Localization and regulation of PKA-phosphorylated AQP2 in response to V₂-receptor agonist/antagonist treatment

Abstract

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.

Acute Hypertonicity Alters Aquaporin-2 Trafficking and Induces a MAPK-dependent Accumulation at the Plasma Membrane of Renal Epithelial Cells

“…Phosphorylation of AQP2 at Ser 256 is essential but not sufficient for steady-state expression of AQP2 at the cell surface (10,12,25,38). We investigated the Ser 256 phosphorylation state of AQP2 that accumulates at the cell surface and in the trans-Golgi region following hypertonic challenge by comparing hypertonicity-induced accumulation of AQP2 in both regions of LLC-PK 1 cells stably expressing mutants that either mimic Ser 256 -phosphorylated (LLC-AQP2 (S256D)) or nonphosphorylated (LLC-AQP2 (S256A)) AQP2 (Fig.…”

PKD1 Haploinsufficiency Causes a Syndrome of Inappropriate Antidiuresis in Mice

“…Rabbit polyclonal antibodies against AQP2 (Sigma-Aldrich), Ser256 phosphorylated AQP2 (p-AQP2) (12), AQP1 (Chemicon, Temecula, CA), AQP3 (a gift from J.-M. Verbavatz, CEA Saclay), extracellular signal-regulated kinase 1/2 (ERK1/2; C16) and Tyr204 p-ERK1/2 (Santa Cruz Biotechnologies, Santa Cruz, CA), mouse monoclonal RhoA and Ser188 p-RhoA (Santa Cruz Biotechnologies), and mouse mAb against ␤-actin (Sigma-Aldrich) were used.…”

“…In normal CD cells, the stimulation of V2R by AVP leads to the phosphorylation of AQP2 on the Ser256 residue and its subsequent insertion in the apical plasma membrane, an essential step to mediate final urine concentration (12,13). A mild impairment in urinary concentrating ability, with increased circulating AVP levels, has been described in patients with ADPKD and cystic kidneys (11,14,15).…”

Increased Apical Targeting of Renal Epithelial Sodium Channel Subunits and Decreased Expression of Type 2 11β-Hydroxysteroid Dehydrogenase in Rats with CCl4-Induced Decompensated Liver Cirrhosis

“…Affinitypurified polyclonal antibodies against the renal sodium transporters/ channels (24) (NHE3, NKCC2, thiazide-sensitive Na-Cl co-transporter [NCC], the ENaC subunits), aquaporin 2 (AQP2) and phosphorylated-AQP2 (p-AQP2) (25) were used. A sheep polyclonal antibody to 11␤HSD2 (Chemicon, Temecula, CA) was commercially obtained.…”

“…Moreover, semiquantitative immunoblotting with antibodies that selectively recognize AQP2 (p-AQP2), which is phosphorylated in the protein kinase A phosphorylation consensus site (Ser256) (25), demonstrated that the abundance of p-AQP2 was also increased in the inner medulla (198 Ϯ 28 versus 100 Ϯ 15%; P Ͻ 0.05) but was not changed in the cortex/OSOM (100 Ϯ 7 versus 100 Ϯ 9%; NS) and ISOM (98 Ϯ 7 versus 100 Ϯ 15%; NS).…”