CDB15:0000449 DLL1 — NOTCH2

ABSTRACT: DLL1 and DLL4 are Notch ligands with high structural similarity but context-dependent functional differences. Here, we analyze their functional divergence using cellular co-culture assays, biochemical studies, and in vivo experiments. DLL1 and DLL4 activate NOTCH1 and NOTCH2 differently in cell-based assays and this discriminating potential lies in the region between the N-terminus and EGF repeat three. Mice expressing chimeric ligands indicate that the ectodomains dictate ligand function during somitogenesis, and that during myogenesis even regions C-terminal to EGF3 are interchangeable. Substitution of NOTCH1-interface residues in the MNNL and DSL domains of DLL1 with the corresponding amino acids of DLL4, however, does not disrupt DLL1 function in vivo. Collectively, our data show that DLL4 preferentially activates NOTCH1 over NOTCH2, whereas DLL1 is equally effective in activating NOTCH1 and NOTCH2, establishing that the ectodomains dictate selective ligand function in vivo, and that features outside the known binding interface contribute to their differences.

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.