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Canonical Notch ligands and Fringes have distinct effects on NOTCH1 and NOTCH2

2020; Elsevier BV; Volume: 295; Issue: 43 Linguagem: Inglês

10.1074/jbc.ra120.014407

ISSN

1083-351X

Autores

Shinako Kakuda, Rachel K. LoPilato, Atsuko Ito, Robert S. Haltiwanger,

Tópico(s)

Congenital heart defects research

Resumo

Notch signaling is a cellular pathway regulating cell-fate determination and adult tissue homeostasis. Little is known about how canonical Notch ligands or Fringe enzymes differentially affect NOTCH1 and NOTCH2. Using cell-based Notch signaling and ligand-binding assays, we evaluated differences in NOTCH1 and NOTCH2 responses to Delta-like (DLL) and Jagged (JAG) family members and the extent to which Fringe enzymes modulate their activity. In the absence of Fringes, DLL4–NOTCH1 activation was more than twice that of DLL4–NOTCH2, whereas all other ligands activated NOTCH2 similarly or slightly more than NOTCH1. However, NOTCH2 showed less sensitivity to the Fringes. Lunatic fringe (LFNG) enhanced NOTCH2 activation by DLL1 and -4, and Manic fringe (MFNG) inhibited NOTCH2 activation by JAG1 and -2. Mass spectral analysis showed that O-fucose occurred at high stoichiometry at most consensus sequences of NOTCH2 and that the Fringe enzymes modified more O-fucose sites of NOTCH2 compared with NOTCH1. Mutagenesis studies showed that LFNG modification of O-fucose on EGF8 and -12 of NOTCH2 was responsible for enhancement of DLL1–NOTCH2 activation, similar to previous reports for NOTCH1. In contrast to NOTCH1, a single O-fucose site mutant that substantially blocked the ability of MFNG to inhibit NOTCH2 activation by JAG1 could not be identified. Interestingly, elimination of the O-fucose site on EGF12 allowed LFNG to inhibit JAG1-NOTCH2 activation, and O-fucosylation on EGF9 was important for trafficking of both NOTCH1 and NOTCH2. Together, these studies provide new insights into the differential regulation of NOTCH1 and NOTCH2 by Notch ligands and Fringe enzymes. Notch signaling is a cellular pathway regulating cell-fate determination and adult tissue homeostasis. Little is known about how canonical Notch ligands or Fringe enzymes differentially affect NOTCH1 and NOTCH2. Using cell-based Notch signaling and ligand-binding assays, we evaluated differences in NOTCH1 and NOTCH2 responses to Delta-like (DLL) and Jagged (JAG) family members and the extent to which Fringe enzymes modulate their activity. In the absence of Fringes, DLL4–NOTCH1 activation was more than twice that of DLL4–NOTCH2, whereas all other ligands activated NOTCH2 similarly or slightly more than NOTCH1. However, NOTCH2 showed less sensitivity to the Fringes. Lunatic fringe (LFNG) enhanced NOTCH2 activation by DLL1 and -4, and Manic fringe (MFNG) inhibited NOTCH2 activation by JAG1 and -2. Mass spectral analysis showed that O-fucose occurred at high stoichiometry at most consensus sequences of NOTCH2 and that the Fringe enzymes modified more O-fucose sites of NOTCH2 compared with NOTCH1. Mutagenesis studies showed that LFNG modification of O-fucose on EGF8 and -12 of NOTCH2 was responsible for enhancement of DLL1–NOTCH2 activation, similar to previous reports for NOTCH1. In contrast to NOTCH1, a single O-fucose site mutant that substantially blocked the ability of MFNG to inhibit NOTCH2 activation by JAG1 could not be identified. Interestingly, elimination of the O-fucose site on EGF12 allowed LFNG to inhibit JAG1-NOTCH2 activation, and O-fucosylation on EGF9 was important for trafficking of both NOTCH1 and NOTCH2. Together, these studies provide new insights into the differential regulation of NOTCH1 and NOTCH2 by Notch ligands and Fringe enzymes. The Notch signaling pathway plays essential roles in development of metazoans, and defects in the Notch pathway result in a wide variety of congenital disorders and cancers (1Kopan R. Ilagan M.X. 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NOTCH2 mutations cause Alagille syndrome, a heterogeneous disorder of the notch signaling pathway.Am. J. Hum. Genet. 2006; 79 (16773578): 169-17310.1086/505332Abstract Full Text Full Text PDF PubMed Scopus (558) Google Scholar) and Hajdu–Cheney syndrome (11Simpson M.A. Irving M.D. Asilmaz E. Gray M.J. Dafou D. Elmslie F.V. Mansour S. Holder S.E. Brain C.E. Burton B.K. Kim K.H. Pauli R.M. Aftimos S. Stewart H. Kim C.A. et al.Mutations in NOTCH2 cause Hajdu-Cheney syndrome, a disorder of severe and progressive bone loss.Nat. Genet. 2011; 43 (21378985): 303-30510.1038/ng.779Crossref PubMed Scopus (234) Google Scholar), and mutations in NOTCH3 cause CADASIL (cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy) (12Joutel A. Corpechot C. Ducros A. Vahedi K. Chabriat H. Mouton P. Alamowitch S. Domenga V. Cécillion M. Maréchal E. Maciazek J. Vayssière C. Cruaud C. Cabanis E.A. 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Ge C. Kakuda S. Wu D. Roein-Peikar M. Haltiwanger R.S. Zhu C. Ha T. Garcia K.C. Notch-Jagged complex structure implicates a catch bond in tuning ligand sensitivity.Science. 2017; 355 (28254785): 1320-132410.1126/science.aaf9739Crossref PubMed Scopus (167) Google Scholar). Elimination of these sites alters Notch activity in vitro and in vivo (22Kakuda S. Haltiwanger R.S. Deciphering the Fringe-mediated Notch code: identification of activating and inhibiting sites allowing discrimination between ligands.Dev. Cell. 2017; 40 (28089369): 193-20110.1016/j.devcel.2016.12.013Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar, 23Pandey A. Harvey B.M. Lopez M.F. Ito A. Haltiwanger R.S. Jafar-Nejad H. Glycosylation of specific Notch EGF repeats by O-Fut1 and Fringe regulates Notch signaling in Drosophila.Cell Rep. 2019; 29 (31722217): 2054-2066.e610.1016/j.celrep.2019.10.027Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar, 24Varshney S. Wei H.X. Batista F. 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Significantly, elongation of the O-fucose residues on Notch by the Fringe family of β3-N-acetylglucosaminyltransferases modulates Notch activity, typically enhancing Notch signaling from Delta-family ligands while inhibiting signaling from Jagged ligands (14Harvey B.M. Haltiwanger R.S. Regulation of Notch function by O-glycosylation.Adv. Exp. Med. Biol. 2018; 1066 (30030822): 59-7810.1007/978-3-319-89512-3_4Crossref PubMed Scopus (33) Google Scholar, 15Varshney S. Stanley P. Multiple roles for O-glycans in Notch signalling.FEBS Lett. 2018; 592 (30207383): 3819-383410.1002/1873-3468.13251Crossref PubMed Scopus (41) Google Scholar, 16Haltom A.R. Jafar-Nejad H. The multiple roles of epidermal growth factor repeat O-glycans in animal development.Glycobiology. 2015; 25 (26175457): 1027-104210.1093/glycob/cwv052Crossref PubMed Scopus (40) Google Scholar). Only one Fringe gene exists in flies, but mammals express three: Lunatic fringe (LFNG), Manic fringe (MFNG) and Radical fringe (RFNG). Elimination of Lfng in mice results in a severe somitogenesis defect (25Zhang N. Gridley T. Defects in somite formation in lunatic fringe-deficient mice.Nature. 1998; 394 (9690472): 374-37710.1038/28625Crossref PubMed Scopus (360) Google Scholar, 26Evrard Y.A. Lun Y. Aulehla A. Gan L. Johnson R.L. Lunatic fringe is an essential mediator of somite segmentation and patterning.Nature. 1998; 394 (9690473): 377-38110.1038/28632Crossref PubMed Google Scholar), and mutations in human LFNG causes a severe vertebral segmentation defect called spondylocostal dysostosis type III (27Sparrow D.B. Chapman G. Wouters M.A. Whittock N.V. Ellard S. Fatkin D. Turnpenny P.D. Kusumi K. Sillence D. Dunwoodie S.L. Mutation of the LUNATIC FRINGE gene in humans causes spondylocostal dysostosis with a severe vertebral phenotype.Am. J. Hum. Genet. 2006; 78 (16385447): 28-3710.1086/498879Abstract Full Text Full Text PDF PubMed Scopus (185) Google Scholar). In this context, LFNG is regulating the activity of NOTCH1 (28Wahi K. Bochter M.S. Cole S.E. The many roles of Notch signaling during vertebrate somitogenesis.Semin. Cell Dev. Biol. 2016; 49 (25483003): 68-7510.1016/j.semcdb.2014.11.010Crossref PubMed Scopus (36) Google Scholar). Although Mfng or Rfng-null mice display no significant developmental phenotypes (29Moran J.L. Shifley E.T. Levorse J.M. Mani S. Ostmann K. Perez-Balaguer A. Walker D.M. Vogt T.F. Cole S.E. Manic fringe is not required for embryonic development, and fringe family members do not exhibit redundant functions in the axial skeleton, limb, or hindbrain.Dev. Dyn. 2009; 238 (19479951): 1803-181210.1002/dvdy.21982Crossref PubMed Scopus (35) Google Scholar), all three Fringes have been implicated in regulation of Notch activity in a variety of contexts, including angiogenesis (30Benedito R. Roca C. Sörensen I. Adams S. Gossler A. Fruttiger M. Adams R.H. The notch ligands Dll4 and Jagged1 have opposing effects on angiogenesis.Cell. 2009; 137 (19524514): 1124-113510.1016/j.cell.2009.03.025Abstract Full Text Full Text PDF PubMed Scopus (773) Google Scholar), B and T cell maturation (31Stanley P. Guidos C.J. Regulation of Notch signaling during T- and B-cell development by O-fucose glycans.Immunol. Rev. 2009; 230 (19594638): 201-21510.1111/j.1600-065X.2009.00791.xCrossref PubMed Scopus (59) Google Scholar, 32Song Y. Kumar V. Wei H.X. Qiu J. Stanley P. Lunatic, Manic, and Radical Fringe each promote T and B cell development.J. Immunol. 2016; 196 (26608918): 232-24310.4049/jimmunol.1402421Crossref PubMed Scopus (31) Google Scholar), bile duct remodeling (33Ryan M.J. Bales C. Nelson A. Gonzalez D.M. Underkoffler L. Segalov M. Wilson-Rawls J. Cole S.E. Moran J.L. Russo P. Spinner N.B. Kusumi K. Loomes K.M. Bile duct proliferation in Jag1/fringe heterozygous mice identifies candidate modifiers of the Alagille syndrome hepatic phenotype.Hepatology. 2008; 48 (19026002): 1989-199710.1002/hep.22538Crossref PubMed Scopus (62) Google Scholar), ventricular chamber development (34D'Amato G. Luxán G. Del Monte-Nieto G. Martínez-Poveda B. Torroja C. Walter W. Bochter M.S. Benedito R. Cole S. Martinez F. Hadjantonakis A.K. Uemura A. Jiménez-Borreguero L.J. de la Pompa J.L. Sequential Notch activation regulates ventricular chamber development.Nat. Cell Biol. 2016; 18 (26641715): 7-2010.1038/ncb3280Crossref PubMed Scopus (117) Google Scholar), and kidney development (35Liu Z. Chen S. Boyle S. Zhu Y. Zhang A. Piwnica-Worms D.R. Ilagan M.X. Kopan R. The extracellular domain of Notch2 increases its cell-surface abundance and ligand responsiveness during kidney development.Dev. Cell. 2013; 25 (23806616): 585-59810.1016/j.devcel.2013.05.022Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar). Fringes have also been implicated in a number of cancers (36López-Arribillaga E. Rodilla V. Colomer C. Vert A. Shelton A. Cheng J.H. Yan B. Gonzalez-Perez A. Junttila M.R. Iglesias M. Torres F. Albanell J. Villanueva A. Bigas A. Siebel C.W. et al.Manic Fringe deficiency imposes Jagged1 addiction to intestinal tumor cells.Nat. Commun. 2018; 9 (30065304): 299210.1038/s41467-018-05385-0Crossref PubMed Scopus (20) Google Scholar, 37Xu K. Usary J. Kousis P.C. Prat A. Wang D.Y. Adams J.R. Wang W. Loch A.J. Deng T. Zhao W. Cardiff R.D. Yoon K. Gaiano N. Ling V. Beyene J. et al.Lunatic fringe deficiency cooperates with the Met/caveolin gene amplicon to induce basal-like breast cancer.Cancer Cell. 2012; 21 (22624713): 626-64110.1016/j.ccr.2012.03.041Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar, 38Zhang S. Chung W.C. Wu G. Egan S.E. Miele L. Xu K. Manic Fringe promotes a claudin-low breast cancer phenotype through Notch-mediated PIK3CG induction.Cancer Res. 2015; 75 (25808869): 1936-194310.1158/0008-5472.CAN-14-3303Crossref PubMed Scopus (44) Google Scholar). Apart from LFNG, which regulates NOTCH1 during somitogenesis, determining which Fringe modulates which Notch receptor in vivo is challenging due to the broad and overlapping expression patterns of the Notch receptors, Notch ligands, and Fringes in embryonic and adult tissues. We and others have studied the effects of individual Fringe enzymes on discrete Notch-ligand pairs (39Brückner K. Perez L. Clausen H. Cohen S. Glycosyltransferase activity of Fringe modulates Notch-Delta interactions.Nature. 2000; 406 (10935637): 411-41510.1038/35019075Crossref PubMed Scopus (592) Google Scholar, 40Moloney D.J. Panin V.M. Johnston S.H. Chen J. Shao L. Wilson R. Wang Y. Stanley P. Irvine K.D. Haltiwanger R.S. Vogt T.F. Fringe is a glycosyltransferase that modifies Notch.Nature. 2000; 406 (10935626): 369-37510.1038/35019000Crossref PubMed Scopus (719) Google Scholar, 41Hicks C. Johnston S.H. DiSibio G. Collazo A. Vogt T.F. Weinmaster G. Fringe differentially modulates Jagged1 and Delta1 signalling through Notch1 and Notch2.Nat. Cell Biol. 2000; 2 (10934472): 515-52010.1038/35019553Crossref PubMed Scopus (336) Google Scholar, 42Shimizu K. Chiba S. Saito T. Kumano K. Takahashi T. Hirai H. Manic fringe and lunatic fringe modify different sites of the Notch2 extracellular region, resulting in different signaling modulation.J. Biol. Chem. 2001; 276 (11346656): 25753-2575810.1074/jbc.M103473200Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar, 43Yang L.T. Nichols J.T. Yao C. Manilay J.O. Robey E.A. Weinmaster G. Fringe glycosyltransferases differentially modulate Notch1 proteolysis induced by Delta1 and Jagged1.Mol. Biol. 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Cell. 2017; 40 (28089369): 193-20110.1016/j.devcel.2016.12.013Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar), we confirmed that all three Fringes enhance NOTCH1 activation by DLL1, and LFNG and MFNG inhibit activation by JAG1. In contrast, RFNG enhances NOTCH1 activation by JAG1. Using mass spectral glycoproteomics methods, we demonstrated that the majority of predicted O-fucose consensus sequences are modified by protein O-fucosyltransferase 1 (POFUT1) at high stoichiometry, that LFNG modifies O-fucose on a subset of these EGF repeats, and that MFNG and RFNG modify a subset of those EGF repeats modified by LFNG. Using cell-based NOTCH1 activation and ligand-binding assays, we showed that O-fucose on EGF8 and -12 of NOTCH1 are the major sites responsible for Fringe-mediated enhancement of NOTCH1 activation by DLL1. More recent studies show that the O-fucose residues on EGF8 and -12 are also important for Fringe-mediated enhancement of Drosophila Notch binding to Delta in vitro and activation during Delta-mediated wing vein development in vivo (23Pandey A. Harvey B.M. Lopez M.F. Ito A. Haltiwanger R.S. Jafar-Nejad H. Glycosylation of specific Notch EGF repeats by O-Fut1 and Fringe regulates Notch signaling in Drosophila.Cell Rep. 2019; 29 (31722217): 2054-2066.e610.1016/j.celrep.2019.10.027Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar). These results are consistent with the importance of O-fucose on EGF8 and -12, as revealed by the recent co-crystal structures of the ligand-binding domain of NOTCH1 and fragments of DLL4 (20Luca V.C. Jude K.M. Pierce N.W. Nachury M.V. Fischer S. Garcia K.C. Structural basis for Notch1 engagement of Delta-like 4.Science. 2015; 347 (25700513): 847-85310.1126/science.1261093Crossref PubMed Scopus (176) Google Scholar) or JAG1 (21Luca V.C. Kim B.C. Ge C. Kakuda S. Wu D. Roein-Peikar M. Haltiwanger R.S. Zhu C. Ha T. Garcia K.C. Notch-Jagged complex structure implicates a catch bond in tuning ligand sensitivity.Science. 2017; 355 (28254785): 1320-132410.1126/science.aaf9739Crossref PubMed Scopus (167) Google Scholar). We also showed that LFNG and MFNG modification of O-fucose on EGF6 and -36 are mainly responsible for inhibition of NOTCH1 activation by JAG1 (22Kakuda S. Haltiwanger R.S. Deciphering the Fringe-mediated Notch code: identification of activating and inhibiting sites allowing discrimination between ligands.Dev. Cell. 2017; 40 (28089369): 193-20110.1016/j.devcel.2016.12.013Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar). In contrast, Fringe modifications of EGF8 and -12 of Drosophila Notch inhibit Serrate (Jagged ortholog in Drosophila) binding in vitro and affect Serrate-mediated wing margin formation in vivo, mainly through blocking the cis-inhibition of Serrate by Notch (23Pandey A. Harvey B.M. Lopez M.F. Ito A. Haltiwanger R.S. Jafar-Nejad H. Glycosylation of specific Notch EGF repeats by O-Fut1 and Fringe regulates Notch signaling in Drosophila.Cell Rep. 2019; 29 (31722217): 2054-2066.e610.1016/j.celrep.2019.10.027Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar). These results suggest that Fringe modifications of O-fucose on EGF8 and -12 play a common role in enhancing Notch binding to Delta-family ligands, but there are differences in how Fringes inhibit signaling from Serrate/Jagged family members. Here we compare how all canonical ligands activate NOTCH1 and NOTCH2, and we examine how the Fringes modulate NOTCH2 activity. Early in vitro studies reported that Fringe differentially modulates JAG1 and DLL1 signaling from NOTCH1 and NOTCH2 (41Hicks C. Johnston S.H. DiSibio G. Collazo A. Vogt T.F. Weinmaster G. Fringe differentially modulates Jagged1 and Delta1 signalling through Notch1 and Notch2.Nat. Cell Biol. 2000; 2 (10934472): 515-52010.1038/35019553Crossref PubMed Scopus (336) Google Scholar). LFNG and MFNG were also shown to modify distinct regions of the NOTCH2 ECD (42Shimizu K. Chiba S. Saito T. Kumano K. Takahashi T. Hirai H. Manic fringe and lunatic fringe modify different sites of the Notch2 extracellular region, resulting in different signaling modulation.J. Biol. Chem. 2001; 276 (11346656): 25753-2575810.1074/jbc.M103473200Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar). Here we compared NOTCH1 and NOTCH2 binding to and activation by canonical Notch ligands (DLL1, DLL4, JAG1, and JAG2), and we examined how LFNG, MFNG, and RFNG affect NOTCH2 binding to and activation by these ligands. We used semiquantitative mass spectral glycoproteomics methods to identify O-fucose modifications on NOTCH2 EGF repeats and assess the relative abundance of Fringe modification at each site by the three Fringe enzymes. We mutated O-fucose sites to determine which are needed for the Fringes to mediate their effects on NOTCH2. Finally, we also examined how O-fucosylation at specific EGF repeats is important for trafficking of both NOTCH1 and NOTCH2. Together, these studies give new insights into the differential regulation of NOTCH1 and NOTCH2 by Notch ligands and Fringe enzymes. Although the extracellular domains of NOTCH1 and NOTCH2 both contain 36 tandem EGF repeats, the location of EGF repeats containing O-fucose consensus sequences is different (Fig. 1A). We compared NOTCH1 and NOTCH2 activities using ligand-coating Notch signaling assays in CHO-K1 cells with extracellular domains of the four canonical ligands: DLL1, DLL4, JAG1, and JAG2 (Fig. 1B and Fig. S1). DLL1 and JAG1 induced higher NOTCH2 activity than NOTCH1, but DLL4 induced NOTCH1 activity much more robustly than NOTCH2. JAG2 induced similar levels of NOTCH1 and NOTCH2 activity. Differences in the C-terminal tags (Fc or His) or species (human, mouse, or rat) of the ligands had little effect on NOTCH1 or NOTCH2 activity (Fig. S1). DLL3 did not activate NOTCH1 or NOTCH2 in trans (Fig. S1E), as reported previously (46Ladi E. Nichols J.T. Ge W. Miyamoto A. Yao C. Yang L.T. Boulter J. Sun Y.E. Kintner C. Weinmaster G. The divergent DSL ligand Dll3 does not activate Notch signaling but cell autonomously attenuates signaling induced by other DSL ligands.J. Cell Biol. 2005; 170 (16144902): 983-99210.1083/jcb.200503113Crossref PubMed Scopus (220) Google Scholar). Relative binding of soluble ligands to NOTCH1- or NOTCH2-expressing HEK293T cells was consistent with their effects on NOTCH1 and NOTCH2 activity (Fig. 1C), suggesting that the ligands have similar effects on NOTCH1 and NOTCH2 in both CHO-K1 and HEK293T cells. NOTCH2 had a higher affinity for DLL1 than NOTCH1, whereas NOTCH1 had both a higher affinity and binding maximum for DLL4 than NOTCH2. These results are in line with the relative affinities of NOTCH1 and NOTCH2 for DLL1 and DLL4 reported previously (47Andrawes M.B. Xu X. Liu H. Ficarro S.B. Marto J.A. Aster J.C. Blacklow S.C. Intrinsic selectivity of Notch 1 for Delta-like 4 over Delta-like 1.J. Biol. Chem. 2013; 288 (23839946): 25477-2548910.1074/jbc.M113.454850Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar, 48Tveriakhina L. Schuster-Gossler K. Jarrett S.M. Andrawes M.B. Rohrbach M. Blacklow S.C. Gossler A. The ectodomains determine ligand function in vivo and selectivity of DLL1 and DLL4 toward NOTCH1 and NOTCH2 in vitro.eLife. 2018; 7 (30289388)10.7554/eLife.40045Crossref PubMed Scopus (21) Google Scholar). In addition, NOTCH2 had higher affinity for JAG1 than NOTCH1, whereas NOTCH1 and NOTCH2 showed similar affinity for JAG2 (Fig. 1C). Using cell-based co-culture assays with NIH3T3 cells, we previously reported that LFNG and MFNG enhance NOTCH1 activation from DLL1 and inhibit activation from JAG1, whereas RFNG enhances NOTCH1 activity from both ligands (22Kakuda S. Haltiwanger R.S. Deciphering the Fringe-mediated Notch code: identification of activating and inhibiting sites allowing discrimination between ligands.Dev. Cell. 2017; 40 (28089369): 193-20110.1016/j.devcel.2016.12.013Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar). We have reproduced the same effects of Fringes on NOTCH1 using ligand-coating assays, also using NIH3T3 cells (Fig. 2A). We and others have previously shown that NIH3T3 cells, HEK293T cells, and CHO-K1 cells express low levels of Fringes and respond similarly in Notch activation assays to expression of the three Fringe enzymes (22Kakuda S. Haltiwanger R.S. Deciphering the Fringe-mediated Notch code: identification of activating and inhibiting sites allowing discrimination between ligands.Dev. Cell. 2017; 40 (28089369): 193-20110.1016/j.devcel.2016.12.013Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar, 45LeBon L. Lee T.V. Sprinzak D. Jafar-Nejad H. Elowitz M.B. Fringe proteins modulate Notch-ligand cis trans interactions to specify signaling states.eLife. 2014; 3 (25255098): e0295010.7554/eLife.02950PubMed Google Scholar). To be complete, we also examined how the Fringes effect NOTCH1 activation by DLL4 and JAG2 (Fig. 2A). Both LFNG and MFNG inhibited JAG2-NOTCH1 activity, similar to their effects on JAG1-NOTCH1. In contrast to DLL1, none of the Fringes caused significant enhancement of DLL4-NOTCH1 activation. These results are consistent with our prior results showing that the Fringe modification of NOTCH1 EGF11-13 significantly enhances binding to DLL1 but not to DLL4 (49Taylor P. Takeuchi H. Sheppard D. Chillakuri C. Lea S.M. Haltiwanger R.S. Handford P.A. Fringe-mediated extension of O-linked fucose in the ligand-binding region of Notch1 increases binding to mammalian Notch ligands.Proc. Natl. Acad. Sci. U. S. A. 2014; 111 (24803430): 7290-729510.107

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