Vasopressin regulation of multisite phosphorylation of UT-A1 in the inner medullary collecting duct.
Vasopressin signaling is critical for the regulation of urea transport in the inner medullary collecting duct (IMCD). Increased urea permeability is driven by a vasopressin-mediated elevation of cAMP that results in the direct phosphorylation of urea transporter (UT)-A1. The identification of cAMP-sensitive phosphorylation sites, Ser(486) and Ser(499), in the rat UT-A1 sequence was the first step in understanding the mechanism of vasopressin action on the phosphorylation-dependent modulation of urea transport. To investigate the significance of multisite phosphorylation of UT-A1 in response to elevated cAMP, we used highly specific and sensitive phosphosite antibodies to Ser(486) and Ser(499) to determine cAMP action at each phosphorylation site. We found that phosphorylation at both sites was rapid and sustained. Furthermore, the rate of phosphorylation of the two sites was similar in both mIMCD3 cells and rat inner medullary tissue. UT-A1 localized to the apical membrane in response to vasopressin was phosphorylated at Ser(486) and Ser(499). We confirmed that elevated cAMP resulted in increased phosphorylation of both sites by PKA but not through the vasopressin-sensitive exchange protein activated by cAMP pathway. These results elucidate the multisite phosphorylation of UT-A1 in response to cAMP, thus providing the beginning of understanding the intracellular factors underlying vasopressin stimulation of urea transport in the IMCD.
Renal Division, Department of Medicine, Emory University School of Medicine, Atlanta, Georgia; and.
Carol A
CA
Hoban
Renal Division, Department of Medicine, Emory University School of Medicine, Atlanta, Georgia; and.
Lauren N
LN
Black
Renal Division, Department of Medicine, Emory University School of Medicine, Atlanta, Georgia; and.
Ronald J
RJ
Ordas
Renal Division, Department of Medicine, Emory University School of Medicine, Atlanta, Georgia; and.
Diane L
DL
Gumina
Renal Division, Department of Medicine, Emory University School of Medicine, Atlanta, Georgia; and.
Fadi E
FE
Pulous
Renal Division, Department of Medicine, Emory University School of Medicine, Atlanta, Georgia; and.
Jae H
JH
Sim
Renal Division, Department of Medicine, Emory University School of Medicine, Atlanta, Georgia; and Department of Physiology, Emory University School of Medicine, Atlanta, Georgia.
Jeff M
JM
Sands
Renal Division, Department of Medicine, Emory University School of Medicine, Atlanta, Georgia; and Department of Physiology, Emory University School of Medicine, Atlanta, Georgia mabloun@emory.edu.
Mitsi A
MA
Blount
D000818:Animals; D002460:Cell Line; D000242:Cyclic AMP / Q000378:metabolism; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism*; D007685:Kidney Tubules, Collecting / Q000378:metabolism*; D008297:Male; D026901:Membrane Transport Proteins / Q000378:metabolism*; D010766:Phosphorylation; D017207:Rats, Sprague-Dawley; D014667:Vasopressins / Q000378:metabolism*; D000096982:Urea Transporters
D000242:Cyclic AMP; D017868:Cyclic AMP-Dependent Protein Kinases; D026901:Membrane Transport Proteins; D014667:Vasopressins; D000096982:Urea Transporters
adenosine 3′,5′-cyclic monophosphate; urea transport; urea transporter-A1; vasopressin