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1 Medicine, Renal Division, Emory University, Atlanta, Georgia, United States
2 Physiology, Emory University, Atlanta, Georgia, United States; , United States
3 Medicine, Renal Division, Emory University, United States
4 Medicine, Renal Division, Emory University, Atlanta, Georgia, United States; , United States; Physiology, Emory University, Atlanta, Georgia, United States
* To whom correspondence should be addressed. E-mail: janet.klein{at}emory.edu.
The UT-A1 urea transporter plays an important role in the urine concentrating mechanism. Vasopressin (or cyclic AMP) increases urea permeability in perfused terminal inner medullary collecting ducts and increases the abundance of phosphorylated UT-A1, suggesting regulation by phosphorylation. We performed a phosphopeptide analysis that strongly suggested that a PKA consensus site(s) in the central loop region of UT-A1 was/were phosphorylated. Serine 486 was most strongly identified, with other potential sites at serine 499 and threonine 524. Phospho-mutation constructs of each residue was made and transiently transfected into LLC-PK1 cells to assay for UT-A1 phosphorylation. The basal level of UT-A1 phosphorylation was unaltered by mutation of these sites. We injected oocytes and assayed [14C]-urea flux and determined that mutation of these sites did not alter basal urea transport activity. Next, we tested the effect of stimulating cAMP production with forskolin. Forskolin increased wild-type UT-A1 and T524A phosphorylation in LLC-PK1 cells and increased urea flux in oocytes. In contrast, the S486A and S499A mutants demonstrated loss of forskolin-stimulated UT-A1 phosphorylation and reduced urea flux. In LLC-PK1 cells, we assessed biotinylated UT-A1. Wild type UT-A1, S486A and S499A accumulated in the membrane in response to forskolin. However, in the S486A/S499A double mutant, forskolin-stimulated UT-A1 membrane accumulation and urea flux were totally blocked. We conclude that the phosphorylation of UT-A1 on both serine 486 and 499 is important for activity and that this phosphorylation may be involved in UT-A1 membrane accumulation.
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