Nedd4-2 interacts with occludin to inhibit tight junction formation and enhance paracellular conductance in collecting duct epithelia.
Nedd4-2, a E3 ubiquitin ligase, regulates epithelial sodium channel-mediated transcellular Na(+) transport in the collecting duct. We investigated the effect of Nedd4-2 on the junctional complex and paracellular conductance in mpkCCD(c14) cells, a collecting duct cell line. We demonstrate that Nedd4-2 coimmunoprecipitated with and reduced the expression of transfected occludin ... in HEK293 cells. This interaction was mediated via a conserved PY motif in the COOH terminus of occludin and mutation of this PY motif increased the half-life of transfected occludin in HEK293 cells from 6.4 to 11.4 h. We demonstrate that Nedd4-2 ubiquitinates occludin, which was not seen when a catalytically inactive form of Nedd4-2 was used. Overexpression of Nedd4-2 in mpkCCD(c14) cells reduced occludin at the tight junction and transiently increased paracellular conductance in a Ca(2+) switch assay consistent with a delay in the formation of tight junctions. Conversely, siRNA-mediated knockdown of Nedd4-2 increased occludin levels and reduced paracellular conductance. In summary, we demonstrate that Nedd4-2 plays a role in tight junction assembly and the regulation of paracellular conductance in the collecting duct.
Mesh Terms:
Animals, Calcium, Cell Line, Electric Impedance, Endosomal Sorting Complexes Required for Transport, Epithelial Cells, Humans, Immunoprecipitation, Kidney Tubules, Collecting, Membrane Potentials, Membrane Proteins, Mice, Permeability, Protein Binding, Protein Interaction Domains and Motifs, Protein Processing, Post-Translational, Protein Stability, RNA Interference, Tight Junctions, Time Factors, Transfection, Ubiquitin-Protein Ligases, Ubiquitination
Animals, Calcium, Cell Line, Electric Impedance, Endosomal Sorting Complexes Required for Transport, Epithelial Cells, Humans, Immunoprecipitation, Kidney Tubules, Collecting, Membrane Potentials, Membrane Proteins, Mice, Permeability, Protein Binding, Protein Interaction Domains and Motifs, Protein Processing, Post-Translational, Protein Stability, RNA Interference, Tight Junctions, Time Factors, Transfection, Ubiquitin-Protein Ligases, Ubiquitination
Am. J. Physiol. Renal Physiol.
Date: Aug. 01, 2010
PubMed ID: 20504882
View in: Pubmed Google Scholar
Download Curated Data For This Publication
125339
Switch View:
- Interactions 1
- PTM Genes 1