Localization and regulation of PKA-phosphorylated AQP2 in response to V(2)-receptor agonist/antagonist treatment

Am J Physiol Renal Physiol. 2000 Jan;278(1):F29-42. doi: 10.1152/ajprenal.2000.278.1.F29.


Phosphorylation of Ser(256), in a PKA consensus site, in AQP2 (p-AQP2) appears to be critically involved in the vasopressin-induced trafficking of AQP2. In the present study, affinity-purified antibodies that selectively recognize AQP2 phosphorylated at Ser(256) were developed. These antibodies were used to determine 1) the subcellular localization of p-AQP2 in rat kidney and 2) changes in distribution and/or levels of p-AQP2 in response to [desamino-Cys(1),D-Arg(8)]vasopressin (DDAVP) treatment or V(2)-receptor blockade. Immunoelectron microscopy revealed that p-AQP2 was localized in both the apical plasma membrane and in intracellular vesicles of collecting duct principal cells. Treatment of rats with V(2)-receptor antagonist for 30 min resulted in almost complete disappearance of p-AQP2 labeling of the apical plasma membrane with only marginal labeling of intracellular vesicles remaining. Immunoblotting confirmed a marked decrease in p-AQP2 levels. In control Brattleboro rats (BB), lacking vasopressin secretion, p-AQP2 labeling was almost exclusively present in intracellular vesicles. Treatment of BB rats with DDAVP for 2 h induced a 10-fold increase in p-AQP2 labeling of the apical plasma membrane. The overall abundance of p-AQP2, however, was not increased, as determined both by immunoelectron microscopy and immunoblotting. Consistent with this, 2 h of DDAVP treatment of normal rats also resulted in unchanged p-AQP2 levels. Thus the results demonstrate that AQP2 phosphorylated in Ser(256) is present in the apical plasma membrane and in intracellular vesicles and that both the intracellular distribution/trafficking, as well as the abundance of p-AQP2, are regulated via V(2) receptors by altering phosphorylation and/or dephosphorylation of Ser(256) in AQP2.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Antibodies / immunology
  • Antidiuretic Hormone Receptor Antagonists
  • Aquaporin 2
  • Aquaporin 6
  • Aquaporins / immunology
  • Aquaporins / metabolism*
  • Benzazepines / pharmacology
  • Binding Sites
  • Cyclic AMP-Dependent Protein Kinases / metabolism*
  • Deamino Arginine Vasopressin / pharmacology
  • Immunoblotting
  • Immunohistochemistry
  • Kidney Tubules / metabolism*
  • Microscopy, Immunoelectron
  • Molecular Sequence Data
  • Phosphorylation
  • Rats
  • Rats, Brattleboro
  • Rats, Wistar
  • Receptors, Vasopressin / agonists
  • Receptors, Vasopressin / drug effects*
  • Subcellular Fractions / metabolism
  • Vasopressins / deficiency


  • Antibodies
  • Antidiuretic Hormone Receptor Antagonists
  • Aqp2 protein, rat
  • Aquaporin 2
  • Aquaporin 6
  • Aquaporins
  • Benzazepines
  • Receptors, Vasopressin
  • Vasopressins
  • mozavaptan
  • Cyclic AMP-Dependent Protein Kinases
  • Deamino Arginine Vasopressin