A mutation in EGF repeat-8 of Notch discriminates between Serrate/Jagged and Delta family ligands

Abstract

Notch signaling affects many developmental and cellular processes and has been implicated in congenital disorders, stroke, and numerous cancers. The Notch receptor binds its ligands Delta and Serrate and is able to discriminate between them in different contexts. However, the specific domains in Notch responsible for this selectivity are poorly defined. Through genetic screens in Drosophila, we isolated a mutation, Notch(jigsaw), that affects Serrate- but not Delta-dependent signaling. Notch(jigsaw) carries a missense mutation in epidermal growth factor repeat-8 (EGFr-8) and is defective in Serrate binding. A homologous point mutation in mammalian Notch2 also exhibits defects in signaling of a mammalian Serrate homolog, Jagged1. Hence, an evolutionarily conserved valine in EGFr-8 is essential for ligand selectivity and provides a molecular handle to study numerous Notch-dependent signaling events.

Fig. 1

N^jigsaw, 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-jκ–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 N^jigsaw 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 N^jigsaw mutant clones exhibit notching. (E) Ectopic wing margin in wings with N^jigsaw mutant clones. (F) N^jigsaw 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 N^jigsaw 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′)] N^jigsaw 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′).

Fig. 2

Ligand-cis-inhibition of Ser is impaired in N^jigsaw mutant cells. (A to B′) N^jigsaw 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 N^jigsaw mutant cells, Ser accumulates at the cell surface and triggers signaling in the neighboring WT cells (E). WT cells are depicted in green, and N^jigsaw homozygous mutant cells are in gray.

Fig. 3

N^jigsaw 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 N^jigsaw. Binding of N^WT and N^jigsaw to Ser is shown in (D), whereas binding of N^WT and N^jigsaw 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.