Vasopressin increases S261 phosphorylation in AQP2-P262L, a mutant in recessive nephrogenic diabetes insipidus.
BACKGROUND
Mutations in the aquaporin-2 (AQP2) gene cause nephrogenic diabetes insipidus (NDI), a renal disorder characterized by polyuria due to a lacking antidiuretic response to vasopressin. While most AQP2 mutants in recessive NDI are misfolded and retained in the endoplasmic reticulum, AQP2-P262L in NDI was impaired in its vasopressin-dependent translocation from vesicles to the plasma membrane.


METHODS
Vasopressin-induced translocation of AQP2 coincides with AQP2 phosphorylation at S256, S264 and T269 and dephosphorylation at S261. Since P262 lies adjacent to S261, we tested whether a changed phosphorylation could underlie AQP-P262L missorting in NDI.


RESULTS
In polarized cells, AQP2-P262L expressed as a double 29/30 kDa band, whereas wt-AQP2 expressed only as a 29 kDa band. Phosphatase treatment revealed that the 30 kDa AQP2-P262L band was due to changed phosphorylation. The use of newly developed phospho-specific antibodies showed that forskolin not only increased pS256 and pT269, but, in contrast to wt-AQP2, also pS261 in AQP2-P262L. The expression of AQP2-P262L proteins in which S261 phosphorylation was prevented (S261A), however, was still missorted to vesicles/basolateral membrane, despite the absence of the 30 kDa band.


CONCLUSIONS
Together, our data reveal that vasopressin induces instead of reduces the phosphorylation of S261 in AQP2-P262L, but it remains to be established whether the changed phosphorylation causes its missorting in NDI.
Department of Physiology (286), RUNMC Nijmegen, Radboud University Nijmegen Medical Centre, Nijmegen, the Netherlands.
Christiane
C
Trimpert
Dennis T M
DT
van den Berg
Robert A
RA
Fenton
Enno
E
Klussmann
Peter M T
PM
Deen
D000818:Animals; D051399:Aquaporin 2 / Q000235:genetics* / Q000378:metabolism*; D002478:Cells, Cultured; D018500:Diabetes Insipidus, Nephrogenic / Q000235:genetics*; D005808:Genes, Recessive; D009154:Mutation; D010766:Phosphorylation; D014667:Vasopressins / Q000502:physiology*
D051399:Aquaporin 2; D014667:Vasopressins