Vasopressin increases phosphorylation of Ser84 and Ser486 in Slc14a2 collecting duct urea transporters

Shelly Hwang, Ruwan Gunaratne, Markus M. Rinschen, Ming Jiun Yu, Trairak Pisitkun, Jason D. Hoffert, Robert A. Fenton, Mark A. Knepper, Chung Lin Chou

Research output: Contribution to journalArticlepeer-review

Abstract

Vasopressin-regulated urea transport in the renal inner medullary collecting duct (IMCD) is mediated by two urea channel proteins, UT-A1 and UT-A3, derived from the same gene (Slc14a2) by alternative splicing. The NH 2-terminal 459 amino acids are the same in both proteins. To study UT-A1/3 phosphorylation, we made phospho-specific antibodies to UT-A sequences targeting phospho-serines at positions 84 and 486, sites identified previously by protein mass spectrometry. Both antibodies proved specific, recognizing only the phosphorylated forms of UT-A1 and -A3. Immunoblotting of rat IMCD suspensions or whole inner medullas showed that the V2R-selective vasopressin analog 1-deamino-8-D-arginine vasopressin (dDAVP) increases phosphorylation at Ser84 (in UT-A1 and UT-A3) and Ser486 (in UT-A1) by about eightfold. Time course studies in rat IMCD suspensions showed maximum phosphorylation within 1 min of dDAVP exposure, consistent with the time course of vasopressin-stimulated phosphorylation of the vasopressin-sensitive water channel aquaporin-2 at Ser256. Confocal immunofluorescence in Brattleboro rat medullary tissue showed labeling limited to the IMCD, which increased markedly in response to dDAVP. Immuno-electron microscopy studies showed that both phosphorylated forms were present mainly in intracellular compartments in the presence of vasopressin. These studies demonstrate regulated phosphorylation of both UT-A1 and UT-A3 in response to vasopressin in a manner consistent with coordinate regulation of UT-A and aquaporin-2 in the renal IMCD. The findings add to prior evidence for vasopressin-induced phosphorylation of UT-A1, providing evidence that UT-A3 may be regulated by phosphorylation as well.

Original languageEnglish (US)
Pages (from-to)F559-F567
JournalAmerican Journal of Physiology - Renal Physiology
Volume299
Issue number3
DOIs
StatePublished - Sep 2010
Externally publishedYes

Keywords

  • Confocal microscopy
  • Immuno-electron microscopy
  • Immunofluorescence immunocytochemistry
  • Phospho-specific antibody

ASJC Scopus subject areas

  • Physiology
  • Urology

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