2004 Global Researcher Conference Proceeding
April 09 - 11, 2004
| Conference: | 2004 Global Researcher Conference |
|---|---|
| Title: | Regulation of AQP2 trafficking and recycling by dopamine, PGE2 and ANP: involvement of AQP2-S256 phosphorylation |
| Authors: | Nejsum, Lene Niemann; Zelenina, Marina; Wang, Weidong; Aperia, Anita; Knepper, Mark; Frokiaer, Jorgen; Nielsen, Soren |
| Institutions: | The Water and Salt Research Center, Karolinska Institutet, University of Aarhus, National Institutes of Health, The Water and Salt Research Center, University of Aarhus |
AQP2 is the predominant vasopressin (AVP)-regulated water channel of the kidney collecting duct, located to the apical plasma membrane, and is critically involved in both long-term and short-term regulation of body water balance. It is hypothesized that S256 phosphorylation is necessary and sufficient for AQP2 to remain in the plasma membrane. PGE2, however, has been shown to inhibit cAMP-induced water permeability of rat terminal inner medullary collecting duct, and to counter-act AVP-induced AQP2 translocation to the plasma membrane in slices of inner medulla independently of AQP2 dephosphorylation. These findings indicate a role of PGE2 in AQP2 shuttling. Dopamine has also been shown to counteract AVP-induced water permeability in the collecting duct; still dopamine’s action on AQP2 trafficking remains unknown. Moreover, it is well established that phosphorylation of S256 in AQP2 is necessary for translocation of AQP2 to the plasma membrane. However, it is undefined whether this phosphorylation is sufficient to maintain AQP2 in the plasma membrane. The present study examined the role of Protein Kinase A (PKA) and AQP2-serine256 (S256) phosphorylation for AQP2 trafficking and recycling in response to dopamine or PGE2 treatment.
In transiently transfected MDCK-C7 or HEK cells, stimulation with forskolin induced translocation of AQP2 wild-type (AQP2-WT) to the plasma membrane. Treatment of AQP2-WT cells with the PKA inhibitor H89 following forskolin stimulation resulted in internalization of AQP2-WT. Moreover, H89 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. Thus, AQP2 internalization can take place independent of AQP2 dephosphorylation. PGE2 or dopamine stimulation both induced endocytosis of AQP2-WT, and importantly of AQP2-S256D, respectively, in forskolin stimulated cells. In contrast ANP stimulation induces exocytosis of WT-AQP2 to the plasma membrane, consistent with previous evidence. 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 rapid internalization of AQP2 independent of AQP2 dephosphorylation, and 4) preceding activation of cAMP production is necessary for PGE2 and dopamine to cause AQP2 internalization.
Aquaporin-2 is a protein that allows water to move across the cell membrane. To perform this function, AQP2 must first travel from the cell interior to the cell membrane. When it is no longer needed at the cell membrane, it travels back to the cell interior. There are other molecular events that must take place in a coordinated fashion for AQP2 to travel back and forth to the cell membrane.
One such event is that a phosphate group must be added to a serine amino acid residue that is the 256th amino acid in the chain of amino acid residues that make up the AQP2. This process is called phosphorylation. Researchers have hypothesized that phosphorylation is both necessary and sufficient and that dopamine causes the AQP2 to move from the cell membrane to the cell interior. This research showed that:
- Phosphorylation at S256 is necessary but not sufficient for AQP2 to travel to the cell membrane,
- PKA is needed for AQP2 to be in the cell membrane,
- PGE2 and dopamine cause AQP2 to return to the cell interior, even if it hasn’t dephosphorylated, and
- cyclic adenosine monophosphate (cAMP) must be activated for PGE2 and dopamine to cause AQP2 to return to the cell interior.