Multiple WW domains, but not the C2 domain, are required for inhibition of the epithelial Na+ channel by human Nedd4.
The epithelial Na+ channel (ENaC) absorbs Na+ across the apical membrane of epithelia. The activity of ENaC is controlled by its interaction with Nedd4; mutations that disrupt this interaction increase Na+ absorption, causing an inherited form of hypertension (Liddle's syndrome). Nedd4 contains an N-terminal C2 domain, a C-terminal ubiquitin ligase domain, and multiple WW domains. The C2 domain is thought to be involved in the Ca2+-dependent localization of Nedd4 at the cell surface. However, we found that the C2 domain was not required for human Nedd4 (hNedd4) to inhibit ENaC in both Xenopus oocytes and Fischer rat thyroid epithelia. Rather, hNedd4 lacking the C2 domain inhibited ENaC more potently than wild-type hNedd4. Earlier work indicated that the WW domains bind to PY motifs in the C terminus of ENaC. However, it is not known which WW domains mediate this interaction. Glutathione S-transferase-fusion proteins of WW domains 2-4 each bound to alpha, beta, and gammaENaC in vitro. The interactions were abolished by mutation of two residues. WW domain 3 (but not the other WW domains) was both necessary and sufficient for the binding of hNedd4 to alphaENaC. WW domain 3 was also required for the inhibition of ENaC by hNedd4; inhibition was nearly abolished when WW domain 3 was mutated. However, the interaction between ENaC and WW domain 3 alone was not sufficient for inhibition. Moreover, inhibition was decreased by mutation of WW domain 2 or WW domain 4. Thus, WW domains 2-4 each participate in the functional interaction between hNedd4 and ENaC in intact cells.
Snyder
P M
PM
Department of Internal Medicine, University of Iowa College of Medicine, Iowa City, Iowa 52242, USA. psynder@blue.uiowa.edu
Olson
D R
DR
McDonald
F J
FJ
Bucher
D B
DB
D000595:Amino Acid Sequence; D000818:Animals; D002118:Calcium / Q000378:metabolism; D002135:Calcium-Binding Proteins / Q000737:chemistry*; D002460:Cell Line; D004247:DNA / Q000378:metabolism; D018076:DNA, Complementary / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D004594:Electrophysiology; D056827:Endosomal Sorting Complexes Required for Transport; D004848:Epithelium / Q000737:chemistry*; D017353:Gene Deletion; D005982:Glutathione Transferase / Q000378:metabolism; D006801:Humans; D008025:Ligases / Q000737:chemistry* / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation; D000075702:Nedd4 Ubiquitin Protein Ligases; D009865:Oocytes / Q000378:metabolism; D011233:Precipitin Tests; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D051381:Rats; D011916:Rats, Inbred F344; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D026941:Sodium Channel Blockers; D013997:Time Factors; D044767:Ubiquitin-Protein Ligases; D014981:Xenopus; D029867:Xenopus Proteins
D002135:Calcium-Binding Proteins; D018076:DNA, Complementary; D056827:Endosomal Sorting Complexes Required for Transport; D011993:Recombinant Fusion Proteins; D026941:Sodium Channel Blockers; D029867:Xenopus Proteins; D004247:DNA; C000618479:NEDD4L protein, rat; D000075702:Nedd4 Ubiquitin Protein Ligases; C000618482:Nedd4 protein, Xenopus; C000618483:Nedd4 protein, human; C000618484:Nedd4 protein, rat; C000618460:nedd4l protein, Xenopus; D044767:Ubiquitin-Protein Ligases; D005982:Glutathione Transferase; D008025:Ligases; D002118:Calcium