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• W2017207705 abstract "Transforming growth factor (TGF)-β family proteins are synthesized as precursors that are cleaved to generate an active ligand. Previous studies suggest that TGF-β activity can be controlled by lysosomal degradation of both precursor proteins and ligands, but how these soluble proteins are trafficked to the lysosome is incompletely understood. The current studies show that sortilin selectively co-immunoprecipitates with the cleaved prodomain and/or precursor form of TGF-β family members. Furthermore, sortilin co-localizes with, and enhances accumulation of a nodal family member in the Golgi. Co-expression of sortilin with TGF-β family members leads to decreased accumulation of precursor proteins and cleavage products and this is attenuated by lysosomal, but not proteosomal inhibitors. In Xenopus embryos, overexpression of sortilin leads to a decrease in phospho-Smad2 levels and phenocopies loss of nodal signaling. Conversely, down-regulation of sortilin expression in HeLa cells leads to an up-regulation of endogenous bone morphogenic protein pathway activation, as indicated by an increase in phospho-Smad1/5/8 levels. Our results suggest that sortilin negatively regulates TGF-β signaling by diverting trafficking of precursor proteins to the lysosome during transit through the biosynthetic pathway. Transforming growth factor (TGF)-β family proteins are synthesized as precursors that are cleaved to generate an active ligand. Previous studies suggest that TGF-β activity can be controlled by lysosomal degradation of both precursor proteins and ligands, but how these soluble proteins are trafficked to the lysosome is incompletely understood. The current studies show that sortilin selectively co-immunoprecipitates with the cleaved prodomain and/or precursor form of TGF-β family members. Furthermore, sortilin co-localizes with, and enhances accumulation of a nodal family member in the Golgi. Co-expression of sortilin with TGF-β family members leads to decreased accumulation of precursor proteins and cleavage products and this is attenuated by lysosomal, but not proteosomal inhibitors. In Xenopus embryos, overexpression of sortilin leads to a decrease in phospho-Smad2 levels and phenocopies loss of nodal signaling. Conversely, down-regulation of sortilin expression in HeLa cells leads to an up-regulation of endogenous bone morphogenic protein pathway activation, as indicated by an increase in phospho-Smad1/5/8 levels. Our results suggest that sortilin negatively regulates TGF-β signaling by diverting trafficking of precursor proteins to the lysosome during transit through the biosynthetic pathway. IntroductionMembers of the TGF-β family, which include TGF-βs, activin, nodal, and bone morphogenic proteins (BMP) 2The abbreviations used are: BMPbone morphogenic proteinXnrXenopus nodal-related proteinVPS10pvacuolar protein sorting 10 proteinERendoplasmic reticulumPCproprotein convertaseGFPgreen fluorescent proteinAPPamyloid precursor proteinGLUT4glucose transporter isoform 4ANOVAanalysis of variance. among others, play critical roles in lineage selection and differentiation of almost all cell types during embryonic development (1Moustakas A. Heldin C.H. Development. 2009; 136: 3699-3714Crossref PubMed Scopus (678) Google Scholar). Given their multifunctional nature, it is not surprising that TGF-β family activity is regulated at many levels, including the level of proteolytic activation (2Sopory S. Christian J.L. Whitman M. Sater A. Methods in Signal Transduction. CRC Press, Boca Raton, FL2006: 37-60Google Scholar). All family members are synthesized as inactive precursor proteins consisting of an amino (N)-terminal prodomain followed by a carboxyl-terminal mature ligand domain. Precursor proteins dimerize in the endoplasmic reticulum (ER) and are cleaved by members of the proprotein convertase (PC) family of serine proteases. Cleavage occurs during transit through the biosynthetic pathway or outside of cells, and is required to generate a mature ligand that can bind and activate its cognate receptor.TGF-β ligands bind to a complex of transmembrane serine-threonine kinase receptors, which activate signaling by phosphorylating receptor-regulated Smad proteins (R-Smads) (1Moustakas A. Heldin C.H. Development. 2009; 136: 3699-3714Crossref PubMed Scopus (678) Google Scholar). Phosphorylated R-Smads bind to the common mediator Smad, Smad4, and this protein complex then moves into the nucleus to regulate transcription of target genes. Although TGF-β ligands bind their receptors at the cell surface, endocytosis of activated receptors is required in most cases for intracellular signal transduction (3Chen Y.G. Cell Res. 2009; 19: 58-70Crossref PubMed Scopus (206) Google Scholar).Receptor-mediated endocytosis is not only required for TGF-β signal transduction, but it also plays an important role in signal termination by targeting the receptor and/or bound ligand to the lysosome for degradation (3Chen Y.G. Cell Res. 2009; 19: 58-70Crossref PubMed Scopus (206) Google Scholar). Several accessory proteins involved in targeting receptors for degradation have been identified. For example, Dapper 2, a late-endosomal protein, binds and facilitates lysosomal degradation of certain TGF-β type I receptors, leading to down-regulation of activin/nodal signaling in zebrafish, Xenopus, and mouse (4Zhang L. Zhou H. Su Y. Sun Z. Zhang H. Zhang L. Zhang Y. Ning Y. Chen Y.G. Meng A. Science. 2004; 306: 114-117Crossref PubMed Scopus (108) Google Scholar). The antagonistic Smad, Smad7, can also promote lysosomal targeting of some TGF-β type I receptors by recruiting the E3 ubiquitin ligases Smurf1 and Smurf2 and inducing ubiquitination (5Ebisawa T. Fukuchi M. Murakami G. Chiba T. Tanaka K. Imamura T. Miyazono K. J. Biol. Chem. 2001; 276: 12477-12480Abstract Full Text Full Text PDF PubMed Scopus (687) Google Scholar, 6Kavsak P. Rasmussen R.K. Causing C.G. Bonni S. Zhu H. Thomsen G.H. Wrana J.L. Mol. Cell. 2000; 6: 1365-1375Abstract Full Text Full Text PDF PubMed Scopus (1090) Google Scholar). Finally, the extracellular protein Bmper (BMP-binding endothelial cell precursor-derived regulator, also known as crossveinless2) binds BMP4 and stimulates receptor-dependent internalization and lysosomal degradation of BMP4 and its receptor (7Kelley R. Ren R. Pi X. Wu Y. Moreno I. Willis M. Moser M. Ross M. Podkowa M. Attisano L. Patterson C. J. Cell Biol. 2009; 184: 597-609Crossref PubMed Scopus (90) Google Scholar).TGF-β family precursor proteins and cleavage products can also be targeted for degradation independent of receptor binding. For example, the BMP4 precursor protein is sequentially cleaved at two sites within the prodomain and this regulates ligand stability (8Cui Y. Hackenmiller R. Berg L. Jean F. Nakayama T. Thomas G. Christian J.L. Genes Dev. 2001; 15: 2797-2802PubMed Google Scholar). Cleavage at the first site generates a non-covalently associated ligand-prodomain complex that is targeted for lysosomal degradation (9Degnin C. Jean F. Thomas G. Christian J.L. Mol. Biol. Cell. 2004; 15: 5012-5020Crossref PubMed Scopus (86) Google Scholar), whereas subsequent cleavage at the second site, which occurs in a tissue-specific fashion (10Goldman D.C. Hackenmiller R. Nakayama T. Sopory S. Wong C. Kulessa H. Christian J.L. Development. 2006; 133: 1933-1942Crossref PubMed Scopus (59) Google Scholar), releases the ligand from the prodomain and thus stabilizes it. The prodomain of the zebrafish nodal-related protein, cyclops, contains a lysosomal targeting signal that destabilizes the precursor and thus restricts the signaling range of the mature ligand (11Tian J. Andrée B. Jones C.M. Sampath K. Development. 2008; 135: 2649-2658Crossref PubMed Scopus (27) Google Scholar). By contrast, the prodomain of mouse nodal functions in cis to stabilize the precursor (12Le Good J.A. Joubin K. Giraldez A.J. Ben-Haim N. Beck S. Chen Y. Schier A.F. Constam D.B. Curr. Biol. 2005; 15: 31-36Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar), whereas PC-mediated cleavage generates a mature ligand that is delivered to lysosomes via multivesicular bodies (13Blanchet M.H. Le Good J.A. Oorschot V. Baflast S. Minchiotti G. Klumperman J. Constam D.B. Sci. Signal. 2008; 1: ra13Crossref PubMed Scopus (34) Google Scholar). The intrinsic instability of mature nodal is dependent on the prodomain because ligand generated from a chimeric precursor containing a dorsalin prodomain is substantially stabilized (14Constam D.B. Robertson E.J. J. Cell Biol. 1999; 144: 139-149Crossref PubMed Scopus (259) Google Scholar). The mechanism by which these soluble proteins are targeted for lysosomal degradation has not been investigated, but the observation that precursor proteins can be destabilized by elements in the prodomain prior to cleavage and receptor binding suggests the involvement of alternate trafficking receptors.Sortilin is a good candidate for an intracellular sorting receptor involved in lysosomal targeting of TGF-β family members (15Willnow T.E. Petersen C.M. Nykjaer A. Nat. Rev. Neurosci. 2008; 9: 899-909Crossref PubMed Scopus (58) Google Scholar, 16Hermey G. Cell Mol. Life Sci. 2009; 66: 2677-2689Crossref PubMed Scopus (142) Google Scholar). It is one of five members of a protein family that are structurally related to yeast vacuolar protein sorting 10 protein (Vps10p), which functions to traffic lysosomal enzymes from the Golgi to the vacuole (17Marcusson E.G. Horazdovsky B.F. Cereghino J.L. Gharakhanian E. Emr S.D. Cell. 1994; 77: 579-586Abstract Full Text PDF PubMed Scopus (397) Google Scholar). Sortilin is synthesized as a precursor molecule that is converted to its mature form by PC-catalyzed removal of its propeptide in a late Golgi compartment (18Munck Petersen C. Nielsen M.S. Jacobsen C. Tauris J. Jacobsen L. Tauris J. Jacobsen L. Gliemann J. Moestrup S.K. Madsen P. EMBO J. 1999; 18: 595-604Crossref PubMed Scopus (168) Google Scholar). The luminal portion of sortilin is composed almost entirely of the Vps10p domain, a 10-bladed β-propeller fold involved in ligand binding (19Quistgaard E.M. Madsen P. Groftehauge M.K. Nissen P. Petersen C.M. Thirup S.S. Nat. Struct. Mol. Biol. 2009; 16: 96-98Crossref PubMed Scopus (124) Google Scholar). This is followed by a transmembrane-spanning region and a short cytoplasmic tail that is homologous to that of the cation independent mannose 6-phosphate receptor, the major protein that routes soluble hydolases to the lysosome. The cytoplasmic tail of sortilin contains motifs that mediate rapid endocytosis and Golgi body to endosome trafficking (20Nielsen M.S. Madsen P. Christensen E.I. Nykjaer A. Gliemann J. Kasper D. Pohlmann R. Petersen C.M. EMBO J. 2001; 20: 2180-2190Crossref PubMed Scopus (345) Google Scholar). Newly synthesized sortilin moves to the cell surface, is internalized and shuttles between the trans-Golgi network (TGN), endosomal compartments, and the plasma membrane thereafter. Only a small fraction of sortilin (∼10%) is found at the cell surface, and yet sortilin has been shown to direct trafficking of substrates not only in the biosynthetic pathway but also during endocytic retrieval from the cell surface (20Nielsen M.S. Madsen P. Christensen E.I. Nykjaer A. Gliemann J. Kasper D. Pohlmann R. Petersen C.M. EMBO J. 2001; 20: 2180-2190Crossref PubMed Scopus (345) Google Scholar, 21Petersen C.M. Nielsen M.S. Nykjaer A. Jacobsen L. Tommerup N. Rasmussen H.H. Roigaard H. Gliemann J. Madsen P. Moestrup S.K. J. Biol. Chem. 1997; 272: 3599-3605Abstract Full Text Full Text PDF PubMed Scopus (337) Google Scholar).In addition to sorting a subset of soluble hydrolases to the lysosome, sortilin modulates trafficking of a variety of non-lysosomal proteins, either restricting or facilitating their function (16Hermey G. Cell Mol. Life Sci. 2009; 66: 2677-2689Crossref PubMed Scopus (142) Google Scholar). For example, within the biosynthetic pathway, sortilin facilitates secretion of apolipoprotein B100-containing lipoproteins, thereby regulating plasma cholesterol levels (22Kjolby M. Andersen O.M. Breiderhoff T. Fjorback A.W. Pedersen K.M. Madsen P. Jansen P. Heeren J. Willnow T.E. Nykjaer A. Cell Metab. 2010; 12: 213-223Abstract Full Text Full Text PDF PubMed Scopus (202) Google Scholar). It also controls sorting of brain-derived neurotrophic factor to the regulated secretory pathway in neurons (23Chen Z.Y. Ieraci A. Teng H. Dall H. Meng C.X. Herrera D.G. Nykjaer A. Hempstead B.L. Lee F.S. J. Neurosci. 2005; 25: 6156-6166Crossref PubMed Scopus (320) Google Scholar) and enhances anterograde transport of Trk family receptors (24Vaegter C.B. Jansen P. Fjorback A.W. Glerup S. Skeldal S. Kjolby M. Richner M. Erdmann B. Nyengaard J.R. Tessarollo L. Lewin G.R. Willnow T.E. Chao M.V. Nykjaer A. Nat. Neurosci. 2011; 14: 54-61Crossref PubMed Scopus (137) Google Scholar). At the cell surface, sortilin negatively regulates progranulin levels by facilitating their endocytosis and degradation (25Hu F. Padukkavidana T. Vaegter C.B. Brady O.A. Zheng Y. Mackenzie I.R. Feldman H.H. Nykjaer A. Strittmatter S.M. Neuron. 2010; 68: 654-667Abstract Full Text Full Text PDF PubMed Scopus (367) Google Scholar). Furthermore, sortilin forms a tripartite complex with the nerve growth factor (NGF) precursor, pro-NGF, and its receptor p75NTR to induce rapid internalization and activate a signaling pathway that leads to neuronal apopotosis (26Nykjaer A. Lee R. Teng K.K. Jansen P. Madsen P. Nielsen M.S. Jacobsen C. Kliemannel M. Schwarz E. Willnow T.E. Hempstead B.L. Petersen C.M. Nature. 2004; 427: 843-848Crossref PubMed Scopus (786) Google Scholar).In the present study, we asked whether sortilin plays a role in intracellular trafficking of TGF-β family proteins. Our results demonstrate that sortilin is a novel inhibitor of TGF-β family signaling that can bind precursor proteins in the biosynthetic pathway and promote their trafficking to the lysosome for degradation.DISCUSSIONThe current studies suggest that sortilin functions as a novel negative regulator of TGF-β family activity. Specifically, our data demonstrate that overexpressed sortilin facilitates trafficking of TGF-β precursor proteins to the lysosome for degradation. The physiological relevance of this negative regulatory mechanism is supported by our data showing that depletion of endogenous sortilin leads to enhanced BMP pathway activation, whereas enhancement of sortilin levels leads to a decrease in endogenous TGF-β signaling.Although our studies support a role for endogenous sortilin as a negative regulator of TGF-β family activity, mice mutant for Sortilin are grossly normal (37Jansen P. Giehl K. Nyengaard J.R. Teng K. Lioubinski O. Sjoegaard S.S. Breiderhoff T. Gotthardt M. Lin F. Eilers A. Petersen C.M. Lewin G.R. Hempstead B.L. Willnow T.E. Nykjaer A. Nat. Neurosci. 2007; 10: 1449-1457Crossref PubMed Scopus (232) Google Scholar), whereas mice carrying mutations in critical negative regulators of TGF-β activity show a variety of defects, in some cases leading to lethality (38Walsh D.W. Godson C. Brazil D.P. Martin F. Trends Cell Biol. 2010; 20: 244-256Abstract Full Text Full Text PDF PubMed Scopus (187) Google Scholar). These phenotypic differences raise the possibility that sortilin functions primarily as a buffer to fine-tune the activity of its many substrates rather than providing an on-off switch for activity. Alternatively, other members of the VPS10 protein family may function redundantly to complement sortilin function in vivo. Consistent with either possibility, mice lacking Sortilin do not phenocopy mice mutant for other known sortilin substrates, although defects can often be uncovered on a sensitized background. For example, Sortilin nullizygous mice do not exhibit signs of lysosomal pathologies despite evidence that sortilin functions as a trafficking receptor for several soluble lysosomal proteins (39Zeng J. Racicott J. Morales C.R. Exp. Cell Res. 2009; 315: 3112-3124Crossref PubMed Scopus (43) Google Scholar). Furthermore, although sortilin is recognized as a crucial component of the proneurotrophin-p75NTR signaling complex that controls apoptotic cell death during neurogenesis (26Nykjaer A. Lee R. Teng K.K. Jansen P. Madsen P. Nielsen M.S. Jacobsen C. Kliemannel M. Schwarz E. Willnow T.E. Hempstead B.L. Petersen C.M. Nature. 2004; 427: 843-848Crossref PubMed Scopus (786) Google Scholar), Sortilin-deficient mice show reduced apoptosis in only a small subset of neurons known to undergo developmentally regulated, p75NTR-dependent apoptosis (37Jansen P. Giehl K. Nyengaard J.R. Teng K. Lioubinski O. Sjoegaard S.S. Breiderhoff T. Gotthardt M. Lin F. Eilers A. Petersen C.M. Lewin G.R. Hempstead B.L. Willnow T.E. Nykjaer A. Nat. Neurosci. 2007; 10: 1449-1457Crossref PubMed Scopus (232) Google Scholar). As a final example, sortilin facilitates hepatic export of lipoproteins in vivo, which is required to prevent hypercholesterolemia. This function is only apparent in Sortilin mutants, however, when plasma low-density lipoprotein levels are elevated above physiological levels by either diet or genetic background (22Kjolby M. Andersen O.M. Breiderhoff T. Fjorback A.W. Pedersen K.M. Madsen P. Jansen P. Heeren J. Willnow T.E. Nykjaer A. Cell Metab. 2010; 12: 213-223Abstract Full Text Full Text PDF PubMed Scopus (202) Google Scholar). Further in-depth analysis in mice mutant for multiple members of the VPS10 protein family is needed to document whether redundancy accounts for the relatively minor defects present in Sortilin mutant mice.Mechanisms that regulate intracellular trafficking, stability, and proteolytic maturation of TGF-β family precursor proteins are poorly understood. Cleavage of pro-BMP4 is proposed to occur within the TGN and/or endosomes as the precursor traverses the biosynthetic pathway (9Degnin C. Jean F. Thomas G. Christian J.L. Mol. Biol. Cell. 2004; 15: 5012-5020Crossref PubMed Scopus (86) Google Scholar). By contrast, the nodal precursor arrives at the cell surface intact via a pathway that bypasses the TGN, but then undergoes rapid endocytic uptake, prodomain removal, and delivery to lysosomes (13Blanchet M.H. Le Good J.A. Oorschot V. Baflast S. Minchiotti G. Klumperman J. Constam D.B. Sci. Signal. 2008; 1: ra13Crossref PubMed Scopus (34) Google Scholar, 32Blanchet M.H. Le Good J.A. Mesnard D. Oorschot V. Baflast S. Minchiotti G. Klumperman J. Constam D.B. EMBO J. 2008; 27: 2580-2591Crossref PubMed Scopus (62) Google Scholar). The receptor involved in cell surface retrieval and lysosomal targeting of nodal is unknown. Interestingly, the precursor form of NGF is also secreted intact and must undergo endocytic uptake and intracellular cleavage to generate the mature ligand (40Boutilier J. Ceni C. Pagdala P.C. Forgie A. Neet K.E. Barker P.A. J. Biol. Chem. 2008; 283: 12709-12716Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). Sortilin binds to the NGF precursor at the cell surface, which results in its rapid internalization and cleavage (26Nykjaer A. Lee R. Teng K.K. Jansen P. Madsen P. Nielsen M.S. Jacobsen C. Kliemannel M. Schwarz E. Willnow T.E. Hempstead B.L. Petersen C.M. Nature. 2004; 427: 843-848Crossref PubMed Scopus (786) Google Scholar, 41Bronfman F.C. Tcherpakov M. Jovin T.M. Fainzilber M. J. Neurosci. 2003; 23: 3209-3220Crossref PubMed Google Scholar), raising the possibility that sortilin might function in a similar manner to direct endocytic retrieval and lysosomal targeting of nodal. Contrary to this prediction, however, we found that ectopic sortilin did not enhance uptake of nodal from the cell surface, but instead appeared to divert and/or retain nodal within the TGN as it trafficked through the biosynthetic pathway. This function is analogous to the role of the sortilin family member, SorLA, in intracellular trafficking of the amyloid precursor protein (APP) (15Willnow T.E. Petersen C.M. Nykjaer A. Nat. Rev. Neurosci. 2008; 9: 899-909Crossref PubMed Scopus (58) Google Scholar). Specifically, SorLA does not affect retrograde trafficking of APP from the cell surface, but instead acts as a retention factor to prolong residence time for nascent APP molecules in the TGN, and may also shuttle APP back to the TGN from endosomes (42Spoelgen R. von Arnim C.A. Thomas A.V. Peltan I.D. Koker M. Deng A. Irizarry M.C. Andersen O.M. Willnow T.E. Hyman B.T. J. Neurosci. 2006; 26: 418-428Crossref PubMed Scopus (149) Google Scholar, 43Schmidt V. Sporbert A. Rohe M. Reimer T. Rehm A. Andersen O.M. Willnow T.E. J. Biol. Chem. 2007; 282: 37906-37912Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar). In the case of APP, this prevents release of the precursor into pathways that promote proteolytic processing into disease causing, amyloidogenic peptides (reviewed in Ref. 15Willnow T.E. Petersen C.M. Nykjaer A. Nat. Rev. Neurosci. 2008; 9: 899-909Crossref PubMed Scopus (58) Google Scholar). Collectively, our results are consistent with a role for endogenous sortilin in enhancing trafficking of nodal and other TGF-β precursors through the TGN to the lysosome. It is possible that assays of endogenous nodal trafficking, which are not currently feasible, and/or analysis of trafficking in cells that express nodal receptors and co-receptors will show that sortilin has a role in endocytic uptake of precursors as well.Our data showing that deletion mutant forms of sortilin lacking most of the VPS10 ligand-binding domain are able to co-immunopurify with TGF-β precursor proteins are unexpected but not unprecedented. For example, following the initial demonstration that SorLA and APP interact directly via the luminal domain of each protein (44Andersen O.M. Reiche J. Schmidt V. Gotthardt M. Spoelgen R. Behlke J. von Arnim C.A. Breiderhoff T. Jansen P. Wu X. Bales K.R. Cappai R. Masters C.L. Gliemann J. Mufson E.J. Hyman B.T. Paul S.M. Nykjaer A. Willnow T.E. Proc. Natl. Acad. Sci. U.S.A. 2005; 102: 13461-13466Crossref PubMed Scopus (485) Google Scholar), subsequent studies demonstrated a secondary binding site between the cytoplasmic domains of each protein (42Spoelgen R. von Arnim C.A. Thomas A.V. Peltan I.D. Koker M. Deng A. Irizarry M.C. Andersen O.M. Willnow T.E. Hyman B.T. J. Neurosci. 2006; 26: 418-428Crossref PubMed Scopus (149) Google Scholar). Similarly, although it has been show that sortilin interacts directly with glucose transporter isoform 4 (GLUT4) and plays a major role in the formation of GLUT4-containing insulin responsive vesicles (45Shi J. Kandror K.V. Dev. Cell. 2005; 9: 99-108Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar), more recent structure-function studies have revealed that this is not a direct binary interaction but instead requires formation of a protein complex consisting of multiple additional partners (46Shi J. Kandror K.V. J. Biol. Chem. 2007; 282: 9008-9016Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar, 47Bogan J.S. Kandror K.V. Curr. Opin. Cell Biol. 2010; 22: 506-512Crossref PubMed Scopus (81) Google Scholar). Our data, which show that deletion mutant forms of sortilin lacking complimentary regions of the luminal domain are able to co-immunopurify with TGF-β family precursors, suggest a similar model in which additional binding partners function as a scaffold to mediate interactions between different domains of sortilin and TGF-β precursor proteins.In overexpression assays, deletion mutant forms of sortilin that lack the cytoplasmic tail and its attendant trafficking signals retain the ability to accelerate degradation of TGF-β precursor proteins. There are several potential explanations for this seemingly anomalous result. First although chimeric analysis shows that signals in the sortilin tail domain are sufficient to direct Golgi to endosome transport and retrieval, additional intrinsic targeting signals appear to be present within the luminal domain because truncated forms of sortilin that lack either specific sorting signals or the entire cytoplasmic domain localize to the late Golgi rather than reaching the plasma membrane (20Nielsen M.S. Madsen P. Christensen E.I. Nykjaer A. Gliemann J. Kasper D. Pohlmann R. Petersen C.M. EMBO J. 2001; 20: 2180-2190Crossref PubMed Scopus (345) Google Scholar, 48Lefrancois S. Zeng J. Hassan A.J. Canuel M. Morales C.R. EMBO J. 2003; 22: 6430-6437Crossref PubMed Scopus (164) Google Scholar). Our observation that SortN is enriched in the Golgi suggests that the extreme N terminus of sortilin contains information sufficient to direct Golgi localization independent of signals in the cytoplasmic tail. Using similar functional assays, previously published studies have shown that a wide range of deletion mutant forms of sortilin, or SorLA, retain function in mediating assembly of GLUT4-containing vesicles and in trafficking of APP (42Spoelgen R. von Arnim C.A. Thomas A.V. Peltan I.D. Koker M. Deng A. Irizarry M.C. Andersen O.M. Willnow T.E. Hyman B.T. J. Neurosci. 2006; 26: 418-428Crossref PubMed Scopus (149) Google Scholar, 46Shi J. Kandror K.V. J. Biol. Chem. 2007; 282: 9008-9016Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar). These findings have been interpreted to suggest the involvement of additional accessory proteins in directing intracellular trafficking and other functions of sortilin containing protein complexes. As one example of this, the neurotrophin receptor p75NTR has been shown to function as a trafficking switch that impairs lysosomal sorting of sortilin and its cargo, pro-NGF, and instead diverts trafficking to the cell surface (49Kim T. Hempstead B.L. EMBO J. 2009; 28: 1612-1623Crossref PubMed Scopus (40) Google Scholar). A second possible explanation for the ability of the deletion mutants to induce degradation of TGF-β precursors is that these mutants are misfolded and aggregate in the ER, leading to ER stress and protein degradation. Sortilin-dependent degradation is selective for TGF-β proteins, however, and is not dependent on proteosomal function for the subset of the proteins that we have tested, demonstrating that the ER-associated degradation pathway has not been activated. Alternatively, it is possible that sortilin mutants aggregate in post-ER compartments, leading to lysosmal targeting similar to that observed for the PC family member, furin. Signals within the cytoplasmic tail of furin direct localization predominantly to the TGN, but in the absence of these signals, determinants in the luminal domain induce aggregation in the TGN followed by lysosomal targeting (50Wolins N. Bosshart H. Küster H. Bonifacino J.S. J. Cell Biol. 1997; 139: 1735-1745Crossref PubMed Scopus (62) Google Scholar). It is possible that similar signals function to direct aggregation and lysosomal trafficking of TGF-β family proteins bound to sortilin mutants. If so, ectopically expressed deletion mutant forms of sortilin may mediate degradation of TGF-β family members through a distinct mechanism (aggregation induced lysosomal targeting) than that used by wild type sortilin (targeting mediated by signals in the cytoplasmic tail). Further studies will be required to determine whether ER-associated degradation, ER stress-induced autophagy (51Lamark T. Johansen T. Curr. Opin. Cell Biol. 2010; 22: 192-198Crossref PubMed Scopus (110) Google Scholar), or aggregation contribute to degradation induced by individual sortilin mutants.Taken together with previously published studies, our results are most consistent with a model in which a trafficking complex, consisting of TGF-β family precursor proteins and multiple other binding partners, assembles and escorts precursor proteins to the cell surface. We propose that sortilin is one component of this trafficking complex that can dominantly route a subset of precursor proteins to the lysosome for degradation. Our results suggest that the level of lysosomal degradation depends on the relative level of sortilin expressed by a given cell type. An alternate model is that TGF-β proteins normally associate with an endogenous trafficking complex that escorts precursors to the cell surface via a TGN-independent route. Sortilin may compete with components of this complex for binding to TGF-β precursors, thereby diverting trafficking through the TGN to the lysosome. This scenario is consistent with our observation that ectopic sortilin can divert trafficking of TGF-β precursor proteins even when expressed at substoichiometric ratios. Future studies will be required to definitively test these models and identify additional components of TGF-β trafficking complexes. IntroductionMembers of the TGF-β family, which include TGF-βs, activin, nodal, and bone morphogenic proteins (BMP) 2The abbreviations used are: BMPbone morphogenic proteinXnrXenopus nodal-related proteinVPS10pvacuolar protein sorting 10 proteinERendoplasmic reticulumPCproprotein convertaseGFPgreen fluorescent proteinAPPamyloid precursor proteinGLUT4glucose transporter isoform 4ANOVAanalysis of variance. among others, play critical roles in lineage selection and differentiation of almost all cell types during embryonic development (1Moustakas A. Heldin C.H. Development. 2009; 136: 3699-3714Crossref PubMed Scopus (678) Google Scholar). Given their multifunctional nature, it is not surprising that TGF-β family activity is regulated at many levels, including the level of proteolytic activation (2Sopory S. Christian J.L. Whitman M. Sater A. Methods in Signal Transduction. CRC Press, Boca Raton, FL2006: 37-60Google Scholar). All family members are synthesized as inactive precursor proteins consisting of an amino (N)-terminal prodomain followed by a carboxyl-terminal mature ligand domain. Precursor proteins dimerize in the endoplasmic reticulum (ER) and are cleaved by members of the proprotein convertase (PC) family of serine proteases. Cleavage occurs during transit through the biosynthetic pathway or outside of cells, and is r" @default.
• W2017207705 created "2016-06-24" @default.
• W2017207705 creator A5008899032 @default.
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• W2017207705 date "2011-06-01" @default.
• W2017207705 modified "2023-09-30" @default.
• W2017207705 title "Sortilin Associates with Transforming Growth Factor-β Family Proteins to Enhance Lysosome-mediated Degradation" @default.
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