Bidirectional Regulation of AQP2 Trafficking and Recycling: Involvement of AQP2-S256 Phosphorylation
| Title: | Bidirectional Regulation of AQP2 Trafficking and Recycling: Involvement of AQP2-S256 Phosphorylation |
|---|---|
| Authors: | Nejsum, Lene Niemann; Zelenina, Marina; Aperia, Anita; Frokiaer, Jorgen; Nielsen, Soren |
| Publisher: | American Journal of Physiology: Renal Physiology |
| Date Published: | May 01, 2005 |
| Reference Number: | 688 |
The present study examined the role of PKA and serine256 (S256) phosphorylation for AQP2 trafficking and recycling using cells transfected with wild-type AQP2 (AQP2-WT) or mutant AQP2 and high-resolution confocal microscopic techniques. In transiently transfected MDCK-C7 cells, stimulation with forskolin induced translocation of AQP2-WT to the plasma membrane. Treatment of AQP2-WT cells with the PKA inhibitor H-89 following forskolin stimulation resulted in internalization of AQP2-WT. Moreover, H-89 treatment of AQP2-S256D (mimicking constitutively phosphorylated AQP2 and hence localized to the plasma membrane) resulted in redistribution of AQP2-S256D to intracellular vesicles, even in the presence of forskolin. Both PGE2 and dopamine stimulation induced endocytosis of AQP2-WT and AQP2-S256D, respectively, in forskolin-stimulated cells. Consistent with this, dopamine in the presence of vasopressin stimulated endocytosis of AQP2 in slices of rat kidney inner medulla without substantial dephosphorylation. In conclusion, these results strongly suggest that 1) S256 phosphorylation is necessary but not sufficient for AQP2 plasma membrane expression, 2) active PKA is required for AQP2 plasma membrane expression, 3) PGE2 and dopamine induce internalization of AQP2 independently of AQP2 dephosphorylation, and 4) preceding activation of cAMP production is necessary for PGE2 and dopamine to cause AQP2 internalization.
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This translation by the NDI Foundation is to assist the lay reader. To provide a clear, accessible interpretation of the original article, we eliminated or simplified some technical detail and complicated scientific language. We concentrated our translation on those aspects of the article dealing directly with NDI. The NDI Foundation thanks the researchers for their work toward understanding and more effectively treating this disorder.
© Copyright NDI Foundation 2007 (JC)
The AQP2’s movement to the cell membrane (exocytosis), and its movement to the cell interior (endocytosis) is governed by the following molecular sequence: The hormone vasopressin (AVP) binds with the vasopressin 2 receptor (V2R). This activates adenylyl cyclase, which leads to an increase of cyclic adenosine monophosphate (cAMP) and an activation of protein kinase A (PKA). PKA initiates a process called phosphorylation, which is the linking of a group of phosphate salts to another molecule. In this case, the molecule is the 256th amino acid residue, which is a serine (S) amino acid, in the chain of amino acid residues that make up the AQP2 protein.
When PKA phosphorylates AQP2 on serine 256 (S256), it stimulates AQP2 to travel from the cell interior to the cell membrane. When AQP2 is in place on the cell membrane, the cell then is able to let water enter. When AVP detaches from V2R, AQP2 returns to the cell interior, and the cell becomes resistant to water entry.
Even such a detailed model of the molecular sequence of AQP2 exocytosis and endocytosis does not provide a complete understanding of the process. For example, even though it is well established that phosphorylation of S256 is necessary for AQP2 to travel to the cell membrane, it is not known whether phosphorylation of S256 is sufficient to keep AQP2 in the cell membrane.
Nejsum, et al., investigated as to:
- how important active PKA was in the AQP2 exocytosis;
- whether phosphorylation of S256 is sufficient to retain AQP2 in the cell membrane; and
- the effect of PGE2, a prostaglandin, and dopamine, a substance found naturally in the body, on AQP2 localization.
The research team treated the cell cultures (stimulated with forskolin) and added PGE2. This stimulated AQP2-S256D endocytosis. Dopamine also stimulated normal AQP2s to internalize, even though the AQP2 did not undergo dephosphorylation.
These results provide deeper understandings of the movement of AQP2 from cell interior to cell membrane and back again. Namely:
- phosphorylation of S256 is necessary but not sufficient for AQP2 exocytosis; other processes must occur;
- PKA must be activated for AQP2 to appear in the cell membrane;
- PGE2 and dopamine stimulate AQP2 endocytosis independently of AQP2 dephosphorylation, demonstrating that dephosphorylation of S256 is not necessary for AQP2 endocytosis; and
- cAMP must be activated before either PGE2 or dopamine can induce AQP2 internalization.