The Molecular Mechanism of Notch Activation

  • Klaus N. Lovendahl
  • Stephen C. Blacklow
  • Wendy R. GordonEmail author
Part of the Advances in Experimental Medicine and Biology book series (AEMB, volume 1066)


Research in the last several years has shown that Notch proteolysis, and thus Notch activation, is conformationally controlled by the extracellular juxtamembrane NRR of Notch, which sterically occludes the S2 protease site until ligand binds. The question of how conformational exposure of the protease site is achieved during physiologic activation, and thus how normal activation is bypassed in disease pathogenesis, has been the subject of intense study in the last several years, and is the subject of this chapter. Here, we summarize the structural features of the NRR domains of Notch receptors that establish the autoinhibited state and then review a number of recent studies aimed at testing the mechanotransduction model for Notch signaling using force spectroscopy and molecular tension sensors.


Mechanotransduction Single molecule force spectroscopy Molecular tension sensors Notch signaling X-ray structure 


  1. Aste-Amézaga M, Zhang N, Lineberger JE et al (2010) Characterization of Notch1 antibodies that inhibit signaling of both normal and mutated Notch1 receptors. PLoS One 5:e9094. CrossRefPubMedPubMedCentralGoogle Scholar
  2. Bernasconi-Elias P, Hu T, Jenkins D, Firestone B, Gans S, Kurth E, Capodieci P, Deplazes-Lauber J, Petropoulos K, Thiel P, Ponsel D, Hee Choi S, LeMotte P, London A, Goetcshkes M, Nolin E, Jones MD, Slocum K, Kluk MJ, Weinstock DM, Christodoulou A, Weinberg O, Jaehrling J, Ettenberg SA, Buckler A, Blacklow SC, Aster JC, Fryer CJ (2016) Characterization of activating mutations of NOTCH3 in T-cell acute lymphoblastic leukemia and anti-leukemic activity of NOTCH3 inhibitory antibodies. Oncogene 35(47):6077–6086CrossRefPubMedPubMedCentralGoogle Scholar
  3. Bray SJ (2006) Notch signalling: a simple pathway becomes complex. Nat Rev Mol Cell Biol 7(9):678–689CrossRefPubMedGoogle Scholar
  4. Brou C, Logeat F, Gupta N, Bessia C, LeBail O, Doedens JR, Cumano A, Roux P, Black RA, Israël A (2000) A novel proteolytic cleavage involved in Notch signaling. Mol Cell 5(2):207–216CrossRefPubMedGoogle Scholar
  5. Chastagner P, Rubinstein E, Brou C (2017) Ligand-activated Notch undergoes DTX4-mediated ubiquitylation and bilateral endocytosis before ADAM10 processing. Sci Signal 10(483):eaag2989CrossRefPubMedGoogle Scholar
  6. Chen N, Greenwald I (2004) The lateral signal for LIN-12/Notch in C. elegans vulval development comprises redundant secreted and transmembrane DSL proteins. Dev Cell 6(2):183–192CrossRefPubMedGoogle Scholar
  7. Chen Y, Ju L, Rushdi M et al (2017) Receptor-mediated cell mechanosensing. Mol Biol Cell 28:3134–3155. CrossRefPubMedPubMedCentralGoogle Scholar
  8. Chowdhury F, Li ITS, Ngo TTM, Leslie BJ, Kim BC, Sokoloski JE, Weiland E, Wang X, Chemla YR, Lohman TM, Ha T (2016) Defining single molecular forces required for Notch activation using Nano Yoyo. Nano Lett 16(6):3892–3897CrossRefPubMedPubMedCentralGoogle Scholar
  9. Gordon WR, Aster JC (2014) Application and evaluation of anti-Notch antibodies to modulate Notch signaling. Methods Mol Biol 1187:323–333. CrossRefPubMedGoogle Scholar
  10. Gordon WR, Vardar-Ulu D, Histen G et al (2007) Structural basis for autoinhibition of Notch. Nat Struct Mol Biol 14:295–300. CrossRefPubMedGoogle Scholar
  11. Gordon WR, Roy M, Vardar-Ulu D et al (2009a) Structure of the Notch1-negative regulatory region: implications for normal activation and pathogenic signaling in T-ALL. Blood 113:4381–4390. CrossRefPubMedPubMedCentralGoogle Scholar
  12. Gordon WR, Vardar-Ulu D, L’Heureux S et al (2009b) Effects of S1 cleavage on the structure, surface export, and signaling activity of human Notch1 and Notch2. PLoS One 4:e6613. CrossRefPubMedPubMedCentralGoogle Scholar
  13. Gordon WR, Zimmerman B, He L et al (2015) Mechanical Allostery: evidence for a force requirement in the proteolytic activation of Notch. Dev Cell 33:729–736. CrossRefPubMedPubMedCentralGoogle Scholar
  14. Klueg KM, Muskavitch MA (1999) Ligand-receptor interactions and trans-endocytosis of delta, serrate and Notch: members of the Notch signalling pathway in Drosophila. J Cell Sci 112(19):3289–3297PubMedGoogle Scholar
  15. Kopan R, Goate A (2000) A common enzyme connects Notch signaling and Alzheimer’s disease. Genes Dev 14(22):2799–2806CrossRefPubMedGoogle Scholar
  16. Kopan R, Ilagan MXG (2009) The canonical Notch signaling pathway: unfolding the activation mechanism. Cell 137(2):216–233CrossRefPubMedPubMedCentralGoogle Scholar
  17. Kovall RA, Gebelein B, Sprinzak D, Kopan R (2017) The canonical Notch signaling pathway: structural and biochemical insights into shape, sugar, and force. Dev Cell 41(3):228–241CrossRefPubMedPubMedCentralGoogle Scholar
  18. Langridge PD, Struhl G (2017) Epsin-dependent ligand endocytosis activates Notch by force. Cell 171(6):1383–1396.e12CrossRefPubMedGoogle Scholar
  19. Li K, Li Y, Wu W et al (2008) Modulation of Notch signaling by antibodies specific for the extracellular negative regulatory region of NOTCH3. J Biol Chem 283:8046–8054. CrossRefPubMedGoogle Scholar
  20. Logeat F, Bessia C, Brou C, LeBail O, Jarriault S, Seidah NG, Israel A (1998) The Notch1 receptor is cleaved constitutively by a furin-like convertase. Proc Natl Acad Sci 95(14):8108–8112CrossRefPubMedGoogle Scholar
  21. Luca VC, Jude KM, Pierce NW, Nachury MV, Fischer S, Garcia KC (2015) Structural basis for Notch1 engagement of Delta-like 4. Science 347(6224):847–853CrossRefPubMedPubMedCentralGoogle Scholar
  22. Luca VC, Kim BC, Ge C, Kakuda S, Di W, Roein-Peikar M, Haltiwanger RS, Zhu C, Ha T, Christopher Garcia K (2017) Notch-Jagged complex structure implicates a catch bond in tuning ligand sensitivity. Science 355(6331):1320–1324CrossRefPubMedPubMedCentralGoogle Scholar
  23. Mallik R, Gross SP (2004) Molecular motors: strategies to get along. Curr Biol 14(22):R971–R982CrossRefPubMedGoogle Scholar
  24. Malecki MJ, Sanchez-Irizarry C, Mitchell JL et al (2006) Leukemia-associated mutations within the NOTCH1 heterodimerization domain fall into at least two distinct mechanistic classes. Mol Cell Biol 26:4642–4651. CrossRefPubMedPubMedCentralGoogle Scholar
  25. Meloty-Kapella L, Shergill B, Kuon J, Botvinick E, Weinmaster G (2012) Notch ligand endocytosis generates mechanical pulling force dependent on dynamin, epsins, and actin. Dev Cell 22(6):1299–1312CrossRefPubMedPubMedCentralGoogle Scholar
  26. Morsut L, Roybal KT, Xiong X, Gordley RM, Coyle SM, Thomson M, Lim WA (2016) Engineering customized cell sensing and response behaviors using synthetic Notch receptors. Cell 164(4):780–791CrossRefPubMedPubMedCentralGoogle Scholar
  27. Mouw JK, Yui Y, Damiano L et al (2014) Tissue mechanics modulate microRNA-dependent PTEN expression to regulate malignant progression. Nat Med 20:360–367. CrossRefPubMedPubMedCentralGoogle Scholar
  28. Mumm JS, Kopan R (2000) Notch signaling: from the outside in. Dev Biol 228(2):151–165CrossRefPubMedGoogle Scholar
  29. Musse AA, Meloty-Kapella L, Weinmaster G (2012) Notch ligand endocytosis: mechanistic basis of signaling activity. Semin Cell Dev Biol 23(4):429–436CrossRefPubMedPubMedCentralGoogle Scholar
  30. Nichols JT, Miyamoto A, Olsen SL, D’Souza B, Yao C, Weinmaster G (2007) DSL ligand endocytosis physically dissociates Notch1 heterodimers before activating proteolysis can occur. J Cell Biol 176(4):445–458CrossRefPubMedPubMedCentralGoogle Scholar
  31. Parks AL et al (2000) Ligand endocytosis drives receptor dissociation and activation in the Notch pathway. Development 127(7):1373–1385PubMedPubMedCentralGoogle Scholar
  32. Rebay I, Fleming RJ, Fehon RG, Cherbas L, Cherbas P, Artavanis-Tsakonas S (1991) Specific EGF repeats of Notch mediate interactions with delta and serrate: implications for notch as a multifunctional receptor. Cell 67(4):687–699CrossRefPubMedGoogle Scholar
  33. Roybal KT, Williams JZ, Morsut L, Rupp LJ, Kolinko I, Choe JH, Walker WJ, McNally KA, Lim WA (2016a) Engineering T cells with customized therapeutic response programs using synthetic Notch receptors. Cell 167(2):419–432.e16CrossRefPubMedPubMedCentralGoogle Scholar
  34. Roybal KT, Rupp LJ, Morsut L, Walker WJ, McNally KA, Park JS, Lim WA (2016b) Precision tumor Vrecognition by T cells with combinatorial antigen-sensing circuits. Cell 164(4):770–779CrossRefPubMedPubMedCentralGoogle Scholar
  35. Seo D, Southard KM, Kim J-w, Lee HJ, Farlow J, Lee J-u, Litt DB, Haas T, Paul Alivisatos A, Cheon J, Gartner ZJ, Jun Y-w (2016) A mechanogenetic toolkit for interrogating cell signaling in space and time. Cell 165(6):1507–1518CrossRefPubMedPubMedCentralGoogle Scholar
  36. Seugnet L, Simpson P, Haenlin M (1997) Requirement for dynamin during Notch signaling in Drosophila neurogenesis. Dev Biol 192:585–598. CrossRefPubMedGoogle Scholar
  37. Shaya O, Binshtok U, Hersch M, Rivkin D, Weinreb S, Amir-Zilberstein L, Khamaisi B, Oppenheim O, Desai RA, Goodyear RJ, Richardson GP, Chen CS, Sprinzak D (2017) Cell-cell contact area affects Notch signaling and Notch-dependent patterning. Dev Cell 40(5):505–511.e6CrossRefPubMedPubMedCentralGoogle Scholar
  38. Shergill B, Meloty-Kapella L, Musse AA, Weinmaster G, Botvinick E (2012) Optical tweezers studies on Notch: single-molecule interaction strength is independent of ligand endocytosis. Dev Cell 22(6):1313–1320CrossRefPubMedPubMedCentralGoogle Scholar
  39. Stabley DR, Jurchenko C, Marshall SS, Salaita KS (2012) Visualizing mechanical tension across membrane receptors with a fluorescent sensor. Nat Methods 9:64–67. CrossRefGoogle Scholar
  40. Stephenson NL, Avis JM (2012) Direct observation of proteolytic cleavage at the S2 site upon forced unfolding of the Notch negative regulatory region. Proc Natl Acad Sci 109(41):E2757–E2765CrossRefPubMedGoogle Scholar
  41. Varnum-Finney B et al (2000) Immobilization of Notch ligand, Delta-1, is required for induction of notch signaling. J Cell Sci 113(23):4313–4318PubMedGoogle Scholar
  42. Vogel V (2006) Mechanotransduction involving multimodular proteins: converting force into biochemical signals. Annu Rev Biophys Biomol Struct 35(1):459–488CrossRefPubMedGoogle Scholar
  43. Wang X, Ha T (2013) Defining single molecular forces required to activate integrin and notch signaling. Science 340:991–994. CrossRefPubMedPubMedCentralGoogle Scholar
  44. Wang W, Struhl G (2005) Distinct roles for Mind bomb, Neuralized and Epsin in mediating DSL endocytosis and signaling in Drosophila. Development 132:2883–2894. CrossRefPubMedGoogle Scholar
  45. Weng AP et al (2004) Activating mutations of NOTCH1 in human T cell acute lymphoblastic leukemia. Science 306(5694):269–271CrossRefPubMedGoogle Scholar
  46. Xiang X, Choi SH, Tiancen H, Tiyanont K, Habets R, Groot AJ, Vooijs M, Aster JC, Chopra R, Fryer C, Blacklow SC (2015) Insights into autoregulation of Notch3 from structural and functional studies of its negative regulatory region. Structure 23(7):1227–1235CrossRefGoogle Scholar
  47. Yan W, Cain-Hom C, Choy L, Hagenbeek TJ, de Leon GP, Chen Y, Finkle D, Venook R, Xiumin W, Ridgway J, Schahin-Reed D, Dow GJ, Shelton A, Stawicki S, Watts RJ, Zhang J, Choy R, Howard P, Kadyk L, Yan M, Zha J, Callahan CA, Hymowitz SG, Siebel CW (2010) Therapeutic antibody targeting of individual Notch receptors. Nature 464(7291):1052–1057CrossRefPubMedGoogle Scholar
  48. Zhang X, Halvorsen K, Zhang C-Z et al (2009) Mechanoenzymatic cleavage of the ultralarge vascular protein von Willebrand factor. Science 324:1330–1334. CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer International Publishing AG, part of Springer Nature 2018

Authors and Affiliations

  • Klaus N. Lovendahl
    • 1
  • Stephen C. Blacklow
    • 2
  • Wendy R. Gordon
    • 1
    Email author
  1. 1.Department of Biochemistry, Molecular Biology and BiophysicsUniversity of MinnesotaMinneapolisUSA
  2. 2.Department of Biological Chemistry and Molecular PharmacologyHarvard Medical SchoolBostonUSA

Personalised recommendations