1999 European Regional Conference Proceeding
May 12 - 16, 1999
| Conference: | 1999 European Regional Conference |
|---|---|
| Title: | Regulation and distribution of phosphorylated aquaporin-2 (AQP2) in rat kidney collecting duct principal cells |
| Authors: | Christensen, Birgitte Monster; Zelenina, Marina; Aperia, Anita; Nielsen, Soren |
| Institutions: | University of Aarhus, Karolinska Institutet |
AQP2 contains a PKA phosphorylation site at Ser-256 which appears to be involved in the vasopressin-induced trafficking of AQP2 between intracellular vesicles and the apical plasma membrane. In order to determine the subcellular localization of phosphorylated AQP2 and to determine the changes in distribution in response to vasopressin-V2 -receptor blockade, antibodies were designed that exclusively recognize phosphorylated AQP2(p-AQP2). Phosphorylated peptides (Ser 256) were used corresponding to amino acids 253-262. Antisera were applied to a column with non-phosphorylated AQP2 peptide (amino acids 250-271) to remove clones recognizing non-phosphorylated AQP2. These cleaned preparations were subsequently applied to a column with the corresponding p-AQP2 peptide for affinity purification. Immunoblotting showed selective labeling of the phosphorylated but not the non-phosphorylated but not the non-phosphorylated peptides, and kidney membranes revealed only 29 and 35-50 kDa AQP2 bands. Immunoblotting confirmed that our previously described AQP2 antibody (LL127) recognizes both non-phosphorylated and phosphorylated AQP2 peptides (amino acids 250-271). Immunoelectron microscopy of kidneys from Wistar rats revealed that p-AQP2 was localized both in the apical plasma membrane and in subapical vesicles of collecting duct principal cells. Treatment with the V2-receptor antagonist OPC31260 i.v. for 30 minutes resulted in almost complete absence of p-AQP2 labeling. The reduced p-AQP2 labeling was confirmed by immunoblotting showing a dramatic decrease in p-AQP2labeling in response to OPC31260 treatment (n=4, P > 0.05). Treatment of BB rats with dDAVP for 2 hours (reducing urine output from 19.3 + 1.1 to 1.4 + 0.5 ml/2 hrs) induced a ten-fold increase in p-aqp2 labeling of the apical plasma membrane (21+2% vs 2+ 1% of total labeling in dDAVP-treated vs. control BB rats, respectively). There was a concomitant reduction in labeling of intracellular vesicles, but the overall abundance of p-AQP2 was not increased as determined both by immunoelectron microscopy and immunoblotting. Consistent with this 2 hours of dDAVP-treatment of normal rats also resulted in unchanged p-AQP2 levels. Thus the results demonstrate that PKA phosphorylate AQP2 in intracellular vesicles, and that both the intracellular distribution/trafficking, and the levels of p-AQP2 are regulated via V2 - receptors by altering phosphorylation and or dephosphorylation of Ser-256 in AQP2.
In order to perform their function as water channels for the principal cells in the kidney collecting duct, aquaporin-2s (AQP2s) must travel from inside the principal cells to the cells' apical membrane. The 256th amino acid in the chain of amino acids that comprise the AQP2 protein is a serine (Ser-256). Ser-256 is a protein kinase A phosphorylation site, i.e. a place at which a phosphate group is introduced into the AQP2 by protein kinase A (PKA). Ser-256 appears to be involved in the vasopressin (VP) induced movement of AQP2 to the principal cells' apical membrane.
Christensen, et al., determined the subcellular location of phosphorylated AQP2s (p-AQP2s). They also determined the changes in p-AQP2 distribution when VP is blocked from binding with its vasopressin-2 receptor (V2R). The researchers report that, in rats, p-AQP2 is distributed inside the collecting duct principals cells - just beneath the apical membrane - as well as in the apical membrane itself.
When VP was blocked from binding with V2R, almost no p-AQP2s could be identified. There appears to be a dramatic decrease in p-AQP2s in response to VP blockage. When the researchers administered a synthetically modified form of VP (dDAVP) to a type of rat that congenitally lacks the ability to produce VP, they found a ten-fold increase in p-AQP2 in the rats' apical membrane. When the researchers treated normal rats with dDAVP, they found no increase in p-AQP2 levels. These results demonstrate that PKA phosphorylates AQP2 inside the principal cell, and that both the intracellular distribution/movement, and the actual numbers of p-AQP2 are regulated via V2-receptors by altering phosphorylation and/or dephosphorylation of Ser-256 in AQP2.