WNT ligand biogenesis and trafficking (Homo sapiens)

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2. Barrott JJ, Cash GM, Smith AP, Barrow JR, Murtaugh LC.; ''Deletion of mouse Porcn blocks Wnt ligand secretion and reveals an ectodermal etiology of human focal dermal hypoplasia/Goltz syndrome.''; PubMed Europe PMC Scholia
3. Belenkaya TY, Wu Y, Tang X, Zhou B, Cheng L, Sharma YV, Yan D, Selva EM, Lin X.; ''The retromer complex influences Wnt secretion by recycling wntless from endosomes to the trans-Golgi network.''; PubMed Europe PMC Scholia
4. Bartscherer K, Pelte N, Ingelfinger D, Boutros M.; ''Secretion of Wnt ligands requires Evi, a conserved transmembrane protein.''; PubMed Europe PMC Scholia
5. Port F, Hausmann G, Basler K.; ''A genome-wide RNA interference screen uncovers two p24 proteins as regulators of Wingless secretion.''; PubMed Europe PMC Scholia
6. Port F, Kuster M, Herr P, Furger E, Bänziger C, Hausmann G, Basler K.; ''Wingless secretion promotes and requires retromer-dependent cycling of Wntless.''; PubMed Europe PMC Scholia
7. Willert K, Brown JD, Danenberg E, Duncan AW, Weissman IL, Reya T, Yates JR, Nusse R.; ''Wnt proteins are lipid-modified and can act as stem cell growth factors.''; PubMed Europe PMC Scholia
8. Eaton S.; ''Release and trafficking of lipid-linked morphogens.''; PubMed Europe PMC Scholia
9. Najdi R, Proffitt K, Sprowl S, Kaur S, Yu J, Covey TM, Virshup DM, Waterman ML.; ''A uniform human Wnt expression library reveals a shared secretory pathway and unique signaling activities.''; PubMed Europe PMC Scholia
10. Buechling T, Chaudhary V, Spirohn K, Weiss M, Boutros M.; ''p24 proteins are required for secretion of Wnt ligands.''; PubMed Europe PMC Scholia
11. Smolich BD, McMahon JA, McMahon AP, Papkoff J.; ''Wnt family proteins are secreted and associated with the cell surface.''; PubMed Europe PMC Scholia
12. Kadowaki T, Wilder E, Klingensmith J, Zachary K, Perrimon N.; ''The segment polarity gene porcupine encodes a putative multitransmembrane protein involved in Wingless processing.''; PubMed Europe PMC Scholia
13. Kurayoshi M, Yamamoto H, Izumi S, Kikuchi A.; ''Post-translational palmitoylation and glycosylation of Wnt-5a are necessary for its signalling.''; PubMed Europe PMC Scholia
14. Coudreuse DY, Roël G, Betist MC, Destrée O, Korswagen HC.; ''Wnt gradient formation requires retromer function in Wnt-producing cells.''; PubMed Europe PMC Scholia
15. MacDonald BT, Tamai K, He X.; ''Wnt/beta-catenin signaling: components, mechanisms, and diseases.''; PubMed Europe PMC Scholia
16. Port F, Basler K.; ''Wnt trafficking: new insights into Wnt maturation, secretion and spreading.''; PubMed Europe PMC Scholia
17. Bänziger C, Soldini D, Schütt C, Zipperlen P, Hausmann G, Basler K.; ''Wntless, a conserved membrane protein dedicated to the secretion of Wnt proteins from signaling cells.''; PubMed Europe PMC Scholia
18. Korkut C, Ataman B, Ramachandran P, Ashley J, Barria R, Gherbesi N, Budnik V.; ''Trans-synaptic transmission of vesicular Wnt signals through Evi/Wntless.''; PubMed Europe PMC Scholia
19. Simons M, Raposo G.; ''Exosomes--vesicular carriers for intercellular communication.''; PubMed Europe PMC Scholia
20. Herr P, Hausmann G, Basler K.; ''WNT secretion and signalling in human disease.''; PubMed Europe PMC Scholia
21. Coombs GS, Yu J, Canning CA, Veltri CA, Covey TM, Cheong JK, Utomo V, Banerjee N, Zhang ZH, Jadulco RC, Concepcion GP, Bugni TS, Harper MK, Mihalek I, Jones CM, Ireland CM, Virshup DM.; ''WLS-dependent secretion of WNT3A requires Ser209 acylation and vacuolar acidification.''; PubMed Europe PMC Scholia
22. Franch-Marro X, Wendler F, Guidato S, Griffith J, Baena-Lopez A, Itasaki N, Maurice MM, Vincent JP.; ''Wingless secretion requires endosome-to-Golgi retrieval of Wntless/Evi/Sprinter by the retromer complex.''; PubMed Europe PMC Scholia
23. Goodman RM, Thombre S, Firtina Z, Gray D, Betts D, Roebuck J, Spana EP, Selva EM.; ''Sprinter: a novel transmembrane protein required for Wg secretion and signaling.''; PubMed Europe PMC Scholia
24. Yang PT, Lorenowicz MJ, Silhankova M, Coudreuse DY, Betist MC, Korswagen HC.; ''Wnt signaling requires retromer-dependent recycling of MIG-14/Wntless in Wnt-producing cells.''; PubMed Europe PMC Scholia
25. Janda CY, Waghray D, Levin AM, Thomas C, Garcia KC.; ''Structural basis of Wnt recognition by Frizzled.''; PubMed Europe PMC Scholia
26. Gasnereau I, Herr P, Chia PZ, Basler K, Gleeson PA.; ''Identification of an endocytosis motif in an intracellular loop of Wntless protein, essential for its recycling and the control of Wnt protein signaling.''; PubMed Europe PMC Scholia
27. Luga V, Zhang L, Viloria-Petit AM, Ogunjimi AA, Inanlou MR, Chiu E, Buchanan M, Hosein AN, Basik M, Wrana JL.; ''Exosomes mediate stromal mobilization of autocrine Wnt-PCP signaling in breast cancer cell migration.''; PubMed Europe PMC Scholia
28. Biechele S, Cox BJ, Rossant J.; ''Porcupine homolog is required for canonical Wnt signaling and gastrulation in mouse embryos.''; PubMed Europe PMC Scholia
29. Proffitt KD, Virshup DM.; ''Precise regulation of porcupine activity is required for physiological Wnt signaling.''; PubMed Europe PMC Scholia
30. Takada R, Satomi Y, Kurata T, Ueno N, Norioka S, Kondoh H, Takao T, Takada S.; ''Monounsaturated fatty acid modification of Wnt protein: its role in Wnt secretion.''; PubMed Europe PMC Scholia
31. Hofmann K.; ''A superfamily of membrane-bound O-acyltransferases with implications for wnt signaling.''; PubMed Europe PMC Scholia
32. Pfeffer SR.; ''Membrane transport: retromer to the rescue.''; PubMed Europe PMC Scholia
33. Strating JR, Martens GJ.; ''The p24 family and selective transport processes at the ER-Golgi interface.''; PubMed Europe PMC Scholia
34. Zhang P, Wu Y, Belenkaya TY, Lin X.; ''SNX3 controls Wingless/Wnt secretion through regulating retromer-dependent recycling of Wntless.''; PubMed Europe PMC Scholia
35. Xu Y, Hortsman H, Seet L, Wong SH, Hong W.; ''SNX3 regulates endosomal function through its PX-domain-mediated interaction with PtdIns(3)P.''; PubMed Europe PMC Scholia
36. Dancourt J, Barlowe C.; ''Protein sorting receptors in the early secretory pathway.''; PubMed Europe PMC Scholia
37. Palmer L, Vincent JP, Beckett K.; ''Wnts need a p(assport)24 to leave the ER.''; PubMed Europe PMC Scholia
38. Gross JC, Chaudhary V, Bartscherer K, Boutros M.; ''Active Wnt proteins are secreted on exosomes.''; PubMed Europe PMC Scholia
39. Ching W, Hang HC, Nusse R.; ''Lipid-independent secretion of a Drosophila Wnt protein.''; PubMed Europe PMC Scholia
40. Galli LM, Burrus LW.; ''Differential palmit(e)oylation of Wnt1 on C93 and S224 residues has overlapping and distinct consequences.''; PubMed Europe PMC Scholia
41. Herr P, Basler K.; ''Porcupine-mediated lipidation is required for Wnt recognition by Wls.''; PubMed Europe PMC Scholia
42. Willert K, Nusse R.; ''Wnt proteins.''; PubMed Europe PMC Scholia
43. Harterink M, Port F, Lorenowicz MJ, McGough IJ, Silhankova M, Betist MC, van Weering JRT, van Heesbeen RGHP, Middelkoop TC, Basler K, Cullen PJ, Korswagen HC.; ''A SNX3-dependent retromer pathway mediates retrograde transport of the Wnt sorting receptor Wntless and is required for Wnt secretion.''; PubMed Europe PMC Scholia
44. van den Heuvel M, Harryman-Samos C, Klingensmith J, Perrimon N, Nusse R.; ''Mutations in the segment polarity genes wingless and porcupine impair secretion of the wingless protein.''; PubMed Europe PMC Scholia
45. Seaman MN.; ''The retromer complex - endosomal protein recycling and beyond.''; PubMed Europe PMC Scholia
46. Liu J, Pan S, Hsieh MH, Ng N, Sun F, Wang T, Kasibhatla S, Schuller AG, Li AG, Cheng D, Li J, Tompkins C, Pferdekamper A, Steffy A, Cheng J, Kowal C, Phung V, Guo G, Wang Y, Graham MP, Flynn S, Brenner JC, Li C, Villarroel MC, Schultz PG, Wu X, McNamara P, Sellers WR, Petruzzelli L, Boral AL, Seidel HM, McLaughlin ME, Che J, Carey TE, Vanasse G, Harris JL.; ''Targeting Wnt-driven cancer through the inhibition of Porcupine by LGK974.''; PubMed Europe PMC Scholia
47. Lippincott-Schwartz J, Roberts TH, Hirschberg K.; ''Secretory protein trafficking and organelle dynamics in living cells.''; PubMed Europe PMC Scholia
48. Johannes L, Wunder C.; ''The SNXy flavours of endosomal sorting.''; PubMed Europe PMC Scholia
49. Saito-Diaz K, Chen TW, Wang X, Thorne CA, Wallace HA, Page-McCaw A, Lee E.; ''The way Wnt works: components and mechanism.''; PubMed Europe PMC Scholia

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External references

DataNodes

Name	Type	Database reference	Comment
Asparagine N-linked glycosylation	Pathway	REACT_22426 (Reactome)	N-linked glycosylation is the most important form of post-translational modification for proteins synthesized and folded in the Endoplasmic Reticulum (Stanley P et al, 2009). An early study in 1999 revealed that about 50% of the proteins in the Swiss-Prot database at the time were N-glycosylated (Apweiler R et al, 1999). It is now established that the majority of the proteins in the secretory pathway require glycosylation in order to achieve proper folding. The addition of an N-glycan to a protein can have several roles (Shental-Bechor D and Levy Y, 2009). First, glycans enhance the solubility and stability of the proteins in the ER, the golgi and on the outside of the cell membrane, where the composition of the medium is strongly hydrophilic and where proteins, that are mostly hydrophobic, have difficulty folding properly. Second, N-glycans are used as signal molecules during the folding and transport process of the protein: they have the role of labels to determine when a protein must interact with a chaperon, be transported to the golgi, or targeted for degradation in case of major folding defects. Third, and most importantly, N-glycans on completely folded proteins are involved in a wide range of processes: they help determine the specificity of membrane receptors in innate immunity or in cell-to-cell interactions, they can change the properties of hormones and secreted proteins, or of the proteins in the vesicular system inside the cell. All N-linked glycans are derived from a common 14-sugar oligosaccharide synthesized in the ER, which is attached co-translationally to a protein while this is being translated inside the reticulum. The process of the synthesis of this glycan, known as Synthesis of the N-glycan precursor or LLO, constitutes one of the most conserved pathways in eukaryotes, and has been also observed in some eubacteria. The attachment usually happens on an asparagine residue within the consensus sequence asparagine-X-threonine by an complex called oligosaccharyl transferase (OST). After being attached to an unfolded protein, the glycan is used as a label molecule in the folding process (also known as Calnexin/Calreticulin cycle) (Lederkremer GZ, 2009). The majority of the glycoproteins in the ER require at least one glycosylated residue in order to achieve proper folding, even if it has been shown that a smaller portion of the proteins in the ER can be folded without this modification. Once the glycoprotein has achieved proper folding, it is transported via the Cis-golgi through all the Golgi compartments, where the glycan is further modified according to the properties of the glycoprotein. This process involves relatively few enzymes but due to its combinatorial nature, can lead to several millions of different possible modifications. The exact topography of this network of reactions has not been established yet, representing one of the major challenges after the sequencing of the human genome (Hossler P et al, 2006). Since N-glycosylation is involved in an great number of different processes, from cell-cell interaction to folding control, mutations in one of the genes involved in glycan assembly and/or modification can lead to severe development problems (often affecting the central nervous system). All the diseases in genes involved in glycosylation are collectively known as Congenital Disorders of Glycosylation (CDG) (Sparks SE et al, 2003), and classified as CDG type I for the genes in the LLO synthesis pathway, and CDG type II for the others.
CoA-SH	Metabolite	CHEBI:15346 (ChEBI)
ER to Golgi Transport	Pathway	REACT_11203 (Reactome)	Secretory cargo destined to be secreted or to arrive at the plasma membrane (PM) leaves the ER via distinct exit sites. This cargo is destined for the Golgi apparatus.
N-glycosylated WNTs	Protein	REACT_164850 (Reactome)
PORCN	Protein	Q9H237 (Uniprot-TrEMBL)
SNX3	Protein	O60493 (Uniprot-TrEMBL)
SNX3	Protein	O60493 (Uniprot-TrEMBL)
TMED5	Protein	Q9Y3A6 (Uniprot-TrEMBL)
VPS26A	Protein	O75436 (Uniprot-TrEMBL)
VPS29	Protein	Q9UBQ0 (Uniprot-TrEMBL)
VPS35 VPS29 VPS26	Complex	REACT_164267 (Reactome)
VPS35	Protein	Q96QK1 (Uniprot-TrEMBL)
WLS WNT	Complex	REACT_164879 (Reactome)
WLS WNT	Complex	REACT_165191 (Reactome)
WLS retromer	Complex	REACT_164829 (Reactome)
WLS retromer	Complex	REACT_164906 (Reactome)
WLS	Protein	Q5T9L3 (Uniprot-TrEMBL)
WLS	Protein	Q5T9L3 (Uniprot-TrEMBL)
WNTs	Protein	REACT_165388 (Reactome)
palmitoleoyl-CoA	Metabolite	CHEBI:53152 (ChEBI)
palmitoleyl-N-glycosylated WNTs	Protein	REACT_164370 (Reactome)
palmitoleyl-N-glycosylated WNTs	Protein	REACT_165503 (Reactome)
unglycosylated WNTs	Protein	REACT_164660 (Reactome)

Annotated Interactions

Source	Target	Type	Database reference	Comment
	CoA-SH	Arrow	REACT_163757 (Reactome)
N-glycosylated WNTs			REACT_163757 (Reactome)
PORCN		mim-catalysis	REACT_163757 (Reactome)
			REACT_163680 (Reactome)	Although the role of retromer in delivering WLS back to the Golgi is reasonably well established (reviewed in Johannes and Wunder, 2011; Willert and Nusse, 2012), the details of how the complex is disassembled at the TGN remain to be determined.
			REACT_163757 (Reactome)	All WNT proteins except Drosophila WntD are lipid modified. Lipid modifications contribute to the hydrophobicity and poor solubility of all known WNT ligands with the exception of Drosophila WntD. Acylation is required for their secretion from the cell and their ability to bind to FRZ receptors (reviewed in MacDonald et al, 2009; Takada et al, 2006; Janda et al, 2012; Herr and Basler, 2012; Ching et al, 2008). Although an initial study suggested that conserved Cys77 in mouse Wnt3a was palmitoylated (Willert et al, 2003), further work showed that mutation of this residue had minimal effect on WNT secretion (Komekado et al, 2007). In contrast, addition of palmitoleic acid to mWnt3a Ser209 is essential for WNT secretion, and mutant S209A is largely retained in the ER (Takada et al, 2006; Galli and Burrus, 2011). This serine residue is conserved at this position in all known WNTs with the exception of Drosophila WntD (Ching et al, 2008; Herr and Basler, 2012). A recent crystal structure of Xenopus WNT8 in complex with a Frizzled cysteine-rich-domain shows a single lipid modification on the conserved serine residue, while the conserved cysteine participates in a disulphide bond (Janda et al, 2012). In addition to being required for secretion, the lipid at S209 also makes direct contact with a groove in the Frizzled receptor and is thus essential for binding (Janda et al, 2012). Porcupine is a conserved multi-pass transmembrane ER protein that has an O-acyl-transferase domain (van den Heuvel et al, 1993; Kadowaki et al, 1996; Hofmann, 2000). First identified in Drosophila, Porcupine is a WNT-specific modulator that is required for Wingless processing and secretion (Kadowaki et al, 1996). In porcn-deficient cells, Wg and WNT3A have decreased palmitoylation at S209 and accumulate in the ER (Takada et al, 2006), and mutations in PORCN eliminate all WNT signalling and cause embryonic lethality in mice (Barrott et al, 2011; Biechele et al, 2011). Recent studies show that PORCN is required for activity of all human WNT ligands (Proffitt et al, 2012; Najdi et al, 2012).
			REACT_163763 (Reactome)	WLS accompanies WNT through the secretory pathway to the cell surface, where the ligand is released into the extracellular space (Bänziger et al, 2006; Bartscherer et al, 2006; Goodman et al, 2006).
			REACT_163799 (Reactome)	All WNT ligands are predicted to be highly glycosylated. By similarity with WNT ligands in mouse, human WNTs are believed to undergo N-linked glycosylation at multiple asparagine residues and this glycosylation is critical for their secretion (Smolich et al, 1993; Willert et al, 2003, Komekado et al, 2006; Kurayoshi et al, 2007). The mechanism of N-linked glycosylation is not shown here. For a more detailed description, please refer to pathway "Asparagine N-linked glycosylation".
			REACT_163866 (Reactome)	Vacuolar acidification is required but not sufficient for the release of WNT ligands from WLS at the cell surface. V-ATPase inhibitors cause the accumulation of WLS-WNT complexes both within the cell and at the plasma membrane (Coombs et al, 2010). Once in the extracellular space, the lipid-modified WNT ligand must be shielded to allow the morphogen to diffuse away from the plasma membrane. Possible mechanisms include interaction with HSPGs, exosomes, multimerization or incorporation into lipoprotein particles (reviewed in Eaton, 2006; Port and Basler, 2010).
			REACT_163876 (Reactome)	WLS endocytosis is a clathrin-dependent process. In Drosophila cells, internalization of WLS has been shown to depend on clathrin, AP-2, dynamin, Rab5 and HRS (Belenkaya et al, 2006; Port et al, 2008), while in HeLa cells, WLS colocalizes with endogenous AP-2, and depletion of AP-2 increases WLS levels at the cell surface (Yang et al, 2008). A recent study identified a conserved YEGL endocytosis motif in the third intracellular loop of WLS that is required for its clathrin- and dynamin-dependent internalization (Gasnereau et al, 2011).
			REACT_163928 (Reactome)	Wntless (WLS) (Evi/Sprinter/GPR177) is a conserved transmembrane protein that is required for the secretion of WNT ligands from the cell (Bänzinger et al, 2006; Bartscherer et al, 2006; Goodman et al, 2006). Notably, WLS is not required for the secretion of Hedgehog, another acylated signaling molecule, and wls-mutants phenocopy wg/wnt mutants (Bänziger et al, 2006; Bartscherer et al, 2006; Goodman et al, 2006), supporting the notion that WLS is a dedicated WNT pathway member. WLS binds directly to WNT ligands in the Golgi in a WNT-acylation dependent manner, as the interaction is abrogated by mutation of either PORCN or the conserved Ser209 residue (Coombs et al, 2010; Herr and Basler, 2012). WLS is thought to contain a lipocalin-family fold (Coombs et al, 2010), a lipid-interacting domain, which may play a role in binding to the lipid adduct on WNT.
			REACT_163930 (Reactome)	Retromer is a conserved multi-protein complex that is required for retrograde transport of transmembrane proteins. It was initially characterized in yeast as a pentameric complex required for the recycling of the transmembrane receptor VPS10 to the trans-Golgi, and was subsequently shown to be conserved in flies, worms and humans. In humans, retromer consists of a cargo-recognition subcomplex made up of VPS35, VPS26 and VPS29 and a membrane-targeting subcomplex containing a heterodimer of SNX proteins (SNX1 or 2 paired with SNX5 or 6). The SNX proteins contain a BAR domain that is believed to promote membrane curvature, and SNX-BAR proteins are thought to aid in the formation of endosomal membrane tubules into which cargo is loaded (reviewed in Pfeffer, 2001; Seaman, 2012). Retromer is required for the recycling of WLS to the Golgi to allow further rounds of WNT-ligand delivery to the plasma membrane (Coudreuse et al 2006; Belenkaya et al 2008; Port et al, 2008). In the absence of essential retromer component VPS35 or VPS26, WLS is diverted to the MVB and degraded, and WNT ligand accumulates inside the cell; overexpression of WLS is sufficient to rescue the vps35 defect in WNT signaling (Belenkaya et al, 2006; Franch-Marro et al, 2008). WLS and retromer colocalize on endosomal structures and WLS and VPS35 co-precipitate in pull down studies (Belenkaya et al, 2006; Port et al, 2008; Franch-Marro et al, 2008). Several recent studies have suggested that WLS recycling depends on a WNT-specific retromer in which the SNX-BAR proteins of the classic complex are replaced by SNX3 (Zhang et al, 2011; Harterink et al, 2011; reviewed in Johannes and Wunder, 2011). Unlike SNX1/2/5/6, SNX3 does not contain a BAR domain, and WLS is suggested to accumulate in endocytic vesicles rather than in the tubular structures of the 'classic' retromer (Harterink et al, 2011; Zhang et al, 2011). SNX3 is recruited from the cytosol to the early endosome through the interaction of its PX domain with PIP3 in the membrane. Mutation of critical residues in the PX domain abolish the interaction with PIP3 and ablate endsomal recruitment of SNX3 (Xu et al, 2001; Zhang at al, 2011; Harterink et al, 2011). SNX3 has been shown to co-immunoprecipitate with VPS35 and VPS26, and some studies have also shown a direct interaction between SNX3 and WLS (Zhang et al, 2011; Harterink et al, 2011).
			REACT_163947 (Reactome)	Retromer is believed to escort WLS from the early endosome back to the Golgi for subsequent rounds of WNT secretion (reviewed in Johannes and Wunder, 2011; Willert and Nusse, 2012 ).
			REACT_164001 (Reactome)	This black box event represents the non-WNT-specific ER-to-Golgi trafficking step of protein secretion (reviewed in Dancourt and Barlowe, 2010; Lippincott Schwatz et al, 2000; for more details, please refer to the pathway "ER to Golgi transport"). Two recent screens in Drosophila have identified members of the p24 family as WNT-specific regulators of ER-to-Golgi transport, although the details have not been elucidated (Port et al, 2011; Buechling et al, 2011; reviewed in Strating and Martens, 2009). Depletion of the Drosophila p24 protein Opossum causes accumulation of WNT ligand in the ER, suggesting a role for Opm in ER-to-Golgi transport of WNTs. WNT-dependent reporter activity was reduced in HEK293 cells that were depleted for the human p24 homologue TMED5, supporting a conserved role for these proteins in WNT signaling (Buechling et al, 2011; reviewed in Palmer et al, 2012).
	SNX3	Arrow	REACT_163680 (Reactome)
SNX3			REACT_163930 (Reactome)
TMED5		Arrow	REACT_164001 (Reactome)
	VPS35 VPS29 VPS26	Arrow	REACT_163680 (Reactome)
VPS35 VPS29 VPS26			REACT_163930 (Reactome)
	WLS	Arrow	REACT_163680 (Reactome)
	WLS	Arrow	REACT_163866 (Reactome)
WLS			REACT_163928 (Reactome)
WLS			REACT_163930 (Reactome)
	WNTs	Arrow	REACT_163866 (Reactome)
palmitoleoyl-CoA			REACT_163757 (Reactome)
	palmitoleyl-N-glycosylated WNTs	Arrow	REACT_163757 (Reactome)
palmitoleyl-N-glycosylated WNTs			REACT_163928 (Reactome)