PMC

Fringe-mediated glycosylation differentially modulates ligand-induced Notch signaling levels. (a) To survive the ligand endocytic pull, ligand–receptor interactions (+++) must be stronger than the force required to unfold the NRR or dissociate the HD (++) for S2 exposure. (b) Fringe glycosylation of Notch enhances Delta–Notch interaction (++++) to increase the number of ligand–receptor pairs that survive the ligand endocytic pull, yielding more NICD for increased signaling. (c) Fringe glycosylation of Notch reduces Jagged/Serrate binding strength (+) to decrease survival of ligand–receptor interactions and consequently lower the level of active NICD for signaling.

Structural basis of Notch autoinhibition. (a) Notch1 NRR ribbon diagram (PBD ID: 3ETO) depicting three LNR modules (magenta) with divalent cations (gray) and the HD [HD-N (yellow) and HD-C (green)]. The S2 site (blue) located in β5 is protected from ADAM cleavage by the LNR modules wrapped over the HD. (b) ADAM10 constitutively interacts with intact Notch but the NRR maintains the OFF state in the absence of ligand. The S1 furin cleavage site is located in the unstructured loop and the ligand-binding site composed of EGF-like repeats 11&12 (red) is indicated.

Mechanistic differences between ligand- and EDTA-induced Notch activation. (a) Pulling force generated by endocytosis of Notch-bound ligand peels the LNR modules away from the HD to expose the S2 site, and induces an ADAM10 conformational change for enzymatic activity. ADAM10 cleavage of the exposed S2 directly generates a γ-secretase substrate (1). Alternatively, physical dissociation of the HD within the intact Notch heterodimer generates an inactive intermediate (2), which requires ADAM10 cleavage of the exposed S2 for further activating γ-secretase proteolysis. (b) Activation of Notch by EDTA-induced chelation of divalent cations critical to LNR structure unravels of the LNR modules (magenta-pink lines) for disengagement from the HD to expose S2 (b). Changes in NRR structure may promote ADAM10 dissociation and recruitment of ADAM17 to effect NECD shedding.

Models for ligand endocytosis in Notch signaling. (a) The “recycling” model proposes endocytosis promotes recycling of ligand ubiquitylated by the E3 ubiquitin (Ub) ligases Mindbomb (Mib) or Neuralized (Neur). Ub recruites epsin, for clathrin/dynamin-dependent ligand endocytosis and subsequent trafficking via early/recycling endosomes to a cell surface microdomain. (b) The “pulling force” model proposes interactions between ligand signal-sending and Notch signal-receiving cells induce ligand ubiquitylation and formation of epsin-dependent endocytic structures for mechanical force (double red arrowheads) to remove NECD from the intact Notch heterodimer for uptake by ligand cells (NECD transendocytosis). The remaining membrane-bound Notch undergoes activating proteolysis by ADAM at S2 and γ-secretase at S3 to release NICD for translocation to the nucleus and transcription of Notch target genes.