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Abstract: SA-PO1034

Quantification of Single and Multi-Site AQP2 Phosphorylation

Session Information

Category: Fluid, Electrolytes, and Acid-Base

  • 702 Water/Urea/Vasopressin, Organic Solutes


  • Yui, Naofumi, Tokyo Medical and Dental University, Sagamihara-shi, Kanagawa-ken, Japan
  • Uchida, Shinichi, Tokyo Medical and Dental University, Sagamihara-shi, Kanagawa-ken, Japan

Vasopressin regulates AQP2 phosphorylation at several serine residues. However, the amounts of the singly and multiply phosphorylated forms in the whole AQP2 population are unclear.


In this study, we developed an immunoprecipitation-based quantification of AQP2 phosphorylation. MDCK cells expressing FLAG-tagged human-AQP2 were grown to confluence, lysed, and precipitated with an anti-pS261 antibody, and then blotted and probed with anti-pS256, anti-pS261, and anti-FLAG-M2 antibodies. The signal intensity of the precipitated pS261-positive AQP2 (IP-pS261; consisting of pS261-single form and pS256-pS261-double form) was normalized to the whole pS261 signal in the lysate (IP-pS261/whole-pS261), and the ratio was used as the control. The ratios of the IP-pS261 signal intensity to the whole pS256 signal intensity (IP-pS261/whole-pS256), and to the whole AQP2 signal intensity (IP-pS261/whole-AQP2) were compared with the control ratio. pS256-positive AQP2 was analyzed in the same way.


pS261-positive AQP2 constituted 21% of the whole AQP2 population under basal condition, this declined to 10% after FK (20 µM, 60 min) treatment. pS256-positive AQP2 constituted 48% of the whole AQP2 population under basal condition, this was at 51% after FK treatment. In the pS256-positive AQP2 population, pS261-positive AQP2 (pS256-pS261-double form) constituted a major part of the pS256-positive AQP2 population at 59% under basal condition, this declined to 27% in the population after FK treatment. IP-pS256/whole-pS256 did not differ significantly from IP-pS256/whole-pS261 with or without FK treatment.


Ser-256 is most highly phosphorylated in the cell; pS256-positive AQP2 constantly constitutes around 50% of the whole AQP2 population with or without FK treatment. Most pS261-positive AQP2 is the pS256-pS261 doubly phosphorylated form. This double phospho-form constitutes about a half population in the pS256-positive AQP2, and decreased by 50% after FK treatment in the population. To the contrary, the pS261-single form may constitute a minor component. This study suggests that relative quantification of pS261 signal may be a good biomarker of vasopressin action on pS256-positive AQP2, and will contribute to develop inspective assays of renal vasopressin action in animal and human samples.


  • Other U.S. Government Support