Affinity and specificity of interactions between Nedd4 isoforms and the epithelial Na+ channel.

Détails

ID Serval
serval:BIB_1B6972DA1533
Type
Article: article d'un périodique ou d'un magazine.
Collection
Publications
Titre
Affinity and specificity of interactions between Nedd4 isoforms and the epithelial Na+ channel.
Périodique
Journal of Biological Chemistry
Auteur(s)
Henry P.C., Kanelis V., O'Brien M.C., Kim B., Gautschi I., Forman-Kay J., Schild L., Rotin D.
ISSN
0021-9258
Statut éditorial
Publié
Date de publication
05/2003
Peer-reviewed
Oui
Volume
278
Numéro
22
Pages
20019-20028
Langue
anglais
Notes
Publication types: Journal Article
Résumé
The epithelial Na+ channel (alphabetagammaENaC) regulates salt and fluid homeostasis and blood pressure. Each ENaC subunit contains a PY motif (PPXY) that binds to the WW domains of Nedd4, a Hect family ubiquitin ligase containing 3-4 WW domains and usually a C2 domain. It has been proposed that Nedd4-2, but not Nedd4-1, isoforms can bind to and suppress ENaC activity. Here we challenge this notion and show that, instead, the presence of a unique WW domain (WW3*) in either Nedd4-2 or Nedd4-1 determines high affinity interactions and the ability to suppress ENaC. WW3* from either Nedd4-2 or Nedd4-1 binds ENaC-PY motifs equally well (e.g. Kd approximately 10 microm for alpha- or betaENaC, 3-6-fold higher affinity than WW4), as determined by intrinsic tryptophan fluorescence. Moreover, dNedd4-1, which naturally contains a WW3* instead of WW2, is able to suppress ENaC function equally well as Nedd4-2. Homology models of the WW3*.betaENaC-PY complex revealed that a Pro and Ala conserved in all WW3*, but not other Nedd4-WW domains, help form the binding pocket for PY motif prolines. Extensive contacts are formed between the betaENaC-PY motif and the Pro in WW3*, and the small Ala creates a large pocket to accommodate the peptide. Indeed, mutating the conserved Pro and Ala in WW3* reduces binding affinity 2-3-fold. Additionally, we demonstrate that mutations in PY motif residues that form contacts with the WW domain based on our previously solved structure either abolish or severely reduce binding affinity to the WW domain and that the extent of binding correlates with the level of ENaC suppression. Independently, we show that a peptide encompassing the PY motif of sgk1, previously proposed to bind to Nedd4-2 and alter its ability to regulate ENaC, does not bind (or binds poorly) the WW domains of Nedd4-2. Collectively, these results suggest that high affinity of WW domain-PY-motif interactions rather than affiliation with Nedd4-1/Nedd-2 is critical for ENaC suppression by Nedd4 proteins.
Mots-clé
Amino Acid Sequence, Animals, Calcium-Binding Proteins/chemistry, Calcium-Binding Proteins/genetics, Epithelial Sodium Channel, Humans, Ligases/chemistry, Ligases/genetics, Models, Molecular, Molecular Sequence Data, Mutagenesis, Protein Binding, Protein Conformation, Protein Isoforms/chemistry, Protein Isoforms/genetics, Sequence Homology, Amino Acid, Sodium Channels/metabolism, Ubiquitin-Protein Ligases
Pubmed
Web of science
Open Access
Oui
Création de la notice
24/01/2008 13:55
Dernière modification de la notice
20/08/2019 13:52
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