• We are sorry, but NCBI web applications do not support your browser and may not function properly. More information
Logo of embojLink to Publisher's site
EMBO J. May 15, 1996; 15(10): 2371–2380.
PMCID: PMC450167

WW domains of Nedd4 bind to the proline-rich PY motifs in the epithelial Na+ channel deleted in Liddle's syndrome.

Abstract

The amiloride-sensitive epithelial sodium channel (ENaC) plays a major role in sodium transport in kidney and other epithelia, and in regulating blood pressure. The channel is composed of three subunits (alphabetagamma) each containing two proline-rich sequences (P1 and P2) at its C-terminus. The P2 regions in human beta and gammaENaC, identical to the rat betagammarENaC, were recently shown to be deleted in patients with Liddle's syndrome (a hereditary form of hypertension), leading to hyperactivation of the channel. Using a yeast two-hybrid screen, we have now identified the rat homologue of Nedd4 (rNedd4) as the binding partner for the P2 regions of beta and gammarENaC. rNedd4 contains a Ca2+ lipid binding (CaLB or C2) domain, three WW domains and a ubiquitin ligase (Hect) domain. Our yeast two-hybrid and in vitro binding studies revealed that the rNedd4-WW domains mediate this association by binding to the P2 regions, which include the PY motifs (XPPXY) of either betarENaC (PPPNY) or gammarENaC (PPPRY). SH3 domains were unable to bind these sequences. Moreover, mutations to Ala of Pro616 or Tyr618 within the betarENaC P2 sequence (to PPANY or PPPNA, respectively), recently described in Liddle's patients, led to abrogation of rNedd4-WW binding. Nedd4-WW domains also bound to the proline-rich C-terminus (containing the sequence PPPAY) of alpharENaC, and endogenous Nedd4 co-immunoprecipitated with alpharENaC expressed in MDCK cells. These results demonstrate that the WW domains of rNedd4 bind to the PY motifs deleted from beta or gammaENaC in Liddle's syndrome patients, and suggest that Nedd4 may be a regulator (suppressor) of the epithelial Na+ channel.

Full text

Full text is available as a scanned copy of the original print version. Get a printable copy (PDF file) of the complete article (2.3M), or click on a page image below to browse page by page. Links to PubMed are also available for Selected References.

Selected References

These references are in PubMed. This may not be the complete list of references from this article.
  • André B, Springael JY. WWP, a new amino acid motif present in single or multiple copies in various proteins including dystrophin and the SH3-binding Yes-associated protein YAP65. Biochem Biophys Res Commun. 1994 Dec 15;205(2):1201–1205. [PubMed]
  • Benos DJ, Awayda MS, Ismailov II, Johnson JP. Structure and function of amiloride-sensitive Na+ channels. J Membr Biol. 1995 Jan;143(1):1–18. [PubMed]
  • Bork P, Sudol M. The WW domain: a signalling site in dystrophin? Trends Biochem Sci. 1994 Dec;19(12):531–533. [PubMed]
  • Breeden L, Nasmyth K. Regulation of the yeast HO gene. Cold Spring Harb Symp Quant Biol. 1985;50:643–650. [PubMed]
  • Canessa CM, Horisberger JD, Rossier BC. Epithelial sodium channel related to proteins involved in neurodegeneration. Nature. 1993 Feb 4;361(6411):467–470. [PubMed]
  • Canessa CM, Schild L, Buell G, Thorens B, Gautschi I, Horisberger JD, Rossier BC. Amiloride-sensitive epithelial Na+ channel is made of three homologous subunits. Nature. 1994 Feb 3;367(6462):463–467. [PubMed]
  • Canessa CM, Merillat AM, Rossier BC. Membrane topology of the epithelial sodium channel in intact cells. Am J Physiol. 1994 Dec;267(6 Pt 1):C1682–C1690. [PubMed]
  • Chen HI, Sudol M. The WW domain of Yes-associated protein binds a proline-rich ligand that differs from the consensus established for Src homology 3-binding modules. Proc Natl Acad Sci U S A. 1995 Aug 15;92(17):7819–7823. [PMC free article] [PubMed]
  • Ciechanover A. The ubiquitin-proteasome proteolytic pathway. Cell. 1994 Oct 7;79(1):13–21. [PubMed]
  • Chien CT, Bartel PL, Sternglanz R, Fields S. The two-hybrid system: a method to identify and clone genes for proteins that interact with a protein of interest. Proc Natl Acad Sci U S A. 1991 Nov 1;88(21):9578–9582. [PMC free article] [PubMed]
  • Clark JD, Lin LL, Kriz RW, Ramesha CS, Sultzman LA, Lin AY, Milona N, Knopf JL. A novel arachidonic acid-selective cytosolic PLA2 contains a Ca(2+)-dependent translocation domain with homology to PKC and GAP. Cell. 1991 Jun 14;65(6):1043–1051. [PubMed]
  • Cohen GB, Ren R, Baltimore D. Modular binding domains in signal transduction proteins. Cell. 1995 Jan 27;80(2):237–248. [PubMed]
  • Coussens L, Parker PJ, Rhee L, Yang-Feng TL, Chen E, Waterfield MD, Francke U, Ullrich A. Multiple, distinct forms of bovine and human protein kinase C suggest diversity in cellular signaling pathways. Science. 1986 Aug 22;233(4766):859–866. [PubMed]
  • Duc C, Farman N, Canessa CM, Bonvalet JP, Rossier BC. Cell-specific expression of epithelial sodium channel alpha, beta, and gamma subunits in aldosterone-responsive epithelia from the rat: localization by in situ hybridization and immunocytochemistry. J Cell Biol. 1994 Dec;127(6 Pt 2):1907–1921. [PMC free article] [PubMed]
  • Feng S, Chen JK, Yu H, Simon JA, Schreiber SL. Two binding orientations for peptides to the Src SH3 domain: development of a general model for SH3-ligand interactions. Science. 1994 Nov 18;266(5188):1241–1247. [PubMed]
  • Garty H. Molecular properties of epithelial, amiloride-blockable Na+ channels. FASEB J. 1994 May;8(8):522–528. [PubMed]
  • Gawler DJ, Zhang LJ, Reedijk M, Tung PS, Moran MF. CaLB: a 43 amino acid calcium-dependent membrane/phospholipid binding domain in p120 Ras GTPase-activating protein. Oncogene. 1995 Mar 2;10(5):817–825. [PubMed]
  • Zhang JZ, Davletov BA, Südhof TC, Anderson RG. Synaptotagmin I is a high affinity receptor for clathrin AP-2: implications for membrane recycling. Cell. 1994 Sep 9;78(5):751–760. [PubMed]
  • Guarente L. Yeast promoters and lacZ fusions designed to study expression of cloned genes in yeast. Methods Enzymol. 1983;101:181–191. [PubMed]
  • Hansson JH, Nelson-Williams C, Suzuki H, Schild L, Shimkets R, Lu Y, Canessa C, Iwasaki T, Rossier B, Lifton RP. Hypertension caused by a truncated epithelial sodium channel gamma subunit: genetic heterogeneity of Liddle syndrome. Nat Genet. 1995 Sep;11(1):76–82. [PubMed]
  • Hansson JH, Schild L, Lu Y, Wilson TA, Gautschi I, Shimkets R, Nelson-Williams C, Rossier BC, Lifton RP. A de novo missense mutation of the beta subunit of the epithelial sodium channel causes hypertension and Liddle syndrome, identifying a proline-rich segment critical for regulation of channel activity. Proc Natl Acad Sci U S A. 1995 Dec 5;92(25):11495–11499. [PMC free article] [PubMed]
  • Hirt RP, Poulain-Godefroy O, Billotte J, Kraehenbuhl JP, Fasel N. Highly inducible synthesis of heterologous proteins in epithelial cells carrying a glucocorticoid-responsive vector. Gene. 1992 Feb 15;111(2):199–206. [PubMed]
  • Hofmann K, Bucher P. The rsp5-domain is shared by proteins of diverse functions. FEBS Lett. 1995 Jan 23;358(2):153–157. [PubMed]
  • Huibregtse JM, Scheffner M, Beaudenon S, Howley PM. A family of proteins structurally and functionally related to the E6-AP ubiquitin-protein ligase. Proc Natl Acad Sci U S A. 1995 Mar 28;92(7):2563–2567. [PMC free article] [PubMed]
  • Kaibuchi K, Fukumoto Y, Oku N, Takai Y, Arai K, Muramatsu M. Molecular genetic analysis of the regulatory and catalytic domains of protein kinase C. J Biol Chem. 1989 Aug 15;264(23):13489–13496. [PubMed]
  • Knopf JL, Lee MH, Sultzman LA, Kriz RW, Loomis CR, Hewick RM, Bell RM. Cloning and expression of multiple protein kinase C cDNAs. Cell. 1986 Aug 15;46(4):491–502. [PubMed]
  • Kozak M. The scanning model for translation: an update. J Cell Biol. 1989 Feb;108(2):229–241. [PMC free article] [PubMed]
  • Kumar S, Tomooka Y, Noda M. Identification of a set of genes with developmentally down-regulated expression in the mouse brain. Biochem Biophys Res Commun. 1992 Jun 30;185(3):1155–1161. [PubMed]
  • Lingueglia E, Voilley N, Waldmann R, Lazdunski M, Barbry P. Expression cloning of an epithelial amiloride-sensitive Na+ channel. A new channel type with homologies to Caenorhabditis elegans degenerins. FEBS Lett. 1993 Feb 22;318(1):95–99. [PubMed]
  • Lingueglia E, Renard S, Waldmann R, Voilley N, Champigny G, Plass H, Lazdunski M, Barbry P. Different homologous subunits of the amiloride-sensitive Na+ channel are differently regulated by aldosterone. J Biol Chem. 1994 May 13;269(19):13736–13739. [PubMed]
  • Mayer BJ, Baltimore D. Signalling through SH2 and SH3 domains. Trends Cell Biol. 1993 Jan;3(1):8–13. [PubMed]
  • McDonald FJ, Snyder PM, McCray PB, Jr, Welsh MJ. Cloning, expression, and tissue distribution of a human amiloride-sensitive Na+ channel. Am J Physiol. 1994 Jun;266(6 Pt 1):L728–L734. [PubMed]
  • Nelson RM, Long GL. A general method of site-specific mutagenesis using a modification of the Thermus aquaticus polymerase chain reaction. Anal Biochem. 1989 Jul;180(1):147–151. [PubMed]
  • O'Brodovich HM. The role of active Na+ transport by lung epithelium in the clearance of airspace fluid. New Horiz. 1995 May;3(2):240–247. [PubMed]
  • Renard S, Lingueglia E, Voilley N, Lazdunski M, Barbry P. Biochemical analysis of the membrane topology of the amiloride-sensitive Na+ channel. J Biol Chem. 1994 Apr 29;269(17):12981–12986. [PubMed]
  • Rotin D, Bar-Sagi D, O'Brodovich H, Merilainen J, Lehto VP, Canessa CM, Rossier BC, Downey GP. An SH3 binding region in the epithelial Na+ channel (alpha rENaC) mediates its localization at the apical membrane. EMBO J. 1994 Oct 3;13(19):4440–4450. [PMC free article] [PubMed]
  • Saumon G, Basset G. Electrolyte and fluid transport across the mature alveolar epithelium. J Appl Physiol (1985) 1993 Jan;74(1):1–15. [PubMed]
  • Scheffner M, Huibregtse JM, Vierstra RD, Howley PM. The HPV-16 E6 and E6-AP complex functions as a ubiquitin-protein ligase in the ubiquitination of p53. Cell. 1993 Nov 5;75(3):495–505. [PubMed]
  • Schiestl RH, Gietz RD. High efficiency transformation of intact yeast cells using single stranded nucleic acids as a carrier. Curr Genet. 1989 Dec;16(5-6):339–346. [PubMed]
  • Schild L, Canessa CM, Shimkets RA, Gautschi I, Lifton RP, Rossier BC. A mutation in the epithelial sodium channel causing Liddle disease increases channel activity in the Xenopus laevis oocyte expression system. Proc Natl Acad Sci U S A. 1995 Jun 6;92(12):5699–5703. [PMC free article] [PubMed]
  • Shimkets RA, Warnock DG, Bositis CM, Nelson-Williams C, Hansson JH, Schambelan M, Gill JR, Jr, Ulick S, Milora RV, Findling JW, et al. Liddle's syndrome: heritable human hypertension caused by mutations in the beta subunit of the epithelial sodium channel. Cell. 1994 Nov 4;79(3):407–414. [PubMed]
  • Snyder PM, McDonald FJ, Stokes JB, Welsh MJ. Membrane topology of the amiloride-sensitive epithelial sodium channel. J Biol Chem. 1994 Sep 30;269(39):24379–24383. [PubMed]
  • Sudol M, Chen HI, Bougeret C, Einbond A, Bork P. Characterization of a novel protein-binding module--the WW domain. FEBS Lett. 1995 Aug 1;369(1):67–71. [PubMed]
  • Voilley N, Lingueglia E, Champigny G, Mattéi MG, Waldmann R, Lazdunski M, Barbry P. The lung amiloride-sensitive Na+ channel: biophysical properties, pharmacology, ontogenesis, and molecular cloning. Proc Natl Acad Sci U S A. 1994 Jan 4;91(1):247–251. [PMC free article] [PubMed]
  • Vojtek AB, Hollenberg SM, Cooper JA. Mammalian Ras interacts directly with the serine/threonine kinase Raf. Cell. 1993 Jul 16;74(1):205–214. [PubMed]
  • Yu H, Chen JK, Feng S, Dalgarno DC, Brauer AW, Schreiber SL. Structural basis for the binding of proline-rich peptides to SH3 domains. Cell. 1994 Mar 11;76(5):933–945. [PubMed]
  • Zhang JZ, Davletov BA, Südhof TC, Anderson RG. Synaptotagmin I is a high affinity receptor for clathrin AP-2: implications for membrane recycling. Cell. 1994 Sep 9;78(5):751–760. [PubMed]

Articles from The EMBO Journal are provided here courtesy of The European Molecular Biology Organization

Formats:

Related citations in PubMed

See reviews...See all...

Cited by other articles in PMC

See all...

Links

  • Cited in Books
    Cited in Books
    PubMed Central articles cited in books
  • Compound
    Compound
    PubChem Compound links
  • Gene
    Gene
    Gene links
  • Gene (nucleotide)
    Gene (nucleotide)
    Records in Gene identified from shared sequence links
  • GEO Profiles
    GEO Profiles
    Related GEO records
  • HomoloGene
    HomoloGene
    HomoloGene links
  • MedGen
    MedGen
    Related information in MedGen
  • Nucleotide
    Nucleotide
    Published Nucleotide sequences
  • Pathways + GO
    Pathways + GO
    Pathways, annotations and biological systems (BioSystems) that cite the current article.
  • Protein
    Protein
    Published protein sequences
  • PubMed
    PubMed
    PubMed citations for these articles
  • Substance
    Substance
    PubChem Substance links