title: Acute regulation of aquaporin-2 phosphorylation at Ser-264 by vasopressin.
abstract: By phosphoproteome analysis, we identified a phosphorylation site, serine 264 (pS264), in the COOH terminus of the vasopressin-regulated water channel, aquaporin-2 (AQP2). In this study, we examined the regulation of AQP2 phosphorylated at serine 264 (pS264-AQP2) by vasopressin, using a phospho-specific antibody (anti-pS264). Immunohistochemical analysis showed pS264-AQP2 labeling of inner medullary collecting duct (IMCD) from control mice, whereas AQP2 knockout mice showed a complete absence of labeling. In rat and mouse, pS264-AQP2 was present throughout the collecting duct system, from the connecting tubule to the terminal IMCD. Immunogold electron microscopy, combined with double-labeling confocal immunofluorescence microscopy with organelle-specific markers, determined that the majority of pS264 resides in compartments associated with the plasma membrane and early endocytic pathways. In Brattleboro rats treated with [deamino-Cys-1, d-Arg-8]vasopressin (dDAVP), the abundance of pS264-AQP2 increased 4-fold over controls. Additionally, dDAVP treatment resulted in a time-dependent change in the distribution of pS264 from predominantly intracellular vesicles, to both the basolateral and apical plasma membranes. Sixty minutes after dDAVP exposure, a proportion of pS264-AQP2 was observed in clathrin-coated vesicles, early endosomal compartments, and recycling compartments, but not lysosomes. Overall, our results are consistent with a dynamic effect of AVP on the phosphorylation and subcellular distribution of AQP2.
authors.affiliation: The Water and Salt Research Center, Institute of Anatomy, University of Aarhus, DK-8000 Aarhus C, Denmark. rofe@ana.au.dk
authors.forename: Robert A
authors.initials: RA
authors.lastname: Fenton
authors.forename: Hanne B
authors.initials: HB
authors.lastname: Moeller
authors.forename: Jason D
authors.initials: JD
authors.lastname: Hoffert
authors.forename: Ming-Jiun
authors.initials: MJ
authors.lastname: Yu
authors.forename: Søren
authors.initials: S
authors.lastname: Nielsen
authors.forename: Mark A
authors.initials: MA
authors.lastname: Knepper
mesh_terms: D000818:Animals; D051399:Aquaporin 2 / Q000235:genetics / Q000378:metabolism*; D003894:Deamino Arginine Vasopressin / Q000378:metabolism* / Q000494:pharmacology; D015151:Immunoblotting; D007150:Immunohistochemistry; D007668:Kidney / Q000378:metabolism* / Q000648:ultrastructure; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D016253:Microscopy, Immunoelectron; D010766:Phosphorylation; D051381:Rats; D017207:Rats, Sprague-Dawley; D014882:Water-Electrolyte Balance / Q000502:physiology*
chemical_list: D051399:Aquaporin 2; D003894:Deamino Arginine Vasopressin