Hrs Controls Sorting of the Epithelial Na+ Channel between Endosomal Degradation and Recycling Pathways
2010; Elsevier BV; Volume: 285; Issue: 40 Linguagem: Inglês
10.1074/jbc.m110.150755
ISSN1083-351X
AutoresRuifeng Zhou, Rajesh Kabra, Diane R. Olson, Robert C. Piper, Peter M. Snyder,
Tópico(s)Genetics and Neurodevelopmental Disorders
ResumoEpithelial Na+ absorption is regulated by Nedd4-2, an E3 ubiquitin ligase that reduces expression of the epithelial Na+ channel (ENaC) at the cell surface. Defects in this regulation cause Liddle syndrome, an inherited form of hypertension. Previous work found that Nedd4-2 functions through two distinct effects on trafficking, enhancing both ENaC endocytosis and ENaC degradation in lysosomes. To investigate the mechanism by which Nedd4-2 targets ENaC to lysosomes, we tested the role of hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs), a component of the endosomal sorting complexes required for transport (ESCRT)-0 complex. We found that α-, β-, and γENaC each interact with Hrs. These interactions were enhanced by Nedd4-2 and were dependent on the catalytic function of Nedd4-2 as well as its WW domains. Mutation of ENaC PY motifs, responsible for inherited hypertension (Liddle syndrome), decreased Hrs binding to ENaC. Moreover, binding of ENaC to Hrs was reduced by dexamethasone/serum- and glucocorticoid-inducible kinase and cAMP, which are signaling pathways that inhibit Nedd4-2. Nedd4-2 bound to Hrs and catalyzed Hrs ubiquitination but did not alter Hrs protein levels. Expression of a dominant negative Hrs lacking its ubiquitin-interacting motif (Hrs-ΔUIM) increased ENaC surface expression and current. This occurred through reduced degradation of the cell surface pool of proteolytically activated ENaC, which enhanced its recycling to the cell surface. In contrast, Hrs-ΔUIM had no effect on degradation of uncleaved inactive channels. The data support a model in which Nedd4-2 induces binding of ENaC to Hrs, which mediates the sorting decision between ENaC degradation and recycling. Epithelial Na+ absorption is regulated by Nedd4-2, an E3 ubiquitin ligase that reduces expression of the epithelial Na+ channel (ENaC) at the cell surface. Defects in this regulation cause Liddle syndrome, an inherited form of hypertension. Previous work found that Nedd4-2 functions through two distinct effects on trafficking, enhancing both ENaC endocytosis and ENaC degradation in lysosomes. To investigate the mechanism by which Nedd4-2 targets ENaC to lysosomes, we tested the role of hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs), a component of the endosomal sorting complexes required for transport (ESCRT)-0 complex. We found that α-, β-, and γENaC each interact with Hrs. These interactions were enhanced by Nedd4-2 and were dependent on the catalytic function of Nedd4-2 as well as its WW domains. Mutation of ENaC PY motifs, responsible for inherited hypertension (Liddle syndrome), decreased Hrs binding to ENaC. Moreover, binding of ENaC to Hrs was reduced by dexamethasone/serum- and glucocorticoid-inducible kinase and cAMP, which are signaling pathways that inhibit Nedd4-2. Nedd4-2 bound to Hrs and catalyzed Hrs ubiquitination but did not alter Hrs protein levels. Expression of a dominant negative Hrs lacking its ubiquitin-interacting motif (Hrs-ΔUIM) increased ENaC surface expression and current. This occurred through reduced degradation of the cell surface pool of proteolytically activated ENaC, which enhanced its recycling to the cell surface. In contrast, Hrs-ΔUIM had no effect on degradation of uncleaved inactive channels. The data support a model in which Nedd4-2 induces binding of ENaC to Hrs, which mediates the sorting decision between ENaC degradation and recycling. IntroductionThe epithelial Na+ channel ENaC 2The abbreviations used are: ENaCepithelial Na+ channelFRTFischer rat thyroidESCRTendosomal sorting complexes required for transportUIMubiquitin-interacting motifHrshepatocyte growth factor-regulated tyrosine kinase substrateSTAMsignal-transducing adaptor moleculeSGKserum- and glucocorticoid-inducible kinaseMTSET[2-(trimethylammonium)ethyl]methanethiosulfonate bromideNi-NTAnickel-nitrilotriacetic acid. is composed of three homologous subunits (α, β, and γ) and functions in Na+ transport across epithelia in the kidney collecting duct and connecting tubule, lung, and distal colon, where it plays a critical role in Na+ homeostasis (reviewed in Refs. 1Schild L. Rev. Physiol. Biochem. Pharmacol. 2004; 151: 93-107Crossref PubMed Scopus (100) Google Scholar and 2Snyder P.M. Endocrinology. 2005; 146: 5079-5085Crossref PubMed Scopus (193) Google Scholar). Defects in ENaC regulation cause most of the identified genetic forms of hypertension. For example, mutations in β- and γENaC cause Liddle syndrome, an autosomal dominant form of hypertension (3Lifton R.P. Science. 1996; 272: 676-680Crossref PubMed Scopus (562) Google Scholar). Conversely, loss of function mutations cause a salt-wasting disorder known as pseudohypoaldosteronism type 1 (3Lifton R.P. Science. 1996; 272: 676-680Crossref PubMed Scopus (562) Google Scholar). In the lung, ENaC controls the composition and quantity of airway surface liquid, and defective ENaC regulation may contribute to lung disease in cystic fibrosis (4Boucher R.C. Stutts M.J. Knowles M.R. Cantley L. Gatzy J.T. J. Clin. Invest. 1986; 78: 1245-1252Crossref PubMed Scopus (438) Google Scholar). Thus, understanding the mechanisms that regulate ENaC may provide new insights into the pathogenesis of hypertension, cystic fibrosis, and other diseases of Na+ homeostasis.Epithelial Na+ absorption is regulated in large part by mechanisms that control ENaC expression at the apical membrane of epithelia. Here, the E3 ubiquitin ligase Nedd4-2 plays an important role (reviewed in Ref. 5Snyder P.M. Sci. Signal. 2009; 2: pe41Crossref PubMed Scopus (53) Google Scholar). Nedd4-2 binds to ENaC channels located at the cell surface (6Zhou R. Patel S.V. Snyder P.M. J. Biol. Chem. 2007; 282: 20207-20212Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar). This interaction occurs through binding of PY motifs located within the C termini of α-, β-, and γENaC to multiple WW domains in Nedd4-2 (6Zhou R. Patel S.V. Snyder P.M. J. Biol. Chem. 2007; 282: 20207-20212Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar, 7Staub O. Dho S. Henry P. Correa J. Ishikawa T. McGlade J. Rotin D. EMBO J. 1996; 15: 2371-2380Crossref PubMed Scopus (734) Google Scholar, 8Snyder 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). Binding is critical for ENaC regulation. Disruption of binding by mutation of the PY motifs causes hypertension in Liddle syndrome (9Snyder 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 (397) Google Scholar, 10Firsov D. Schild L. Gautschi I. Mérillat A.M. Schneeberger E. Rossier B.C. Proc. Natl. Acad. Sci. U.S.A. 1996; 93: 15370-15375Crossref PubMed Scopus (395) Google Scholar, 11Knight K.K. Olson D.R. Zhou R. Snyder P.M. Proc. Natl. Acad. Sci. U.S.A. 2006; 103: 2805-2808Crossref PubMed Scopus (123) Google Scholar). Binding is also important for the hormonal regulation of ENaC. Aldosterone and vasopressin activate signaling pathways that lead to Nedd4-2 phosphorylation by serum- and glucocorticoid-inducible kinase (SGK) and cAMP-dependent protein kinase (PKA), respectively (5Snyder P.M. Sci. Signal. 2009; 2: pe41Crossref PubMed Scopus (53) Google Scholar). Phosphorylation reduces Nedd4-2 binding to ENaC, which enhances epithelial Na+ transport (12Snyder P.M. Olson D.R. Thomas B.C. J. Biol. Chem. 2002; 277: 5-8Abstract Full Text Full Text PDF PubMed Scopus (385) Google Scholar, 13Debonneville C. Flores S.Y. Kamynina E. Plant P.J. Tauxe C. Thomas M.A. Münster C. Chraïbi A. Pratt J.H. Horisberger J.D. Pearce D. Loffing J. Staub O. EMBO J. 2001; 20: 7052-7059Crossref PubMed Scopus (574) Google Scholar, 14Snyder P.M. Olson D.R. Kabra R. Zhou R. Steines J.C. J. Biol. Chem. 2004; 279: 45753-45758Abstract Full Text Full Text PDF PubMed Scopus (196) Google Scholar, 15Soundararajan R. Zhang T.T. Wang J. Vandewalle A. Pearce D. J. Biol. Chem. 2005; 280: 39970-39981Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar).Following binding, Nedd4-2 catalyzes mono- and polyubiquitination of each ENaC subunit (6Zhou R. Patel S.V. Snyder P.M. J. Biol. Chem. 2007; 282: 20207-20212Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar), which reduces ENaC surface expression through two distinct mechanisms. First, Nedd4-2 increases the rate of ENaC endocytosis (16Kabra R. Knight K.K. Zhou R. Snyder P.M. J. Biol. Chem. 2008; 283: 6033-6039Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar). This process involves epsin, which links ubiquitinated ENaC to the clathrin-based endocytic pathway (17Wang H. Traub L.M. Weixel K.M. Hawryluk M.J. Shah N. Edinger R.S. Perry C.J. Kester L. Butterworth M.B. Peters K.W. Kleyman T.R. Frizzell R.A. Johnson J.P. J. Biol. Chem. 2006; 281: 14129-14135Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar). Second, once in endosomes, Nedd4-2 increases ENaC degradation in lysosomes, which reduces ENaC recycling back to the cell surface (16Kabra R. Knight K.K. Zhou R. Snyder P.M. J. Biol. Chem. 2008; 283: 6033-6039Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar, 18Lu C. Pribanic S. Debonneville A. Jiang C. Rotin D. Traffic. 2007; 8: 1246-1264Crossref PubMed Scopus (97) Google Scholar).A key unanswered question is what are the endosomal proteins that recognize ubiquitinated ENaC and direct it to the lysosomal degradation pathway? Proteins that comprise the endosomal sorting complexes required for transport (ESCRT)-0 complex are strong candidates. ESCRT-0 is a heterodimer of hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs) and signal-transducing adaptor molecule (STAM) (reviewed in Refs. 20Piper R.C. Luzio J.P. Curr. Opin. Cell Biol. 2007; 19: 459-465Crossref PubMed Scopus (133) Google Scholar and 21Raiborg C. Stenmark H. Nature. 2009; 458: 445-452Crossref PubMed Scopus (978) Google Scholar). In yeast, where many of the details of the pathway have been elucidated, the corresponding proteins are vacuolar protein sorting 27p (Vps27p) and Hbp, STAM, and EAST (Hse1p) (22Piper R.C. Cooper A.A. Yang H. Stevens T.H. J. Cell Biol. 1995; 131: 603-617Crossref PubMed Scopus (340) Google Scholar, 23Bilodeau P.S. Urbanowski J.L. Winistorfer S.C. Piper R.C. Nat. Cell Biol. 2002; 4: 534-539Crossref PubMed Scopus (278) Google Scholar). The complex is localized to endosomes through the binding of a Fab1, YotB, Vacl, and EEA1 (FYVE) domain in Hrs to phosphatidylinositol 3-phosphate in the surface of endosomes (24Raiborg C. Bremnes B. Mehlum A. Gillooly D.J. D'Arrigo A. Stang E. Stenmark H. J. Cell Sci. 2001; 114: 2255-2263Crossref PubMed Google Scholar). Both Hrs and STAM contain ubiquitin-interacting motifs (UIMs), which allow them to bind to ubiquitinated proteins (25Hirano S. Kawasaki M. Ura H. Kato R. Raiborg C. Stenmark H. Wakatsuki S. Nat. Struct. Mol. Biol. 2006; 13: 272-277Crossref PubMed Scopus (144) Google Scholar, 26Mizuno E. Kawahata K. Kato M. Kitamura N. Komada M. Mol. Biol. Cell. 2003; 14: 3675-3689Crossref PubMed Scopus (96) Google Scholar). In concert with additional ESCRT complexes, Hrs and STAM are thought to direct ubiquitinated membrane proteins into the endosomal lumen, forming multivesicular bodies that fuse with lysosomes to induce protein degradation. Consistent with this model, disruption of the Hrs-STAM complex or yeast homologues reduced lysosomal/vacuolar degradation of several cargo proteins, including the epithelial growth factor receptor (27Stern K.A. Visser Smit G.D. Place T.L. Winistorfer S. Piper R.C. Lill N.L. Mol. Cell. Biol. 2007; 27: 888-898Crossref PubMed Scopus (54) Google Scholar), the G protein-coupled receptor CXCR4 (28Marchese A. Raiborg C. Santini F. Keen J.H. Stenmark H. Benovic J.L. Dev. Cell. 2003; 5: 709-722Abstract Full Text Full Text PDF PubMed Scopus (313) Google Scholar), and the yeast α-factor receptor Ste3p (23Bilodeau P.S. Urbanowski J.L. Winistorfer S.C. Piper R.C. Nat. Cell Biol. 2002; 4: 534-539Crossref PubMed Scopus (278) Google Scholar, 29Shih S.C. Prag G. Francis S.A. Sutanto M.A. Hurley J.H. Hicke L. EMBO J. 2003; 22: 1273-1281Crossref PubMed Scopus (238) Google Scholar). Targeting to the endosomal lumen is dependent on the UIMs in Hrs/Vps27p and STAM/Hse1p (23Bilodeau P.S. Urbanowski J.L. Winistorfer S.C. Piper R.C. Nat. Cell Biol. 2002; 4: 534-539Crossref PubMed Scopus (278) Google Scholar, 29Shih S.C. Prag G. Francis S.A. Sutanto M.A. Hurley J.H. Hicke L. EMBO J. 2003; 22: 1273-1281Crossref PubMed Scopus (238) Google Scholar). Because the control of ENaC surface expression is critical for regulating epithelial Na+ transport, we tested the role of Hrs in the sorting decision between ENaC degradation and recycling.DISCUSSIONThe sorting decision between ENaC degradation in lysosomes and ENaC recycling is a critical step in the regulation of epithelial Na+ transport. Signals that increase degradation (e.g. Nedd4-2) reduce Na+ transport, whereas signals that increase recycling (e.g. cAMP) increase Na+ transport. In this work, we identified an important role for Hrs in this trafficking decision. We found that ENaC bound to Hrs, an interaction that was augmented by the E3 ubiquitin ligase Nedd4-2. Importantly, disruption of Hrs activity reduced degradation of the cell surface pool of ENaC and enhanced ENaC recycling to the cell surface. Together with previous work, these findings suggest a model in which Nedd4-2 induces binding of ENaC to Hrs in endosomes, which in turn sorts ENaC to lysosomes for degradation.There are three potential mechanisms by which Nedd4-2 might increase ENaC binding to Hrs. First, previous work indicates that Nedd4-2 causes the redistribution of ENaC from the cell surface to endosomes (18Lu C. Pribanic S. Debonneville A. Jiang C. Rotin D. Traffic. 2007; 8: 1246-1264Crossref PubMed Scopus (97) Google Scholar), the site of localization of Hrs. This colocalization within a common compartment would increase the amount of ENaC available to bind to Hrs. Second, Nedd4-2 catalyzes ENaC ubiquitination (6Zhou R. Patel S.V. Snyder P.M. J. Biol. Chem. 2007; 282: 20207-20212Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar), which would mediate binding through an interaction with the Hrs ubiquitin-interacting motif. Third, Nedd4-2 could function as an adaptor to strengthen the interaction between ENaC and Hrs. Consistent with this possibility, we found that Nedd4-2 bound to Hrs. In this regard, ubiquitination of ENaC alone may not be the only feature that induces its association with Hrs. In addition to Nedd4-2, other HECT-type E3 ligases such as AIP4 and the yeast Rsp5 have been shown to interact with a wide variety of adaptors, some of which associate with ESCRT-0 (38Malik R. Marchese A. Mol. Biol. Cell. 2010; 21: 2529-2541Crossref PubMed Scopus (74) Google Scholar, 39Léon S. Erpapazoglou Z. Haguenauer-Tsapis R. Mol. Biol. Cell. 2008; 19: 2379-2388Crossref PubMed Scopus (69) Google Scholar, 40Ren J. Kee Y. Huibregtse J.M. Piper R.C. Mol. Biol. Cell. 2007; 18: 324-335Crossref PubMed Scopus (59) Google Scholar, 41Lin C.H. MacGurn J.A. Chu T. Stefan C.J. Emr S.D. Cell. 2008; 135: 714-725Abstract Full Text Full Text PDF PubMed Scopus (361) Google Scholar). Thus, a convergence of several mechanisms including multiple protein-protein interactions is likely to drive the ENaC-Hrs association we see here.ENaC gating is regulated in part by proteolytic cleavage of its extracellular domains, which converts inactive channels into active ones. Both proteolytically cleaved (active) and uncleaved (inactive) channels are present at the cell surface. Here we found that Hrs selectively controls trafficking of proteolytically cleaved channels. Expression of Hrs-ΔUIM dramatically slowed degradation of the cell surface pool of cleaved channels but not the surface pool of uncleaved channels. This observation is interesting because it provides evidence that proteolytically cleaved and uncleaved channels are trafficked differently within the endocytic pathway. In addition, it indicates that some membrane proteins are degraded through a mechanism independent of the Hrs UIM. The reason for this difference in degradation of cleaved and uncleaved channels is unclear. We found that both forms of αENaC coprecipitated with Hrs (Fig. 1A), indicating that both are delivered to the compartment where Hrs resides and that both bind to Hrs. Other parts of the ESCRT-0 complex are known to interact with ubiquitin, including the UIM of STAM and the Vps-27, Hrs, and STAM (VHS) domains of both Hrs and STAM (20Piper R.C. Luzio J.P. Curr. Opin. Cell Biol. 2007; 19: 459-465Crossref PubMed Scopus (133) Google Scholar, 21Raiborg C. Stenmark H. Nature. 2009; 458: 445-452Crossref PubMed Scopus (978) Google Scholar). Thus, the cleaved ENaC might be specifically reliant on recognition by the Hrs UIM, whereas other cargo, such as the uncleaved ENaC, might be recognized and sorted efficiently by other UIMs that reside in ESCRT-0.In previous work, we found that the kinases SGK and PKA reduce ENaC endocytosis by inhibiting its binding of Nedd4-2 (12Snyder P.M. Olson D.R. Thomas B.C. J. Biol. Chem. 2002; 277: 5-8Abstract Full Text Full Text PDF PubMed Scopus (385) Google Scholar, 14Snyder P.M. Olson D.R. Kabra R. Zhou R. Steines J.C. J. Biol. Chem. 2004; 279: 45753-45758Abstract Full Text Full Text PDF PubMed Scopus (196) Google Scholar). As a result, these kinases and their upstream activators (aldosterone and vasopressin, respectively) increase ENaC surface expression, and hence, epithelial Na+ absorption. cAMP (presumably via PKA) also increased ENaC surface expression through enhanced recycling (42Butterworth M.B. Edinger R.S. Johnson J.P. Frizzell R.A. J. Gen. Physiol. 2005; 125: 81-101Crossref PubMed Scopus (144) Google Scholar). Our current work provides a potential mechanism by which PKA (cAMP) and SGK could increase ENaC recycling. We found that SGK and cAMP reduced ENaC binding to Hrs. By decreasing ENaC sorting to lysosomes, this would increase the pool of ENaC available to recycle to the cell surface.The regulation of epithelial Na+ absorption is critical to maintain Na+ homeostasis and to control blood pressure. Defects in Na+ transport cause hypertension and lung diseases including cystic fibrosis. Our data suggest that the delivery of ENaC to the ESCRT machinery is a critical step in controlling Na+ transport. Mutations that cause inherited hypertension (Liddle syndrome) reduced ENaC binding to Hrs, as did signaling pathways critical for the regulation of Na+ transport. Thus, discovery of additional pathways that regulate ENaC binding to Hrs and identification of additional ESCRT proteins that modulate ENaC trafficking will provide new insight into mechanisms that regulate epithelial Na+ absorption. IntroductionThe epithelial Na+ channel ENaC 2The abbreviations used are: ENaCepithelial Na+ channelFRTFischer rat thyroidESCRTendosomal sorting complexes required for transportUIMubiquitin-interacting motifHrshepatocyte growth factor-regulated tyrosine kinase substrateSTAMsignal-transducing adaptor moleculeSGKserum- and glucocorticoid-inducible kinaseMTSET[2-(trimethylammonium)ethyl]methanethiosulfonate bromideNi-NTAnickel-nitrilotriacetic acid. is composed of three homologous subunits (α, β, and γ) and functions in Na+ transport across epithelia in the kidney collecting duct and connecting tubule, lung, and distal colon, where it plays a critical role in Na+ homeostasis (reviewed in Refs. 1Schild L. Rev. Physiol. Biochem. Pharmacol. 2004; 151: 93-107Crossref PubMed Scopus (100) Google Scholar and 2Snyder P.M. Endocrinology. 2005; 146: 5079-5085Crossref PubMed Scopus (193) Google Scholar). Defects in ENaC regulation cause most of the identified genetic forms of hypertension. For example, mutations in β- and γENaC cause Liddle syndrome, an autosomal dominant form of hypertension (3Lifton R.P. Science. 1996; 272: 676-680Crossref PubMed Scopus (562) Google Scholar). Conversely, loss of function mutations cause a salt-wasting disorder known as pseudohypoaldosteronism type 1 (3Lifton R.P. Science. 1996; 272: 676-680Crossref PubMed Scopus (562) Google Scholar). In the lung, ENaC controls the composition and quantity of airway surface liquid, and defective ENaC regulation may contribute to lung disease in cystic fibrosis (4Boucher R.C. Stutts M.J. Knowles M.R. Cantley L. Gatzy J.T. J. Clin. Invest. 1986; 78: 1245-1252Crossref PubMed Scopus (438) Google Scholar). Thus, understanding the mechanisms that regulate ENaC may provide new insights into the pathogenesis of hypertension, cystic fibrosis, and other diseases of Na+ homeostasis.Epithelial Na+ absorption is regulated in large part by mechanisms that control ENaC expression at the apical membrane of epithelia. Here, the E3 ubiquitin ligase Nedd4-2 plays an important role (reviewed in Ref. 5Snyder P.M. Sci. Signal. 2009; 2: pe41Crossref PubMed Scopus (53) Google Scholar). Nedd4-2 binds to ENaC channels located at the cell surface (6Zhou R. Patel S.V. Snyder P.M. J. Biol. Chem. 2007; 282: 20207-20212Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar). This interaction occurs through binding of PY motifs located within the C termini of α-, β-, and γENaC to multiple WW domains in Nedd4-2 (6Zhou R. Patel S.V. Snyder P.M. J. Biol. Chem. 2007; 282: 20207-20212Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar, 7Staub O. Dho S. Henry P. Correa J. Ishikawa T. McGlade J. Rotin D. EMBO J. 1996; 15: 2371-2380Crossref PubMed Scopus (734) Google Scholar, 8Snyder 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). Binding is critical for ENaC regulation. Disruption of binding by mutation of the PY motifs causes hypertension in Liddle syndrome (9Snyder 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 (397) Google Scholar, 10Firsov D. Schild L. Gautschi I. Mérillat A.M. Schneeberger E. Rossier B.C. Proc. Natl. Acad. Sci. U.S.A. 1996; 93: 15370-15375Crossref PubMed Scopus (395) Google Scholar, 11Knight K.K. Olson D.R. Zhou R. Snyder P.M. Proc. Natl. Acad. Sci. U.S.A. 2006; 103: 2805-2808Crossref PubMed Scopus (123) Google Scholar). Binding is also important for the hormonal regulation of ENaC. Aldosterone and vasopressin activate signaling pathways that lead to Nedd4-2 phosphorylation by serum- and glucocorticoid-inducible kinase (SGK) and cAMP-dependent protein kinase (PKA), respectively (5Snyder P.M. Sci. Signal. 2009; 2: pe41Crossref PubMed Scopus (53) Google Scholar). Phosphorylation reduces Nedd4-2 binding to ENaC, which enhances epithelial Na+ transport (12Snyder P.M. Olson D.R. Thomas B.C. J. Biol. Chem. 2002; 277: 5-8Abstract Full Text Full Text PDF PubMed Scopus (385) Google Scholar, 13Debonneville C. Flores S.Y. Kamynina E. Plant P.J. Tauxe C. Thomas M.A. Münster C. Chraïbi A. Pratt J.H. Horisberger J.D. Pearce D. Loffing J. Staub O. EMBO J. 2001; 20: 7052-7059Crossref PubMed Scopus (574) Google Scholar, 14Snyder P.M. Olson D.R. Kabra R. Zhou R. Steines J.C. J. Biol. Chem. 2004; 279: 45753-45758Abstract Full Text Full Text PDF PubMed Scopus (196) Google Scholar, 15Soundararajan R. Zhang T.T. Wang J. Vandewalle A. Pearce D. J. Biol. Chem. 2005; 280: 39970-39981Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar).Following binding, Nedd4-2 catalyzes mono- and polyubiquitination of each ENaC subunit (6Zhou R. Patel S.V. Snyder P.M. J. Biol. Chem. 2007; 282: 20207-20212Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar), which reduces ENaC surface expression through two distinct mechanisms. First, Nedd4-2 increases the rate of ENaC endocytosis (16Kabra R. Knight K.K. Zhou R. Snyder P.M. J. Biol. Chem. 2008; 283: 6033-6039Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar). This process involves epsin, which links ubiquitinated ENaC to the clathrin-based endocytic pathway (17Wang H. Traub L.M. Weixel K.M. Hawryluk M.J. Shah N. Edinger R.S. Perry C.J. Kester L. Butterworth M.B. Peters K.W. Kleyman T.R. Frizzell R.A. Johnson J.P. J. Biol. Chem. 2006; 281: 14129-14135Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar). Second, once in endosomes, Nedd4-2 increases ENaC degradation in lysosomes, which reduces ENaC recycling back to the cell surface (16Kabra R. Knight K.K. Zhou R. Snyder P.M. J. Biol. Chem. 2008; 283: 6033-6039Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar, 18Lu C. Pribanic S. Debonneville A. Jiang C. Rotin D. Traffic. 2007; 8: 1246-1264Crossref PubMed Scopus (97) Google Scholar).A key unanswered question is what are the endosomal proteins that recognize ubiquitinated ENaC and direct it to the lysosomal degradation pathway? Proteins that comprise the endosomal sorting complexes required for transport (ESCRT)-0 complex are strong candidates. ESCRT-0 is a heterodimer of hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs) and signal-transducing adaptor molecule (STAM) (reviewed in Refs. 20Piper R.C. Luzio J.P. Curr. Opin. Cell Biol. 2007; 19: 459-465Crossref PubMed Scopus (133) Google Scholar and 21Raiborg C. Stenmark H. Nature. 2009; 458: 445-452Crossref PubMed Scopus (978) Google Scholar). In yeast, where many of the details of the pathway have been elucidated, the corresponding proteins are vacuolar protein sorting 27p (Vps27p) and Hbp, STAM, and EAST (Hse1p) (22Piper R.C. Cooper A.A. Yang H. Stevens T.H. J. Cell Biol. 1995; 131: 603-617Crossref PubMed Scopus (340) Google Scholar, 23Bilodeau P.S. Urbanowski J.L. Winistorfer S.C. Piper R.C. Nat. Cell Biol. 2002; 4: 534-539Crossref PubMed Scopus (278) Google Scholar). The complex is localized to endosomes through the binding of a Fab1, YotB, Vacl, and EEA1 (FYVE) domain in Hrs to phosphatidylinositol 3-phosphate in the surface of endosomes (24Raiborg C. Bremnes B. Mehlum A. Gillooly D.J. D'Arrigo A. Stang E. Stenmark H. J. Cell Sci. 2001; 114: 2255-2263Crossref PubMed Google Scholar). Both Hrs and STAM contain ubiquitin-interacting motifs (UIMs), which allow them to bind to ubiquitinated proteins (25Hirano S. Kawasaki M. Ura H. Kato R. Raiborg C. Stenmark H. Wakatsuki S. Nat. Struct. Mol. Biol. 2006; 13: 272-277Crossref PubMed Scopus (144) Google Scholar, 26Mizuno E. Kawahata K. Kato M. Kitamura N. Komada M. Mol. Biol. Cell. 2003; 14: 3675-3689Crossref PubMed Scopus (96) Google Scholar). In concert with additional ESCRT complexes, Hrs and STAM are thought to direct ubiquitinated membrane proteins into the endosomal lumen, forming multivesicular bodies that fuse with lysosomes to induce protein degradation. Consistent with this model, disruption of the Hrs-STAM complex or yeast homologues reduced lysosomal/vacuolar degradation of several cargo proteins, including the epithelial growth factor receptor (27Stern K.A. Visser Smit G.D. Place T.L. Winistorfer S. Piper R.C. Lill N.L. Mol. Cell. Biol. 2007; 27: 888-898Crossref PubMed Scopus (54) Google Scholar), the G protein-coupled receptor CXCR4 (28Marchese A. Raiborg C. Santini F. Keen J.H. Stenmark H. Benovic J.L. Dev. Cell. 2003; 5: 709-722Abstract Full Text Full Text PDF PubMed Scopus (313) Google Scholar), and the yeast α-factor receptor Ste3p (23Bilodeau P.S. Urbanowski J.L. Winistorfer S.C. Piper R.C. Nat. Cell Biol. 2002; 4: 534-539Crossref PubMed Scopus (278) Google Scholar, 29Shih S.C. Prag G. Francis S.A. Sutanto M.A. Hurley J.H. Hicke L. EMBO J. 2003; 22: 1273-1281Crossref PubMed Scopus (238) Google Scholar). Targeting to the endosomal lumen is dependent on the UIMs in Hrs/Vps27p and STAM/Hse1p (23Bilodeau P.S. Urbanowski J.L. Winistorfer S.C. Piper R.C. Nat. Cell Biol. 2002; 4: 534-539Crossref PubMed Scopus (278) Google Scholar, 29Shih S.C. Prag G. Francis S.A. Sutanto M.A. Hurley J.H. Hicke L. EMBO J. 2003; 22: 1273-1281Crossref PubMed Scopus (238) Google Scholar). Because the control of ENaC surface expression is critical for regulating epithelial Na+ transport, we tested the role of Hrs in the sorting decision between ENaC degradation and recycling.
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