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Affinity and Specificity of Interactions between Nedd4 Isoforms and the Epithelial Na+ Channel

2003; Elsevier BV; Volume: 278; Issue: 22 Linguagem: Inglês

10.1074/jbc.m211153200

ISSN

1083-351X

Autores

Pauline Henry, Voula Kanelis, M. Christine O'Brien, Brian W. Kim, Ivan Gautschi, Julie D. Forman‐Kay, Laurent Schild, Daniela Rotin,

Tópico(s)

Neuroscience of respiration and sleep

Resumo

The epithelial Na+ channel (αβγENaC) 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 ∼10 μm for α- or βENaC, 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*·βENaC-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 βENaC-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. The epithelial Na+ channel (αβγENaC) 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 ∼10 μm for α- or βENaC, 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*·βENaC-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 βENaC-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. The epithelial sodium channel (ENaC) 1The abbreviations used are: ENaC, epithelial Na+ channel; x-, Xenopus; m-, mouse; d-, Drosophila; h-, human; WT, wild type; PDB, Protein Data Bank. is an apically located ion channel found in absorptive epithelia of organs involved in fluid and electrolyte homeostasis such as the kidney, lung, distal colon, and ducts of exocrine glands (1Garty H. Palmer L.G. Physiol. Rev. 1997; 77: 359-396Crossref PubMed Scopus (1043) Google Scholar, 2Rossier B.C. Pradervand S. Schild L. Hummler E. Annu. Rev. Physiol. 2002; 64: 877-897Crossref PubMed Scopus (325) Google Scholar). In the kidney, ENaC participates in the unidirectional transport of Na+ ions into epithelial cells of the distal nephron in response to hormonal signaling, particularly aldosterone and vasopressin. ENaC is composed of three homologous subunits (α, β, and γ), each comprised of intracellular N and C termini, two transmembrane domains, and a glycosylated extracellular loop (3Canessa C.M. Merillat A.M. Rossier B.C. Am. J. Physiol. 1994; 267: C1682-C1690Crossref PubMed Google Scholar, 4Canessa C.M. Schild L. Buell G. Thorens B. Gautschi I. Horisberger J.D. Rossier B.C. Nature. 1994; 367: 463-467Crossref PubMed Scopus (1789) Google Scholar). The stoichiometry of ENaC subunit assembly conforms to a α2βγ configuration (5Firsov D. Gautschi I. Merillat A.M. Rossier B.C. Schild L. EMBO J. 1998; 17: 344-352Crossref PubMed Scopus (370) Google Scholar, 6Kosari F. Sheng S. Li J. Mak D.O. Foskett J.K. Kleyman T.R. J. Biol. Chem. 1998; 273: 13469-13474Abstract Full Text Full Text PDF PubMed Scopus (202) Google Scholar). Each ENaC chain contains two short proline-rich sequences (P1 and P2) at its C terminus, where the P2 includes the sequence PPXY, which conforms to a PY motif known to be a target for WW domains (7Rotin D. Bar-Sagi D. O'Brodovich H. Merilainen J. Lehto V.P. Canessa C.M. Rossier B.C. Downey G.P. EMBO J. 1994; 13: 4440-4450Crossref PubMed Scopus (218) Google Scholar, 8Schild L. Lu Y. Gautschi I. Schneeberger E. Lifton R.P. Rossier B.C. EMBO J. 1996; 15: 2381-2387Crossref PubMed Scopus (362) Google Scholar, 9Staub O. Dho S. Henry P. Correa J. Ishikawa T. McGlade J. Rotin D. EMBO J. 1996; 15: 2371-2380Crossref PubMed Scopus (741) Google Scholar). The PY motif of β or γ ENaC is deleted or mutated in patients with Liddle's syndrome (10Shimkets R.A. Warnock D.G. Bositis C.M. Nelson-Williams C. Hansson J.H. Schambelan M. Gill Jr., J.R. Ulick S. Milora R.V. Findling J.W. Canessa M.C. Rossier B.C. 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S. A. 1996; 93: 15370-15375Crossref PubMed Scopus (399) Google Scholar). There are several hormones, regulatory proteins, and ions known to regulate ENaC (1Garty H. Palmer L.G. Physiol. Rev. 1997; 77: 359-396Crossref PubMed Scopus (1043) Google Scholar). A well established suppressor of ENaC, which has received much attention in recent years, is Nedd4. Nedd4 is a ubiquitin protein ligase (E3) comprised of a C2 domain, 3 or 4 WW domains, and a ubiquitin ligase Hect domain (9Staub O. Dho S. Henry P. Correa J. Ishikawa T. McGlade J. Rotin D. EMBO J. 1996; 15: 2371-2380Crossref PubMed Scopus (741) Google Scholar, 20Kumar S. Tomooka Y. Noda M. Biochem. Biophys. Res. Commun. 1992; 185: 1155-1161Crossref PubMed Scopus (450) Google Scholar, 21Rotin D. Staub O. Haguenauer-Tsapis R. J. Membr. Biol. 2000; 176: 1-17Crossref PubMed Google Scholar). The C2 domain is involved in membrane targeting (22Plant P.J. Lafont F. Lecat S. Verkade P. Simons K. Rotin D. J. Cell Biol. 2000; 149: 1473-1484Crossref PubMed Scopus (124) Google Scholar, 23Plant P.J. Yeger H. Staub O. Howard P. Rotin D. J. Biol. Chem. 1997; 272: 32329-32336Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar), the WW domains are involved in protein-protein interactions and substrate recognition (24Bork P. Sudol M. Trends Biochem. Sci. 1994; 19: 531-533Abstract Full Text PDF PubMed Scopus (348) Google Scholar) (see below), and the Hect domain provides the catalytic ubiquitin ligase (E3) activity (25Huibregtse J.M. Scheffner M. Beaudenon S. Howley P.M. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 5249Crossref PubMed Scopus (162) Google Scholar) responsible for ubiquitination and degradation (or endocytosis) of target proteins. The interaction between Nedd4 and ENaC takes place between the WW domains of Nedd4 and the PY motifs of ENaC (9Staub O. Dho S. Henry P. Correa J. Ishikawa T. McGlade J. Rotin D. EMBO J. 1996; 15: 2371-2380Crossref PubMed Scopus (741) Google Scholar). WW domains are small modules of ∼40 residues in length containing two highly conserved tryptophan residues and an invariant proline and bind proline-rich sequences (26Chen H.I. Sudol M. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 7819-7823Crossref PubMed Scopus (489) Google Scholar). Nedd4 WW domains have been grouped as Class I WW domains by virtue of their specificity for binding the PY motif, most recently defined as (L/P)PXY (27Kasanov J. Pirozzi G. Uveges A.J. Kay B.K. Chem. Biol. (Lond.). 2001; 8: 231-241Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar). We have recently determined the solution structure of the third WW domain of rat Nedd4 (homologous to WW4 of human Nedd4s, hereafter called rNedd4-1 WW4) in complex with the PY motif-containing (P2 region) of βENaC (28Kanelis V. Rotin D. Forman-Kay J.D. Nat. Struct. Biol. 2001; 8: 407-412Crossref PubMed Scopus (187) Google Scholar). This WW domain of Nedd4 adopts a three-stranded anti-parallel β-sheet with a hydrophobic binding surface, similar to other WW domains (28Kanelis V. Rotin D. Forman-Kay J.D. Nat. Struct. Biol. 2001; 8: 407-412Crossref PubMed Scopus (187) Google Scholar, 29Macias M.J. Hyvonen M. Baraldi E. Schultz J. Sudol M. Saraste M. Oschkinat H. Nature. 1996; 382: 646-649Crossref PubMed Scopus (362) Google Scholar, 30Verdecia M.A. Bowman M.E. Lu K.P. Hunter T. Noel J.P. Nat. Struct. Biol. 2000; 7: 639-643Crossref PubMed Scopus (428) Google Scholar, 31Huang X. Poy F. Zhang R. Joachimiak A. Sudol M. Eck M.J. Nat. Struct. Biol. 2000; 7: 634-638Crossref PubMed Scopus (225) Google Scholar, 32Pires J.R. Taha-Nejad F. Toepert F. Ast T. Hoffmuller U. Schneider-Mergener J. Kuhne R. Macias M.J. Oschkinat H. J. Mol. Biol. 2001; 314: 1147-1156Crossref PubMed Scopus (93) Google Scholar). A polyproline type II (PPII) helix is formed by residues Pro-614′–Asn-617′, which bind the XP grove of the WW domain (28Kanelis V. Rotin D. Forman-Kay J.D. Nat. Struct. Biol. 2001; 8: 407-412Crossref PubMed Scopus (187) Google Scholar), a pocket found in all WW domains and SH3 domains. (ENaC residues are indicated with a "′" to distinguish from WW domain residues.) Similar interactions are observed for the PY motifs of β-dystroglycan and WBP1 that bind the dystrophin and YAP65 WW domains, respectively (31Huang X. Poy F. Zhang R. Joachimiak A. Sudol M. Eck M.J. Nat. Struct. Biol. 2000; 7: 634-638Crossref PubMed Scopus (225) Google Scholar, 32Pires J.R. Taha-Nejad F. Toepert F. Ast T. Hoffmuller U. Schneider-Mergener J. Kuhne R. Macias M.J. Oschkinat H. J. Mol. Biol. 2001; 314: 1147-1156Crossref PubMed Scopus (93) Google Scholar), or the phospho-Ser-Pro sequence of RNA polymerase II that binds to the WW domain of Pin1 (30Verdecia M.A. Bowman M.E. Lu K.P. Hunter T. Noel J.P. Nat. Struct. Biol. 2000; 7: 639-643Crossref PubMed Scopus (428) Google Scholar). However, unique to the Nedd4 WW domain-βENaC PY complex, a helical turn is adopted by residues Tyr-618′–Leu-621′ (C-terminal to the PPII helix), which is stabilized by intrapeptide and peptide-domain interactions involving both Tyr-618′ and Leu-621′ (28Kanelis V. Rotin D. Forman-Kay J.D. Nat. Struct. Biol. 2001; 8: 407-412Crossref PubMed Scopus (187) Google Scholar). This suggests that an elongated (extended) PY motif sequence (PPXYXXL), conserved in all ENaC chains, is responsible for binding to Nedd4 WW domains. Thus, our tertiary structure reveals contact points between side chains of Pro-615′, Pro-616′, Tyr-618′, and Leu-621′ of the extended PY motif of βENaC (615PPNYDSL621) and the WW4 domain of rNedd4-1 (28Kanelis V. Rotin D. Forman-Kay J.D. Nat. Struct. Biol. 2001; 8: 407-412Crossref PubMed Scopus (187) Google Scholar). One focus of our present work is the investigation of the contribution of these contacts to the affinity of binding between ENaC and Nedd4. Moreover, we have probed the importance of the unique contribution of Leu-621′ to binding to the Nedd4 WW domain for the regulation of channel activity. Nedd4 is a suppressor of ENaC that regulates the number of channels at the plasma membrane (33Abriel H. Loffing J. Rebhun J.F. Pratt J.H. Schild L. Horisberger J.D. Rotin D. Staub O. J. Clin. Invest. 1999; 103: 667-673Crossref PubMed Scopus (329) Google Scholar, 34Goulet C.C. Volk K.A. Adams C.M. Prince L.S. Stokes J.B. Snyder P.M. J. Biol. Chem. 1998; 273: 30012-30017Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar) in agreement with the role of ubiquitination in regulating cell surface stability of ENaC (35Staub O. Gautschi I. Ishikawa T. Breitschopf K. Ciechanover A. Schild L. Rotin D. EMBO J. 1997; 16: 6325-6336Crossref PubMed Scopus (601) Google Scholar). Accordingly, Liddle's syndrome mutations, which eliminate all or part of the PY motif of β or γ ENaC and, thus (at least partially), impair binding of the channel to the WW domains of Nedd4, lead to increased cell surface stability of ENaC (8Schild L. Lu Y. Gautschi I. Schneeberger E. Lifton R.P. Rossier B.C. EMBO J. 1996; 15: 2381-2387Crossref PubMed Scopus (362) Google Scholar, 19Snyder P.M. Price M.P. McDonald F.J. Adams C.M. Volk K.A. Zeiher B.G. Stokes J.B. Welsh M.J. Cell. 1995; 83: 969-978Abstract Full Text PDF PubMed Scopus (401) Google Scholar, 33Abriel H. Loffing J. Rebhun J.F. Pratt J.H. Schild L. Horisberger J.D. Rotin D. Staub O. J. Clin. Invest. 1999; 103: 667-673Crossref PubMed Scopus (329) Google Scholar, 34Goulet C.C. Volk K.A. Adams C.M. Prince L.S. Stokes J.B. Snyder P.M. J. Biol. Chem. 1998; 273: 30012-30017Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar). There are several Nedd4 isoforms and relatives (21Rotin D. Staub O. Haguenauer-Tsapis R. J. Membr. Biol. 2000; 176: 1-17Crossref PubMed Google Scholar), and in particular Nedd4-1 and Nedd4-2 isoforms have been studied with regard to ENaC binding and regulation (36Kamynina E. Debonneville C. Bens M. Vandewalle A. Staub O. FASEB J. 2001; 15: 204-214Crossref PubMed Scopus (250) Google Scholar, 37Kamynina E. Tauxe C. Staub O. Am. J. Physiol. Renal Physiol. 2001; 281: 469-477Crossref PubMed Google Scholar). Although called "isoforms," Nedd4-1 and -2 are encoded by different genes. Recent work has proposed that Nedd4-2 can bind to and regulate ENaC activity much better than Nedd4-1 (33Abriel H. Loffing J. Rebhun J.F. Pratt J.H. Schild L. Horisberger J.D. Rotin D. Staub O. J. Clin. Invest. 1999; 103: 667-673Crossref PubMed Scopus (329) Google Scholar, 36Kamynina E. Debonneville C. Bens M. Vandewalle A. Staub O. FASEB J. 2001; 15: 204-214Crossref PubMed Scopus (250) Google Scholar, 37Kamynina E. Tauxe C. Staub O. Am. J. Physiol. Renal Physiol. 2001; 281: 469-477Crossref PubMed Google Scholar, 38Harvey K.F. Dinudom A. Cook D.I. Kumar S. J. Biol. Chem. 2001; 276: 8597-8601Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar), and moreover, the Nedd4-2 isoform contains sgk1 phosphorylation sites (missing from Nedd4-1), possibly involved in the regulation of ENaC by aldosterone (39Debonneville C. Flores S.Y. Kamynina E. Plant P.J. Tauxe C. Thomas M.A. Munster C. Chraibi A. Pratt J.H. Horisberger J.D. Pearce D. Loffing J. Staub O. EMBO J. 2001; 20: 7052-7059Crossref PubMed Scopus (583) Google Scholar, 40Snyder P.M. Olson D.R. Thomas B.C. J. Biol. Chem. 2002; 277: 5-8Abstract Full Text Full Text PDF PubMed Scopus (389) Google Scholar). However, a puzzling observation demonstrating that human Nedd4-1 (hNedd4-1) is a potent inhibitor of ENaC when lacking its N-terminal region (37Kamynina E. Tauxe C. Staub O. Am. J. Physiol. Renal Physiol. 2001; 281: 469-477Crossref PubMed Google Scholar, 41Snyder P.M. Olson D.R. McDonald F.J. Bucher D.B. J. Biol. Chem. 2001; 276: 28321-28326Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar) suggests that the suppression of ENaC by the different Nedd4 isoforms and family members may be more complicated than originally anticipated and requires further investigations. All Nedd4-2 isoforms (including those from Xenopus, mouse, and human, i.e. x/m/hNedd4-2) contain an "extra" WW domain, WW3*, located between WW2 and WW4, which is lacking from rat and mouse Nedd4-1 isoforms but is found in hNedd4-1 and Drosophila Nedd4-1 (dNedd4-1) (see Fig. 1). Both WW3* and WW4 appear to play key roles in regulating ENaC activity (36Kamynina E. Debonneville C. Bens M. Vandewalle A. Staub O. FASEB J. 2001; 15: 204-214Crossref PubMed Scopus (250) Google Scholar, 37Kamynina E. Tauxe C. Staub O. Am. J. Physiol. Renal Physiol. 2001; 281: 469-477Crossref PubMed Google Scholar). Although we (28Kanelis V. Rotin D. Forman-Kay J.D. Nat. Struct. Biol. 2001; 8: 407-412Crossref PubMed Scopus (187) Google Scholar) and others (42Asher C. Chigaev A. Garty H. Biochem. Biophys. Res. Commun. 2001; 286: 1228-1231Crossref PubMed Scopus (23) Google Scholar, 43Lott J.S. Coddington-Lawson S.J. Teesdale-Spittle P.H. McDonald F.J. Biochem. J. 2002; 361: 481-488Crossref PubMed Scopus (43) Google Scholar) have quantified binding between the Nedd4 WW domains and the ENaC PY motifs, a comparison between the affinity of interactions with ENaC, particularly of the third and fourth WW domains from the different Nedd4 isoforms, and the consequence for ENaC suppression, have been lacking and, hence, is a second focus of our work. Our results presented here demonstrate that contrary to previous suggestions (that only Nedd4-2 proteins can regulate ENaC), it is the presence of WW3* in either Nedd4-1 or —2 that provides high affinity binding to the ENaC PY motifs, promoting suppression of the channel by the WW3*-containing Nedd4 protein. Peptides—Peptides representing wild-type or mutant sequences of rat ENaC subunits α, β, and γ were synthesized by the Hospital for Sick Children/Advanced Protein Technology Centre (Toronto, Canada). All peptides were purified by reverse-phase high performance liquid chromatography using a C18 column with an acetonitrile gradient. Mass and purity of the peptides were confirmed by electrospray mass spectrometry. Wild-type peptide sequences were: α, MTPPLALTAPPPAYATLG (residues 660–677); β, TLPIPGTPPPNYDSL (residues 607–621); and γ, GSTVPGTPPPRYNTLR (residues 617–632), with the indicated substitutions for the β peptides (see Table I). The sgk1 peptide sequence was LYGLPPFYSRNTAE. Lyophilized peptides were re-suspended in 150 mm KCl, 10 mm K+ phosphate, pH 6.5, or where indicated, with 150 mm NaCl, 10 mm Na+ phosphate, pH 6.5. Peptide concentrations were measured in 6.0 m guanidine HCl at A280 (44Pace C.N. Vajdos F. Fee L. Grimsley G. Gray T. Protein Sci. 1995; 4: 2411-2423Crossref PubMed Scopus (3472) Google Scholar). In the case of the Y618′A mutant peptide, the concentration was measured at A215 and compared with a standard curve of A215versus concentration of wild-type peptide.Table IDissociation constants (Kd) of rNedd4-1 WW4 complexed with WT or mutant ENaC βP2 peptidesβP2 Peptide sequenceKdRelative to WT (adjusted p value)n (No. of experiments)μMTLPIPGTPPPNYDSL (WT)54 ± 819TLPIPGTPAPNYDSL (P615′A)302 ± 445.6 × (p < 0.001)10TLPIPGTPPANYDSL (P616′A)NB5TLPIPGTPPPNADSL (Y618′A)NB3TLPIPGTPPPNYASL (D619′A)85 ± 131.6 × (p = 0.20)10TLPIPGTPPPNYDSA (L621′A)298 ± 465.5 × (p < 0.001)9TLPIPGTPPPNYDpSL (S(P)620′)30 ± 60.6 × (p = 0.16)6 Open table in a new tab Expression and Purification of Proteins—The third WW domain of rat Nedd4-1 (renamed WW4) (accession number AAB48949, residues 451–498), Xenopus Nedd4-2 WW3* (accession number CAA03915, residues 489–528), human Nedd4-1 WW3* (accession number BAA07655, residues 362–411), and Drosophila Nedd4-1 WW3* (dNedd4-1(short), accession number CG7555(RD), residues 430–469) were sub-cloned into pQE-30 and expressed as N-terminal MRGS His6-tagged proteins in Escherichia coli M15 pREP4 at 37 °C in LB (Sigma). Bacteria were induced with 1 mm isopropyl-1-thio-β-d-galactopyranoside (Promega) at an A600 of 0.6 for an additional 3 h at 37 °C, and cells were harvested by centrifugation at 6000 × g for 10 min. Each poly-His-tagged protein was purified after lysing cells by sonication and applying the soluble supernatant (cleared by 10,000 × g centrifugation) over a Ni2+-nitrilotriacetic acid-charged resin column (as described by the manufacturer, Qiagen). The protein was dialyzed overnight into 10 mm K+ phosphate, pH 6.5, plus 0.1 mm EDTA, 0.15 μg/ml aprotinin, 0.15 μg/ml leupeptin, 0.1 mm benzamidine at 4 °C before concentrating and purification on a Superdex 75 gel filtration column (Amersham Biosciences) in 150 mm KCl, 10 mm K+ phosphate, pH 6.5 (or Na+ replacing K+, where indicated). Protein concentrations were measured in 6.0 m guanidine HCl at A280 (44Pace C.N. Vajdos F. Fee L. Grimsley G. Gray T. Protein Sci. 1995; 4: 2411-2423Crossref PubMed Scopus (3472) Google Scholar). rNedd4-1 WW4 His-Thr → Ala-Pro (H470A, T471P) and hNedd4-1 WW3* Ala-Pro → His-Thr (A381H, P382T) substitutions were generated using the QuikChange site-directed mutagenesis kit (Stratagene). His-470 and Thr-471 in rNedd4-1 WW4 and Ale-381 and Pro-382 in hNedd4-1 WW3* are equivalent to residues Ala-504 and Pro-505, respectively, in xNedd4-2 WW3* (Fig. 1B). Equilibrium Dissociation Constant (Kd) Measurement—Intrinsic tryptophan fluorescence of the WW domains was used to monitor peptide binding (28Kanelis V. Rotin D. Forman-Kay J.D. Nat. Struct. Biol. 2001; 8: 407-412Crossref PubMed Scopus (187) Google Scholar, 45Viguera A.R. Arrondo J.L. Musacchio A. Saraste M. Serrano L. Biochemistry. 1994; 33: 10925-10933Crossref PubMed Scopus (129) Google Scholar). Fluorescence measurements were obtained using a Hitachi F-2500 fluorescence spectrophotometer at 25 °C with excitation and emission wavelengths of 298 and 333 nm, respectively, and slit widths of 2.5 nm. Experiments were measured in 150 mm KCl, 10 mm K+ phosphate, pH 6.5, or 150 mm NaCl, 10 mm Na+ phosphate, pH 6.5, with WW domain concentrations kept constant at 3 μm (βP2 mutant studies) or 1 μm (Nedd4 isoform study). ENaC peptides were added at concentrations ranging from 0 to 2 mm. Kd measurements for n experiments, where n is indicated in Tables I and II, were log-transformed such that residuals were normally distributed. Reported Kd means and S.E. were then calculated from the transformed data using the Δ method conversion. A one-way analysis of variance was performed, and pair-wise differences were examined using the Tukey adjustment to determine adjusted p values (46Selvin S. Epidemiologic Analysis. Oxford University Press, Oxford2001Crossref Scopus (19) Google Scholar).Table IIDissociation constants (Kd) of rNedd4-1 WW4, x/m/hNedd4-2 WW3*, or hNedd4-1 WW3* domains binding to ENaC αP2, βP2, or γP2-PY motif peptidesProteinαP2 (MTPPLALTAPPPAYATLG)βP2 (TLPIPGTPPPNYDSL)βP2 (150 mM NaCl)γP2 (GSTTVPGTPPPRYNTLR)rNedd4-1-WW438 ± 3 (4Canessa C.M. Schild L. Buell G. Thorens B. Gautschi I. Horisberger J.D. Rossier B.C. Nature. 1994; 367: 463-467Crossref PubMed Scopus (1789) Google Scholar)44 ± 3 (4Canessa C.M. Schild L. Buell G. Thorens B. Gautschi I. Horisberger J.D. Rossier B.C. Nature. 1994; 367: 463-467Crossref PubMed Scopus (1789) Google Scholar)ND141 ± 33 (5Firsov D. Gautschi I. Merillat A.M. Rossier B.C. Schild L. EMBO J. 1998; 17: 344-352Crossref PubMed Scopus (370) Google Scholar)x/m/hNedd4-2 WW3*12 ± 3 (5Firsov D. Gautschi I. Merillat A.M. Rossier B.C. Schild L. EMBO J. 1998; 17: 344-352Crossref PubMed Scopus (370) Google Scholar)9 ± 1 (8Schild L. Lu Y. Gautschi I. Schneeberger E. Lifton R.P. Rossier B.C. EMBO J. 1996; 15: 2381-2387Crossref PubMed Scopus (362) Google Scholar)8 ± 2 (4Canessa C.M. Schild L. Buell G. Thorens B. Gautschi I. Horisberger J.D. Rossier B.C. Nature. 1994; 367: 463-467Crossref PubMed Scopus (1789) Google Scholar)24 ± 3 (5Firsov D. Gautschi I. Merillat A.M. Rossier B.C. Schild L. EMBO J. 1998; 17: 344-352Crossref PubMed Scopus (370) Google Scholar)hNedd4-1 WW3*10 ± 2 (4Canessa C.M. Schild L. Buell G. Thorens B. Gautschi I. Horisberger J.D. Rossier B.C. Nature. 1994; 367: 463-467Crossref PubMed Scopus (1789) Google Scholar)14 ± 1 (4Canessa C.M. Schild L. Buell G. Thorens B. Gautschi I. Horisberger J.D. Rossier B.C. Nature. 1994; 367: 463-467Crossref PubMed Scopus (1789) Google Scholar)ND26 ± 4 (4Canessa C.M. Schild L. Buell G. Thorens B. Gautschi I. Horisberger J.D. Rossier B.C. Nature. 1994; 367: 463-467Crossref PubMed Scopus (1789) Google Scholar)rNedd4-1 WW4 (His-Thr → Ala-Pro)ND65 ± 6 (4Canessa C.M. Schild L. Buell G. Thorens B. Gautschi I. Horisberger J.D. Rossier B.C. Nature. 1994; 367: 463-467Crossref PubMed Scopus (1789) Google Scholar)NDNDhNedd4-1 WW3* (Ala-Pro → His-Thr)ND38 ± 3 (6Kosari F. Sheng S. Li J. Mak D.O. Foskett J.K. Kleyman T.R. J. Biol. Chem. 1998; 273: 13469-13474Abstract Full Text Full Text PDF PubMed Scopus (202) Google Scholar)NDND Open table in a new tab Electrophysiological Measurement of Mutant ENaC Activity—Site-directed mutagenesis was performed on rat ENaC cDNA as described previously (8Schild L. Lu Y. Gautschi I. Schneeberger E. Lifton R.P. Rossier B.C. EMBO J. 1996; 15: 2381-2387Crossref PubMed Scopus (362) Google Scholar). Complementary RNAs of each subunit (WT or mutants) and of xNedd4-2 (WT or CS mutant) or dNedd4-1 (WT or CA mutant) were synthesized in vitro. Healthy stage V and VI Xenopus oocytes were pressure-injected with 100 nl of a solution containing equal amounts of α, β, and γ ENaC complementary RNA at a total concentration of 100 ng/μl. The oocytes were kept in modified low sodium Barth's saline containing 10 mm NaCl, 2 mm KCl, 80 mmn-methyl-d-glucamine chloride, 0.4 mm CaCl2, 0.3 mm CaNO3, 0.8 mm MgSO4, 5 mmn-methyl-d-glucamine-Hepes, pH 7.4. Standard electrophysiological measurements were taken 16–20h after injection. Macroscopic amiloride-sensitive Na+ currents, defined as the difference between Na+ currents obtained in the presence and absence of 5 μm amiloride (Sigma), in the bath were recorded using the two-electrode voltage-clamp technique. For current measurements the oocytes were voltage-clamped to —100 mV. The bath solution was a standard oocyte Ringer solution containing 120 mm NaCl, 2.5 mm KCl, 1.8 mm CaCl2, and 10 mm Hepes. Oocytes were initially placed in a bath solution containing amiloride (10—6m) to prevent changes in intracellular Na+ concentration, and current was measured after washout of amiloride. Currents were recorded with a Dagan TEV-200 amplifier (Minneapolis, MN). Homology Modeling of Nedd4-WW3* Domain Complexed to βP2 ENaC Peptide—Homology models of complexes between the xNedd4-2 or hNedd4-1 WW3* domain and the βENaC PY motif were obtained using the program Modeler (47Sali A. Blundell T.L. J. Mol. Biol. 1993; 234: 779-815Crossref PubMed Scopus (10636) Google Scholar). Note that the WW3* domain in the Nedd4-2 isoforms (xNedd4-2, mNedd4-2, and hNedd4-2) is identical between species and differs by four residues from that in hNedd4-1. WW domains from Nedd4 and other proteins were aligned based on the previously determined structures of WW domains from rNedd4-1 (PDB code 1I5H) (28Kanelis V. Rotin D. Forman-Kay J.D. Nat. Struct. Biol. 2001; 8: 407-412Crossref PubMed Scopus (187) Google Scholar), hYAP65 (PDB code 1JMQ) (32Pires J.R. Taha-Nejad F. Toepert F. Ast T. Hoffmuller U. Schneider-Mergener J. Kuhne R. Macias M.J. Oschkinat H. J. Mol. Biol. 2001; 314: 1147-1156Crossref PubMed Scopus (93) Google Scholar), h-dystrophin (PDB code 1EG4) (31Huang X. Poy F. Zhang R. Joachimiak A. Sudol M. Eck M.J. Nat. Struct. Biol. 2000; 7: 634-638Crossref PubMed Scopus (225

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