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Science 338 (6111): 1229-1232

Copyright © 2012 by the American Association for the Advancement of Science

A Mutation in EGF Repeat-8 of Notch Discriminates Between Serrate/Jagged and Delta Family Ligands

Shinya Yamamoto1, Wu-Lin Charng1, Nadia A. Rana2, Shinako Kakuda2, Manish Jaiswal3,4, Vafa Bayat1,5, Bo Xiong1, Ke Zhang6, Hector Sandoval3, Gabriela David1, Hao Wang3, Robert S. Haltiwanger2, and Hugo J. Bellen1,3,4,6,7,8,*

1 Program in Developmental Biology, Baylor College of Medicine, Houston, TX 77030, USA.
2 Department of Biochemistry and Cell Biology, Stony Brook University, Stony Brook, NY 11794, USA.
3 Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX 77030, USA.
4 Howard Hughes Medical Institute, Baylor College of Medicine, Houston, TX 77030, USA.
5 Medical Scientist Training Program, Baylor College of Medicine, Houston, TX 77030, USA.
6 Program in Structural and Computational Biology and Molecular Biophysics, Baylor College of Medicine, Houston, TX 77030, USA.
7 Department of Neuroscience, Baylor College of Medicine, Houston, TX 77030, USA.
8 Jan and Dan Duncan Neurological Research Institute, Baylor College of Medicine, Houston, TX 77030, USA.

Figure 1
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Fig. 1. Njigsaw, a V361M mutation in a conserved domain in EGFr-8, is defective in Ser-mediated signaling. (A) Schematic representation of N. LNR, Lin12/Notch repeats; TMD, transmembrane domain; RAM, RBP-j{kappa}–associated molecule domain; NLS, nuclear localization signal; ANK, ankyrin repeat domain; TAD, transactivation domain; PEST, Pro-Glu-Ser-Thr rich domain. (B) Valine 361 mutated in Njigsaw is conserved in Notch paralogs. (C) Schematic diagram of EGFr-8. [Single-letter abbreviations for the amino acid residues are as follows: A, Ala; C, Cys; D, Asp; E, Glu; F, Phe; G, Gly; H, His; I, Ile; K, Lys; L, Leu; M, Met; N, Asn; P, Pro; Q, Gln; R, Arg; S, Ser; T, Thr; V, Val; W, Trp; and Y, Tyr.] (D) Wings with Njigsaw mutant clones exhibit notching. (E) Ectopic wing margin in wings with Njigsaw mutant clones. (F) Njigsaw homozygous mutant bristles, marked by yellow, are present in their normal stereotypic pattern. Homozygous WT bristles are singed, and heterozygous bristles are yellow+ singed+. (G) Diagram of wing margin formation. (H to I’) N activation at the dorsal-ventral boundary of WT and Njigsaw mutant discs. [(H) and (I)] Low and [(H’) and (I’)] high magnification images of third instar wing imaginal discs of [(H) and (H’)] WT and [(I) and (I’)] Njigsaw hemizygous mutant larva. The dorsal domain is labeled with β-galactosidase (LacZ) driven by the msh enhancer (msh-lacZ: green). N activation is shown by Cut (red). Scale bars, 100 μm (D) to (F); 20 μm (H) to (I’).


Figure 2
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Fig. 2. Ligand-cis-inhibition of Ser is impaired in Njigsaw mutant cells. (A to B’) Njigsaw clones in larval wing discs (marked by the absence of GFP, green). Cell-nonautonomous ectopic N activation is indicated by arrows [Wingless (Wg) in (A)] and arrowheads [Cut in (B)]. Clones were induced by (A) Ubx-FLP or (B) hs-FLP. (C and C') Expression of Ser (blue) is elevated in mutant clones, leading to cell-nonautonomous N activation (monitored by Cut) in surrounding WT neighboring cells. Scale bars, 20 μm. (D and E) Schematic diagrams of ligand-cis-inhibition and cell-nonautonomous N activation. In Njigsaw mutant cells, Ser accumulates at the cell surface and triggers signaling in the neighboring WT cells (E). WT cells are depicted in green, and Njigsaw homozygous mutant cells are in gray.


Figure 3
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Fig. 3. Njigsaw is defective in Ser binding in fly cells and Jagged1 signaling in mammalian cells, independent of Fng. (A and B) Multiple reaction monitoring (MRM) traces of ions show relative levels of unmodified (black line), O-fucose monosaccharide (red line), or O-fucose disaccharide (blue line) glycoforms of a peptide from EGFr-8 generated in the presence of Fng. Red triangle, fucose; blue square, GlcNAc; black rectangle, peptide. (C) Analysis of relative levels of monosaccharide versus disaccharide forms of the peptide from EGFr-8 based on the average of multiple MRM analyses (fig. S10, C and D). (D and E) Ligand-receptor binding assays in Drosophila S2 cells reveal loss of N-Ser binding in Njigsaw. Binding of NWT and Njigsaw to Ser is shown in (D), whereas binding of NWT and Njigsaw to Dl is shown in (E). (F and G) Mammalian cell–based assays show that V327M in mouse Notch2 reduces receptor activation by Jagged1 (F) but not Dll1 (G). The V327A reduces Notch2-Jagged1 signaling even more (F), whereas a mutation in the O-fut1/Fng modification site of EGFr-8 (T314A) does not affect Jagged1 or Dll1 signaling [(F) and (G)]. Error bars indicate SEM.


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