Abstract
UT-A1 is the primary urea transporter in the apical plasma membrane responsible for urea reabsorption in the inner medullary collecting duct. Although the physiological function of UT-A1 has been well established, the molecular mechanisms that regulate its activity are less well understood. Analysis of the UT-A1 amino acid sequence revealed a potential MDM2 E3 ubiquitin ligase-binding motif in the large intracellular loop of UT-A1, suggesting that UT-A1 urea transporter protein may be regulated by the ubiquitin-proteasome pathway. Here, we report that UT-A1 is ubiquitinated and degraded by the proteasome but not the lysosome proteolytic pathway. Inhibition of proteasome activity causes UT-A1 cell surface accumulation and concomitantly increases urea transport activity. UT-A1 interacts directly with MDM2; the binding site is located in the NH2-terminal p53-binding region of MDM2. MDM2 mediates UT-A1 ubiquitination both in vivo and in vitro. Overexpression of MDM2 promotes UT-A1 degradation. The mechanism is likely to be physiologically important as UT-A1 ubiquitination was identified in kidney inner medullary tissue. The ubiquitin-proteasome degradation pathway provides an important novel mechanism for UT-A1 regulation.
Publication types
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Research Support, N.I.H., Extramural
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Research Support, Non-U.S. Gov't
MeSH terms
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Animals
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Binding Sites
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Cell Line
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Cell Membrane / metabolism
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Cycloheximide / pharmacology
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Dogs
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Enzyme Inhibitors / pharmacology
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Humans
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Imidazoles / pharmacology
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Kidney Medulla / drug effects
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Kidney Medulla / metabolism
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Leupeptins / pharmacology
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Male
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Membrane Transport Proteins / genetics
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Membrane Transport Proteins / metabolism*
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Mice
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Piperazines / pharmacology
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Protease Inhibitors / pharmacology
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Proteasome Endopeptidase Complex / metabolism
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Proteasome Inhibitors
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Protein Binding
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Proto-Oncogene Proteins c-mdm2 / antagonists & inhibitors
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Proto-Oncogene Proteins c-mdm2 / genetics
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Proto-Oncogene Proteins c-mdm2 / metabolism*
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Rats
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Rats, Sprague-Dawley
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Sequence Deletion
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Ubiquitin-Protein Ligases / antagonists & inhibitors
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Ubiquitin-Protein Ligases / genetics
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Ubiquitin-Protein Ligases / metabolism*
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Ubiquitination / drug effects
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Urea / metabolism
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Urea Transporters
Substances
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Enzyme Inhibitors
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Imidazoles
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Leupeptins
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Membrane Transport Proteins
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Piperazines
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Protease Inhibitors
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Proteasome Inhibitors
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nutlin 3
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Urea
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Cycloheximide
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Proto-Oncogene Proteins c-mdm2
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Ubiquitin-Protein Ligases
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Proteasome Endopeptidase Complex
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benzyloxycarbonylleucyl-leucyl-leucine aldehyde