Bidirectional regulation of AQP2 trafficking and recycling: involvement of AQP2-S256 phosphorylation.
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.
Nejsum
Lene N
LN
The Water and Salt Research Center, University of Aarhus, DK-8000 Aarhus, Denmark.
Zelenina
Marina
M
Aperia
Anita
A
Frøkiaer
Jørgen
J
Nielsen
Søren
S
D000818:Animals; D000918:Antibody Specificity; D051399:Aquaporin 2; D020346:Aquaporins / Q000276:immunology / Q000378:metabolism*; D002478:Cells, Cultured; D005576:Colforsin / Q000494:pharmacology; D000242:Cyclic AMP / Q000378:metabolism; D017868:Cyclic AMP-Dependent Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism; D015232:Dinoprostone / Q000378:metabolism; D004285:Dogs; D004298:Dopamine / Q000378:metabolism; D004705:Endocytosis / Q000187:drug effects / Q000502:physiology; D007546:Isoquinolines / Q000494:pharmacology; D007668:Kidney / Q000166:cytology / Q000378:metabolism*; D008297:Male; D010766:Phosphorylation; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D021381:Protein Transport / Q000187:drug effects / Q000502:physiology*; D051381:Rats; D017207:Rats, Sprague-Dawley; D012076:Renal Agents / Q000494:pharmacology; D012694:Serine / Q000378:metabolism; D013449:Sulfonamides / Q000494:pharmacology; D014667:Vasopressins / Q000494:pharmacology
C496025:Aqp2 protein, rat; D051399:Aquaporin 2; D020346:Aquaporins; D007546:Isoquinolines; D047428:Protein Kinase Inhibitors; D012076:Renal Agents; D013449:Sulfonamides; D014667:Vasopressins; D005576:Colforsin; D012694:Serine; D000242:Cyclic AMP; D017868:Cyclic AMP-Dependent Protein Kinases; D015232:Dinoprostone; C063509:N-(2-(4-bromocinnamylamino)ethyl)-5-isoquinolinesulfonamide; D004298:Dopamine