Phosphorylation of UT-A1 urea transporter at serines 486 and 499 is important for vasopressin-regulated activity and membrane accumulation

Blount, Mitsi A.; Mistry, Abinash C.; Fröhlich, Otto; Price, S. Russ; Chen, Guangping; Sands, Jeff M.; Klein, Janet D.

doi:10.1152/ajprenal.00102.2008

Cited by 81 publications

(94 citation statements)

References 16 publications

(23 reference statements)

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“…IMCD membrane proteins were biotinylated, and the biotinylated protein fraction was collected as described previously. [13][14][15] We previously showed that this biotinylation protocol identifies the apical plasma membrane. 13 …”

Section: Biotinylationmentioning

confidence: 99%

“…Fresh suspensions of rat IMCDs were prepared as described previously. [12][13][14][15] After washing the IMCDs free of digestion enzymes, they were resuspended in 1 ml of treatment buffer (biotinylation buffer without biotin: 215 mM NaCl, 4 mM KCl, 1.2 mM MgSO 4 , 2 mM CaCl 2 , 5.5 mM glucose, 10 mM triethanolamine, 2.5 mM Na 2 HPO 4 ) and treated with the Epac activator (75 M) or vehicle for 30 min at 37°C. IMCD membrane proteins were biotinylated, and the biotinylated protein fraction was collected as described previously.…”

Section: Biotinylationmentioning

confidence: 99%

“…10 Vasopressin stimulates urea transport across perfused rat terminal IMCDs by increasing UT-A1 phosphorylation and apical plasma membrane accumulation. [11][12][13][14][15] Vasopressin acts by binding to V 2 receptors in the basolateral plasma membrane, stimulating adenylyl cyclase, increasing cAMP production, and increasing urea transport. 11,16 -18 Forskolin, which directly activates adenylyl cyclase, 19 also increases urea transport in perfused rat terminal IMCDs.…”

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confidence: 99%

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Epac Regulates UT-A1 to Increase Urea Transport in Inner Medullary Collecting Ducts

Wang

Klein

Blount

et al. 2009

Journal of the American Society of Nephrology

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Urea plays a critical role in the concentration of urine, thereby regulating water balance. Vasopressin, acting through cAMP, stimulates urea transport across rat terminal inner medullary collecting ducts (IMCD) by increasing the phosphorylation and accumulation at the apical plasma membrane of UT-A1. In addition to acting through protein kinase A (PKA), cAMP also activates Epac (exchange protein activated by cAMP). In this study, we tested whether the regulation of urea transport and UT-A1 transporter activity involve Epac in rat IMCD. Functional analysis showed that an Epac activator significantly increased urea permeability in isolated, perfused rat terminal IMCD. Similarly, stimulating Epac by adding forskolin and an inhibitor of PKA significantly increased urea permeability. Incubation of rat IMCD suspensions with the Epac activator significantly increased UT-A1 phosphorylation and its accumulation in the plasma membrane. Furthermore, forskolin-stimulated cAMP significantly increased ERK 1/2 phosphorylation, which was not prevented by inhibiting PKA, indicating that Epac mediated this phosphorylation of ERK 1/2. Inhibition of MEK 1/2 phosphorylation decreased the forskolin-stimulated UT-A1 phosphorylation. Taken together, activation of Epac increases urea transport, accumulation of UT-A1 at the plasma membrane, and UT-A1 phosphorylation, the latter of which is mediated by the MEK-ERK pathway.

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Section: Biotinylationmentioning

confidence: 99%

Section: Biotinylationmentioning

confidence: 99%

mentioning

confidence: 99%

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Epac Regulates UT-A1 to Increase Urea Transport in Inner Medullary Collecting Ducts

Wang

Klein

Blount

et al. 2009

Journal of the American Society of Nephrology

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“…Vasopressin increases the phosphorylation and apical plasma membrane accumulation of UT-A1 and UT-A3 (14,26). Vasopressin phosphorylates serines 486 and 499 in UT-A1, and both must be mutated to eliminate vasopressin stimulation (27). Vasopressin increases urea transport, urea transporter phosphorylation, and apical plasma membrane accumulation through two cAMP-dependent pathways: protein kinase A and exchange protein activated by cAMP (28) ( Figure 6).…”

Section: Rapid Regulation Of Urea Transporter Proteinsmentioning

confidence: 99%

Urea and Ammonia Metabolism and the Control of Renal Nitrogen Excretion

Weiner

Mitch

Sands

2015

Clinical Journal of the American Society of Nephrology

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330

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Renal nitrogen metabolism primarily involves urea and ammonia metabolism, and is essential to normal health. Urea is the largest circulating pool of nitrogen, excluding nitrogen in circulating proteins, and its production changes in parallel to the degradation of dietary and endogenous proteins. In addition to serving as a way to excrete nitrogen, urea transport, mediated through specific urea transport proteins, mediates a central role in the urine concentrating mechanism. Renal ammonia excretion, although often considered only in the context of acidbase homeostasis, accounts for approximately 10% of total renal nitrogen excretion under basal conditions, but can increase substantially in a variety of clinical conditions. Because renal ammonia metabolism requires intrarenal ammoniagenesis from glutamine, changes in factors regulating renal ammonia metabolism can have important effects on glutamine in addition to nitrogen balance. This review covers aspects of protein metabolism and the control of the two major molecules involved in renal nitrogen excretion: urea and ammonia. Both urea and ammonia transport can be altered by glucocorticoids and hypokalemia, two conditions that also affect protein metabolism. Clinical conditions associated with altered urine concentrating ability or water homeostasis can result in changes in urea excretion and urea transporters. Clinical conditions associated with altered ammonia excretion can have important effects on nitrogen balance.

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“…Vasopressin binds to V 2 receptors on the basolateral plasma membrane (PM) of the IMCD, stimulates adenylyl cyclase, generates cAMP, and activates protein kinase A (PKA), which in turn, phosphorylates UT-A1 proteins, mainly at serines 486 and 499, and regulates urea permeability. 11,12 In addition to the cAMP-PKA signaling pathway, UT-A1 is also found to be regulated by protein kinase C (PKC). This connection was based on the observation that a PKC-a knockout mouse has a urine-concentrating defect.…”

mentioning

confidence: 99%

Activation of Protein Kinase C-α and Src Kinase Increases Urea Transporter A1 α-2, 6 Sialylation

Yang

Chen³

et al. 2015

Journal of the American Society of Nephrology

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The urea transporter A1 (UT-A1) is a glycosylated protein with two glycoforms: 117 and 97 kD. In diabetes, the increased abundance of the heavily glycosylated 117-kD UT-A1 corresponds to an increase of kidney tubule urea permeability. We previously reported that diabetes not only causes an increase of UT-A1 protein abundance but also, results in UT-A1 glycan changes, including an increase of sialic acid content. Because activation of the diacylglycerol (DAG)-protein kinase C (PKC) pathway is elevated in diabetes and PKC-a regulates UT-A1 urea transport activity, we explored the role of PKC in UT-A1 glycan sialylation. We found that activation of PKC specifically promotes UT-A1 glycan sialylation in both UT-A1-MDCK cells and rat kidney inner medullary collecting duct suspensions, and inhibition of PKC activity blocks high glucose-induced UT-A1 sialylation. Overexpression of PKC-a promoted UT-A1 sialylation and membrane surface expression. Conversely, PKC-a-deficient mice had significantly less sialylated UT-A1 compared with wild-type mice. Furthermore, the effect of PKC-a-induced UT-A1 sialylation was mainly mediated by Src kinase but not Raf-1 kinase. Functionally, increased UT-A1 sialylation corresponded with enhanced urea transport activity. Thus, our results reveal a novel mechanism by which PKC regulates UT-A1 function by increasing glycan sialylation through Src kinase pathways, which may have an important role in preventing the osmotic diuresis caused by glucosuria under diabetic conditions.

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Phosphorylation of UT-A1 urea transporter at serines 486 and 499 is important for vasopressin-regulated activity and membrane accumulation

Cited by 81 publications

References 16 publications

Epac Regulates UT-A1 to Increase Urea Transport in Inner Medullary Collecting Ducts

Epac Regulates UT-A1 to Increase Urea Transport in Inner Medullary Collecting Ducts

Urea and Ammonia Metabolism and the Control of Renal Nitrogen Excretion

Activation of Protein Kinase C-α and Src Kinase Increases Urea Transporter A1 α-2, 6 Sialylation

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