Pflügers Archiv - European Journal of Physiology

, Volume 455, Issue 6, pp 1041–1054 | Cite as

Missorting of the Aquaporin-2 mutant E258K to multivesicular bodies/lysosomes in dominant NDI is associated with its monoubiquitination and increased phosphorylation by PKC but is due to the loss of E258

  • Erik-Jan Kamsteeg
  • Paul J. M. Savelkoul
  • Giel Hendriks
  • Irene B. M. Konings
  • Nicole M. I. Nivillac
  • Anne Karine Lagendijk
  • Peter van der Sluijs
  • Peter M. T. Deen
Cell and Molecular Physiology


To stimulate renal water reabsorption, vasopressin induces phosphorylation of Aquaporin-2 (AQP2) water channels at S256 and their redistribution from vesicles to the apical membrane, whereas vasopressin removal results in AQP2 ubiquitination at K270 and its internalization to multivesicular bodies (MVB). AQP2-E258K causes dominant nephrogenic diabetes insipidus (NDI), but its subcellular location is unclear, and the molecular reason for its involvement in dominant NDI is unknown. To unravel these, AQP2-E258K was studied in transfected polarized Madin–Darby canine kidney (MDCK) cells. In MDCK cells, AQP2-E258K mainly localized to MVB/lysosomes (Lys). Upon coexpression, wild-type (wt) AQP2 and AQP2-E258K formed multimers, which also localized to MVB/Lys, independent of forskolin stimulation. Orthophosphate labeling revealed that forskolin increased phosphorylation of wt-AQP2 and AQP2-E258K but not AQP2-S256A, indicating that the E258K mutation does not interfere with the AQP2 phosphorylation at S256. In contrast to wt-AQP2 but consistent with the introduced protein kinase C (PKC) consensus site, AQP2-E258K was phosphorylated by phorbol esters. Besides the 29-kDa band, however, an additional band of about 35 kDa was observed for AQP2-E258K only, which represented AQP2-E258K uniquely monoubiquitinated at K228 only. Analysis of several mutants interfering with AQP2-E258K phosphorylation, and/or ubiquitination, however, revealed that the MVB/lysosomal sorting of AQP2-E258K occurred independent of its monoubiquitination or phosphorylation by PKC. Instead, our data reveal that the loss of the E258 in AQP2-E258K is fundamental to its missorting to MVB/Lys and indicate that this amino acid has an important role in the proper structure formation of the C-terminal tail of AQP2.


Water channel AQP2 Phosphorylation Membrane trafficking Ubiquitination 



This research was supported by grants from the Dutch Organization of Scientific Research (NWO-MW 902-18-292) to PMTD and PvdS and to EJK (NWO; 916.36.122) and from the European Union (QLRT-2000-00778, QLK3-CT-2001-00987) to PMTD.


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Copyright information

© Springer-Verlag 2007

Authors and Affiliations

  • Erik-Jan Kamsteeg
    • 1
  • Paul J. M. Savelkoul
    • 1
  • Giel Hendriks
    • 2
  • Irene B. M. Konings
    • 1
  • Nicole M. I. Nivillac
    • 1
  • Anne Karine Lagendijk
    • 1
  • Peter van der Sluijs
    • 2
  • Peter M. T. Deen
    • 1
  1. 1.Department of PhysiologyRadboud University Nijmegen Medical CenterNijmegenThe Netherlands
  2. 2.Department of Cell BiologyUMC UtrechtUtrechtThe Netherlands

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