Loss of N-Linked Glycosylation Reduces Urea Transporter UT-A1 Response to Vasopressin

Chen, Guangping; Fröhlich, Otto; Yang, Yuan; Klein, Janet D.; Sands, Jeff M.

doi:10.1074/jbc.m605525200

Cited by 51 publications

(57 citation statements)

References 31 publications

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“…Cells were grown on Transwell filters to confluence. Before immunostaining, cells were rinsed twice with PBS, fixed with 4% formalin in PBS for 10 -15 min at 4°C, washed with PBS three times, permeabilized in PBS containing 0.1% Triton X-100 for 10 min, and blocked with 1% bovine serum albumin in PBS (blocking buffer) for 30 min at 25°C (27). Cells were then incubated with primary antibody, anti-UT-A1 (C-terminal), diluted 1:4000 in blocking buffer for 1 h at room temperature.…”

Section: Methodsmentioning

confidence: 99%

“…Plasma Membrane Isolation-Plasma membrane isolation of UT-A1-MDCK cells was performed as described previously (27) with some modification. Ad-snapin-infected UT-A1-MDCK confluent cells (in 6-well plates) were treated with 10 M forskolin (FSK, Sigma) for 20 min and then frozen at Ϫ80°C for 1 h. The cell pellets were collected and resuspended in icecold homogenization buffer (250 mM sucrose, 2 mM EDTA, 10 mM Tris, pH 7.4) containing protease inhibitor mixture (Sigma) at 4°C.…”

Section: Methodsmentioning

confidence: 99%

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The UT-A1 Urea Transporter Interacts with Snapin, a SNARE-associated Protein

Mistry

Mallick²,

Fröhlich

et al. 2007

Journal of Biological Chemistry

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The UT-A1 urea transporter mediates rapid transepithelial urea transport across the inner medullary collecting duct and plays a major role in the urinary concentrating mechanism. To transport urea, UT-A1 must be present in the plasma membrane. The purpose of this study was to screen for UT-A1-interacting proteins and to study the interactions of one of the identified potential binding partners with UT-A1. Using a yeast two-hybrid screen of a human kidney cDNA library with the UT-A1 intracellular loop (residues 409 -594) as bait, we identified snapin, a ubiquitously expressed SNARE-associated protein, as a novel UT-A1 binding partner. Deletion analysis indicated that the C-terminal coiled-coil domain (H2) of snapin is required for UT-A1 interaction. Snapin binds to the intracellular loop of UT-A1 but not to the N-or C-terminal fragments. Glutathione S-transferase pulldown experiments and co-immunoprecipitation studies verified that snapin interacts with native UT-A1, SNAP23, and syntaxin-4 (t-SNARE partners), indicating that UT-A1 participates with the SNARE machinery in rat kidney inner medulla. Confocal microscopic analysis of immunofluorescent UT-A1 and snapin showed co-localization in both the cytoplasm and in the plasma membrane. When we co-injected UT-A1 with snapin cRNA in Xenopus oocytes, urea influx was significantly increased. In the absence of snapin, the influx was decreased when UT-A1 was combined with t-SNARE components syntaxin-4 and SNAP23. We conclude that UT-A1 may be linked to the SNARE machinery via snapin and that this interaction may be functionally and physiologically important for urea transport.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

The UT-A1 Urea Transporter Interacts with Snapin, a SNARE-associated Protein

Mistry

Mallick²,

Fröhlich

et al. 2007

Journal of Biological Chemistry

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“…Reminiscent of its effect on several other membrane transport proteins, such as AQP2, 23 NKCC2, 24 and ENaC, 25 N-linked glycosylation is critical for UT-A1 membrane trafficking, stability, and activity. 8,10 Animal studies revealed a functional link between UT-A1 activity and its glycosylation state. In particular, the highly glycosylated 117-kD form of UT-A1 is increased in several conditions associated with decreased urine concentration, such as STZ-induced diabetes mellitus, 19 a low-protein diet, 26 hypercalcemia, 27 water diuresis, 26 and furosemide administration.…”

Section: Discussionmentioning

confidence: 99%

“…Only Asn279 and Asn742 in the two large extracellular loops are proven to be sites for N-linked glycosylation. 10 Importantly, loss of glycosylation by mutating the two glycosylation sites significantly impairs the response of UT-A1 to vasopressin. 10 Vasopressin is the primary hormone regulating UT-A1 transport activity in vivo.…”

mentioning

confidence: 99%

“…10 Importantly, loss of glycosylation by mutating the two glycosylation sites significantly impairs the response of UT-A1 to vasopressin. 10 Vasopressin is the primary hormone regulating UT-A1 transport activity in vivo. 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.…”

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confidence: 99%

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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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Urea Transport in the Kidney

Klein

Blount

Sands

2011

Comprehensive Physiology

124

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Urea transport proteins were initially proposed to exist in the kidney in the late 1980s when studies of urea permeability revealed values in excess of those predicted by simple lipid-phase diffusion and paracellular transport. Less than a decade later, the first urea transporter was cloned. Currently, the SLC14A family of urea transporters contains two major subgroups: SLC14A1, the UT-B urea transporter originally isolated from erythrocytes; and SLC14A2, the UT-A group with six distinct isoforms described to date. In the kidney, UT-A1 and UT-A3 are found in the inner medullary collecting duct; UT-A2 is located in the thin descending limb, and UT-B is located primarily in the descending vasa recta; all are glycoproteins. These transporters are crucial to the kidney's ability to concentrate urine. UT-A1 and UT-A3 are acutely regulated by vasopressin. UT-A1 has also been shown to be regulated by hypertonicity, angiotensin II, and oxytocin. Acute regulation of these transporters is through phosphorylation. Both UT-A1 and UT-A3 rapidly accumulate in the plasma membrane in response to stimulation by vasopressin or hypertonicity. Long-term regulation involves altering protein abundance in response to changes in hydration status, low protein diets, adrenal steroids, sustained diuresis, or antidiuresis. Urea transporters have been studied using animal models of disease including diabetes mellitus, lithium intoxication, hypertension, and nephrotoxic drug responses. Exciting new animal models are being developed to study these transporters and search for active urea transporters. Here we introduce urea and describe the current knowledge of the urea transporter proteins, their regulation, and their role in the kidney.

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Loss of N-Linked Glycosylation Reduces Urea Transporter UT-A1 Response to Vasopressin

Cited by 51 publications

References 31 publications

The UT-A1 Urea Transporter Interacts with Snapin, a SNARE-associated Protein

The UT-A1 Urea Transporter Interacts with Snapin, a SNARE-associated Protein

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

Urea Transport in the Kidney

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