Nedd4-2 isoforms differentially associate with ENaC and regulate its activity.
Mutations that disrupt a PY motif in epithelial Na(+) channel (ENaC) subunits increase surface expression of Na(+) channels in the collecting duct, resulting in greater Na(+) reabsorption. Nedd4 and Nedd4-2 have been identified as ubiquitin ligases that can interact with ENaC via its PY motifs to regulate channel activity. We recently reported that human Nedd4-2 (hNedd4-2) is expressed as many isoforms because of alternative promoter usage and/or variable splicing. To understand the relevance of hNedd4-2 isoforms for collecting duct Na(+) transport, we studied the interaction with ENaC and the intracellular localization and function of the following three naturally occurring hNedd4-2 isoforms: full-length Nedd4-2 (Nedd4-2), Nedd4-2 lacking the NH(2)-terminal C2 domain (Nedd4-2DeltaC2), and Nedd4-2 lacking the C2 domain and WW domains 2 and 3 (Nedd4-2DeltaWW2,3). Nedd4-2 and Nedd4-2DeltaC2 associate with ENaC and robustly reduce Na(+) transport in Xenopus oocytes, whereas the interaction with and functional effect of Nedd4-2DeltaWW2,3 on ENaC is weak. Nedd4-2 is expressed in the mouse collecting duct, and overexpression of Nedd4-2 reduces endogenous ENaC activity in a collecting duct cell line. This reduction in ENaC activity can be reversed early with exposure to dexamethasone, an effect that is associated with an increase in sgk1 abundance. The C2 domain is required to target Nedd4-2 to the plasma membrane in response to elevation of intracellular Ca(2+) concentration ([Ca(2+)](i)) in MDCK cells, although it does not appear to mediate the inhibitory effect of [Ca(2+)](i) on Na(+) transport. Our data illustrate that naturally occurring hNedd4-2 isoforms differentially associate with ENaC to regulate its activity.
Itani
Omar A
OA
Department of Internal Medicine, University of Iowa, Iowa City, 52242, USA.
Stokes
John B
JB
Thomas
Christie P
CP
D000256:Adenoviridae / Q000235:genetics; D000818:Animals; D015153:Blotting, Western; D019556:COS Cells; D002522:Chlorocebus aethiops; D003593:Cytoplasm / Q000378:metabolism; D056827:Endosomal Sorting Complexes Required for Transport; D053503:Epithelial Sodium Channels; D005260:Female; D005455:Fluorescent Antibody Technique; D005938:Glucocorticoids / Q000494:pharmacology; D006706:Homeostasis / Q000502:physiology; D047468:Immunoprecipitation; D066298:In Vitro Techniques; D007536:Isomerism; D007685:Kidney Tubules, Collecting / Q000378:metabolism; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D008969:Molecular Sequence Data; D000075702:Nedd4 Ubiquitin Protein Ligases; D023282:Nuclease Protection Assays; D009865:Oocytes / Q000378:metabolism; D012313:RNA / Q000096:biosynthesis / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015222:Sodium Channels / Q000378:metabolism* / Q000502:physiology*; D014162:Transfection; D044767:Ubiquitin-Protein Ligases / Q000378:metabolism*; D014981:Xenopus; D029867:Xenopus Proteins
D056827:Endosomal Sorting Complexes Required for Transport; D053503:Epithelial Sodium Channels; D005938:Glucocorticoids; D015222:Sodium Channels; D029867:Xenopus Proteins; D012313:RNA; D000075702:Nedd4 Ubiquitin Protein Ligases; C000618482:Nedd4 protein, Xenopus; C000618483:Nedd4 protein, human; C000618485:Nedd4L protein, human; C000618461:Nedd4l protein, mouse; C000618460:nedd4l protein, Xenopus; D044767:Ubiquitin-Protein Ligases