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Detection of Protein Ubiquitination
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Detection of Protein Ubiquitination

Published on: August 19, 2009

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An Essential Role of Nedd4-2 in the Ubiquitination, Expression, and Function of Organic Anion Transporter-3

Da Xu1, Haoxun Wang1, Guofeng You1

  • 1Department of Pharmaceutics, Rutgers University , Piscataway, New Jersey 08854, United States.

Molecular Pharmaceutics
|December 15, 2015
PubMed

Insights

Neural precursor cell expressed, developmentally downregulated 4-2 (Nedd4-2) regulates organic anion transporter-3 (OAT3) activity. Nedd4-2 enhances OAT3 ubiquitination, leading to decreased cell surface expression and inhibited transport activity.

Area of Science:

  • Pharmacology
  • Molecular Biology
  • Cell Biology

Background:

  • Organic anion transporter-3 (OAT3) is crucial for drug disposition.
  • Protein kinase C (PKC) activation inhibits OAT3 activity via internalization.
  • The precise molecular mechanisms underlying PKC-mediated OAT3 regulation require further elucidation.

Purpose of the Study:

  • To investigate the role of Nedd4-2 (neural precursor cell expressed, developmentally downregulated 4-2) in regulating OAT3 ubiquitination and transport activity.
  • To determine if Nedd4-2 mediates the PKC-induced inhibition of OAT3.

Main Methods:

  • Overexpression of Nedd4-2 and its mutant in cell cultures.
  • siRNA-mediated knockdown of endogenous Nedd4-2.
  • Immunoprecipitation assays in cultured cells and rat kidney slices.
  • Measurement of OAT3 ubiquitination, cell surface expression, and transport activity.

Main Results:

  • Overexpression of Nedd4-2 increased OAT3 ubiquitination, reduced cell surface OAT3 levels, and inhibited OAT3 transport.
  • Nedd4-2 ubiquitin ligase-dead mutant overexpression or Nedd4-2 knockdown reversed these effects.
  • Physical interaction between OAT3 and Nedd4-2 was confirmed.

Conclusions:

  • Nedd4-2 directly ubiquitinates OAT3, promoting its internalization and reducing its transport function.
  • Nedd4-2 acts as a key regulator of OAT3 expression and activity.
  • These findings elucidate a novel mechanism for OAT3 regulation, relevant to drug disposition and kidney physiology.
Keywords:
drug transportermembrane transporterregulationubiquitin ligaseubiquitination

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