Identification of phosphorylation-dependent binding partners of aquaporin-2 using protein mass spectrometry

J Proteome Res. 2009 Mar;8(3):1540-54. doi: 10.1021/pr800894p.


Vasopressin-mediated control of water permeability in the renal collecting duct occurs in part through regulation of the distribution of aquaporin-2 (AQP2) between the apical plasma membrane and intracellular membrane compartments. Phosphorylation of Ser-256 at AQP2's cytoplasmic COOH-terminus is well-accepted as a critical step for translocation. The aim of this study was to identify binding partners to phosphorylated versus nonphosphorylated forms of the AQP2 COOH-terminus via a targeted comparative proteomic approach. Cytosol from inner medullary collecting ducts isolated from rat kidneys was incubated with "bait" peptides, representing the COOH-terminal AQP2 tail in its nonphosphorylated and phosphorylated forms, to capture differentially bound proteins prior to LC-MS/MS analysis. Mass spectrometric results were confirmed by immunoblotting. Immunoprecipitation was performed using an AQP2 COOH-terminal antibody combined with immunblotting against the proposed binding partners to demonstrate interactions with native AQP2. Our studies confirmed previously identified interactions between AQP2 and hsc70, hsp70-1 and -2, as well as annexin II. These proteins were found to bind less to the Ser-256-phosphorylated AQP2 than to the nonphosphorylated form. In contrast, another heat shock protein, hsp70-5 (BiP/grp78), bound to phosphorylated AQP2 more avidly than to nonphosphorylated AQP2. Immunogold EM studies demonstrated that BiP is present not only in the ER but also in the cytoplasm and apical plasma membrane of rat collecting duct cells. Furthermore, confocal immunofluorescence studies showed partial colocalization of BiP with AQP2 in non-ER compartments. These results suggest that phosphorylation of AQP2 at Ser-256 may regulate AQP2 trafficking in part by mediating differential binding of hsp70 family proteins to the COOH-terminal tail.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, N.I.H., Intramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Aquaporin 2 / physiology*
  • Chromatography, Liquid
  • Kidney / metabolism
  • Male
  • Molecular Sequence Data
  • Phosphorylation
  • Protein Binding
  • Protein Transport
  • Rats
  • Rats, Sprague-Dawley
  • Tandem Mass Spectrometry


  • Aqp2 protein, rat
  • Aquaporin 2