Localization and regulation of PKA-phosphorylated AQP2 in response to V(2)-receptor agonist/antagonist treatment.
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.
Christensen
B M
BM
Department of Cell Biology, Institute of Anatomy, University of Aarhus, DK-8000 Aarhus, Denmark.
Zelenina
M
M
Aperia
A
A
Nielsen
S
S
D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies / Q000276:immunology; D065092:Antidiuretic Hormone Receptor Antagonists; D051399:Aquaporin 2; D051403:Aquaporin 6; D020346:Aquaporins / Q000276:immunology / Q000378:metabolism*; D001552:Benzazepines / Q000494:pharmacology; D001665:Binding Sites; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism*; D003894:Deamino Arginine Vasopressin / Q000494:pharmacology; D015151:Immunoblotting; D007150:Immunohistochemistry; D007684:Kidney Tubules / Q000378:metabolism*; D016253:Microscopy, Immunoelectron; D008969:Molecular Sequence Data; D010766:Phosphorylation; D051381:Rats; D011910:Rats, Brattleboro; D017208:Rats, Wistar; D017483:Receptors, Vasopressin / Q000819:agonists / Q000187:drug effects*; D013347:Subcellular Fractions / Q000378:metabolism; D014667:Vasopressins / Q000172:deficiency
D000906:Antibodies; D065092:Antidiuretic Hormone Receptor Antagonists; C496025:Aqp2 protein, rat; D051399:Aquaporin 2; D051403:Aquaporin 6; D020346:Aquaporins; D001552:Benzazepines; D017483:Receptors, Vasopressin; D014667:Vasopressins; C076251:mozavaptan; D017868:Cyclic AMP-Dependent Protein Kinases; D003894:Deamino Arginine Vasopressin