CDB15:0000952 JAG1 — NOTCH1

ABSTRACT: 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.

ABSTRACT: Here we report the isolation of a rat cDNA clone, Jagged, which we show encodes a ligand for vertebrate Notch. Our conclusion is based on three observations. First, sequence analysis reveals substantial homology between Jagged and invertebrate ligands for the LIN-12/Notch proteins. Second, in situ hybridization of rat embryos identifies both distinct and overlapping patterns of gene expression for Jagged with those for Notch1, Notch2, and Notch3. Finally, the biological activity of Jagged was tested using a cell culture assay in which Jagged activates rat Notch1 expressed in myoblasts and prevents muscle cell differentiation. Our data support the hypothesis that Notch-ligand interactions function in maintaining mammalian cells in an undifferentiated state.