S256 phosphorylation was not increased upon forskolin treatment, which coincided with an unchanged subcellular localization. Upon co-expression, both mutants interacted with and retained wt-AQP2 in intracellular vesicles. These data indicated that in these families, dominant NDI is caused by the inability to phosphorylate the mutants at S256 by PKA and, as such, they represent the first AQP2 mutants in which dominant NDI is caused by a loss of function instead of a gain of function mutation. Since they mimic non-phosphorylated AQP2, the wt/mutant AQP2 complexes in the patients are, as found for wt-AQP2 though to a lesser extent, also expected to cycle to the apical membrane, which provides an explanation for the significant increase in urine concentrating ability in the NDI patient upon long-term dehydration or restored water intake ad libitum and short term dDAVP administration. These data indicate that a balanced dDAVP treatment might relieve NDI in these patients. These results provide the first in vivo evidence of the importance of S256 phosphorylation for AQP2 translocation and underscore the relevance of the identification of the molecular cause of a disease in order to provide a rationally-based treatment to the patient.

Phosphorylation is the process wherein a phosphate group is added onto an organic molecule. On the aquaporin 2 (AQP2) protein, phosphorylation occurs at Ser256, a serine amino acid that is the 256th amino acid on the chain of amino acids that makes up the AQP2. When phosphorylation occurs at Ser256, it induces the AQP2 to translocate from the cell interior to the cell membrane, a movement necessary for the kidney to concentrate urine. This phosphorylation process is accomplished by protein kinase A (PKA), which recognizes a PKA consensus site located in the section of the AQP2 called the C-tail.

Here, De Mattia, et al., identified two families, in which dominant NDI was caused by R254L and R254Q mutations in AQP2, which destroy the PKA consensus site. These AQP2 mutants are functional water channels, but are unable to travel to the cell membrane. Instead, they are retained in intracellular vesicles in a distribution pattern that was similar to that of non-phosphorylated normal AQP2 (wt-AQP2). Although these AQP2 mutants were phosphorylated at a low basal level at Ser256, their level of phosphorylation was not increased upon forskolin treatment, and the forskolin did not induce the mutant AQP2s to move to the cell membrane. Upon co-expression, both mutants interacted with and retained wt-AQP2 in intracellular vesicles.

These data indicated that in these families, dominant NDI is caused by the inability to phosphorylate the mutants at S256 by PKA and, as such, they represent the first AQP2 mutants in which dominant NDI is caused by a loss of function instead of a gain of function.