title: Quantitative analysis of aquaporin-2 phosphorylation.
abstract: The action of vasopressin in rodent collecting ducts to regulate water permeability depends in part on increases in phosphorylation of the water channel aquaporin-2 (AQP2) at three sites: Ser256, Ser264, and Ser269. Previous studies of AQP2 phosphorylation have depended largely on qualitative data using protein mass spectrometry and phospho-specific antibodies. Here, we use a new method employing phospho-specific antibodies to determine the percentage of total AQP2 phosphorylated at each site in the presence and absence of the V2-receptor-selective vasopressin analog dDAVP in rat renal inner medullary collecting duct (IMCD) and cultured mpkCCD cells. Phosphorylation of Ser269, a site previously implicated in plasma membrane retention, was found to increase from 3 to 26% of total AQP2 in rat IMCD cells following dDAVP. Quantification of immunogold labeling of the opposite kidneys from the same rats estimated that 11% of total AQP2 is present in the apical plasma membrane (APM) without injection of dDAVP and 25% is present in the APM after dDAVP. Surprisingly, the baseline level of Ser256 phosphorylation was constitutively high, and there was no increase with dDAVP (confirmed in 2 more sets of rats). In general, Ser264 phosphorylation remained below 5% of total. The pattern of response was similar in cultured mpkCCD cells (large increase in Ser269 phosphorylation following dDAVP, but constitutively high levels of Ser256 phosphorylation). We suggest from these studies that Ser269 phosphorylation may be a more consistent indicator of vasopressin action and AQP2 membrane abundance than is Ser256 phosphorylation.
authors.affiliation: Epithelial Systems Biology Laboratory, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda,MD 20892, USA.
authors.forename: Luke
authors.initials: L
authors.lastname: Xie
authors.forename: Jason D
authors.initials: JD
authors.lastname: Hoffert
authors.forename: Chung-Lin
authors.initials: CL
authors.lastname: Chou
authors.forename: Ming-Jiun
authors.initials: MJ
authors.lastname: Yu
authors.forename: Trairak
authors.initials: T
authors.lastname: Pisitkun
authors.forename: Mark A
authors.initials: MA
authors.lastname: Knepper
authors.forename: Robert A
authors.initials: RA
authors.lastname: Fenton
mesh_terms: D000818:Animals; D000906:Antibodies; D000918:Antibody Specificity; D051399:Aquaporin 2 / Q000235:genetics / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism; D003894:Deamino Arginine Vasopressin / Q000494:pharmacology; D005786:Gene Expression Regulation / Q000187:drug effects / Q000502:physiology; D007679:Kidney Medulla / Q000502:physiology; D007685:Kidney Tubules, Collecting / Q000166:cytology / Q000502:physiology; D008297:Male; D010766:Phosphorylation; D021381:Protein Transport; D051381:Rats; D017207:Rats, Sprague-Dawley; D014667:Vasopressins / Q000378:metabolism
chemical_list: D000906:Antibodies; C496025:Aqp2 protein, rat; D051399:Aquaporin 2; D014667:Vasopressins; D003894:Deamino Arginine Vasopressin