title: Vasopressin increases phosphorylation of Ser84 and Ser486 in Slc14a2 collecting duct urea transporters.
abstract: Vasopressin-regulated urea transport in the renal inner medullary collecting duct (IMCD) is mediated by two urea channel proteins, UT-A1 and UT-A3, derived from the same gene (Slc14a2) by alternative splicing. The NH(2)-terminal 459 amino acids are the same in both proteins. To study UT-A1/3 phosphorylation, we made phospho-specific antibodies to UT-A sequences targeting phospho-serines at positions 84 and 486, sites identified previously by protein mass spectrometry. Both antibodies proved specific, recognizing only the phosphorylated forms of UT-A1 and -A3. Immunoblotting of rat IMCD suspensions or whole inner medullas showed that the V2R-selective vasopressin analog 1-deamino-8-d-arginine vasopressin (dDAVP) increases phosphorylation at Ser84 (in UT-A1 and UT-A3) and Ser486 (in UT-A1) by about eightfold. Time course studies in rat IMCD suspensions showed maximum phosphorylation within 1 min of dDAVP exposure, consistent with the time course of vasopressin-stimulated phosphorylation of the vasopressin-sensitive water channel aquaporin-2 at Ser256. Confocal immunofluorescence in Brattleboro rat medullary tissue showed labeling limited to the IMCD, which increased markedly in response to dDAVP. Immuno-electron microscopy studies showed that both phosphorylated forms were present mainly in intracellular compartments in the presence of vasopressin. These studies demonstrate regulated phosphorylation of both UT-A1 and UT-A3 in response to vasopressin in a manner consistent with coordinate regulation of UT-A and aquaporin-2 in the renal IMCD. The findings add to prior evidence for vasopressin-induced phosphorylation of UT-A1, providing evidence that UT-A3 may be regulated by phosphorylation as well.
authors.affiliation: Laboratory of Kidney and Electrolyte Metabolism, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland, USA.
authors.forename: Shelly
authors.initials: S
authors.lastname: Hwang
authors.forename: Ruwan
authors.initials: R
authors.lastname: Gunaratne
authors.forename: Markus M
authors.initials: MM
authors.lastname: Rinschen
authors.forename: Ming-Jiun
authors.initials: MJ
authors.lastname: Yu
authors.forename: Trairak
authors.initials: T
authors.lastname: Pisitkun
authors.forename: Jason D
authors.initials: JD
authors.lastname: Hoffert
authors.forename: Robert A
authors.initials: RA
authors.lastname: Fenton
authors.forename: Mark A
authors.initials: MA
authors.lastname: Knepper
authors.forename: Chung-Lin
authors.initials: CL
authors.lastname: Chou
mesh_terms: D000818:Animals; D000906:Antibodies / Q000276:immunology; D000918:Antibody Specificity; D050034:Antidiuretic Agents / Q000494:pharmacology*; D007685:Kidney Tubules, Collecting / Q000166:cytology / Q000378:metabolism*; D008297:Male; D026901:Membrane Transport Proteins / Q000276:immunology / Q000378:metabolism*; D023421:Models, Animal; D010766:Phosphorylation / Q000187:drug effects; D051381:Rats; D017207:Rats, Sprague-Dawley; D012694:Serine / Q000378:metabolism; D014667:Vasopressins / Q000494:pharmacology*; D000096982:Urea Transporters
chemical_list: D000906:Antibodies; D050034:Antidiuretic Agents; D026901:Membrane Transport Proteins; D012694:Serine; D014667:Vasopressins; D000096982:Urea Transporters