Nedd4-2 interacts with occludin to inhibit tight junction formation and enhance paracellular conductance in collecting duct epithelia

Nandita S Raikwar; Alain Vandewalle; Christie P Thomas

doi:10.1152/ajprenal.00674.2009

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Nedd4-2 interacts with occludin to inhibit tight junction formation and enhance paracellular conductance in collecting duct epithelia

Journal article

Open access

Peer reviewed

Nedd4-2 interacts with occludin to inhibit tight junction formation and enhance paracellular conductance in collecting duct epithelia

Nandita S Raikwar, Alain Vandewalle and Christie P Thomas

American journal of physiology. Renal physiology, Vol.299(2), pp.F436-444

08/2010

DOI: 10.1152/ajprenal.00674.2009

PMCID: PMC2928518

PMID: 20504882

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Abstract

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.

Epithelial Cells - metabolism

Immunoprecipitation

Calcium - metabolism

Humans

Endosomal Sorting Complexes Required for Transport - genetics

Occludin

Ubiquitination

Transfection

RNA Interference

Time Factors

Nedd4 Ubiquitin Protein Ligases

Membrane Proteins - metabolism

Protein Interaction Domains and Motifs

Protein Stability

Tight Junctions - metabolism

Cell Line

Membrane Proteins - genetics

Endosomal Sorting Complexes Required for Transport - metabolism

Ubiquitin-Protein Ligases - metabolism

Electric Impedance

Permeability

Kidney Tubules, Collecting - metabolism

Animals

Membrane Potentials

Protein Binding

Mice

Protein Processing, Post-Translational

Ubiquitin-Protein Ligases - genetics

Details

Title: Subtitle: Nedd4-2 interacts with occludin to inhibit tight junction formation and enhance paracellular conductance in collecting duct epithelia
Creators: Nandita S Raikwar - Department of Internal Medicine, University of Iowa College of Medicine, Iowa City, 52242, USA
Alain Vandewalle
Christie P Thomas
Resource Type: Journal article
Publication Details: American journal of physiology. Renal physiology, Vol.299(2), pp.F436-444
DOI: 10.1152/ajprenal.00674.2009
PMID: 20504882
PMCID: PMC2928518
NLM abbreviation: Am J Physiol Renal Physiol
ISSN: 0363-6127
eISSN: 1522-1466
Publisher: United States
Grant note: HL-71664 / NHLBI NIH HHS R01 HL071664 / NHLBI NIH HHS
Language: English
Date published: 08/2010
Academic Unit: Stead Family Department of Pediatrics; Obstetrics and Gynecology; Internal Medicine
Record Identifier: 9983985902602771

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