Figure 4: Integration of Notch signaling circuitry:

The Notch pathway generates a short-range communication channel utilized throughout skin development and elsewhere in the adult to regulate multiple cellular processes (e.g., proliferation, stem cell and stem cell niche maintenance, cell fate specification, differentiation and cell death). Notch signals in a unique mechanism mediated by proteolysis and without any secondary messengers. Post-translational modifications and trafficking of the ligands and receptors can regulate the amplitude and timing of Notch activity. 1–2) Receptor Maturation. Upon translation, the Notch protein is fucosylated by the Pofut1. The fucose can be extended by the glycosyltransferase activity of Fringe. This impacts the ability of specific ligands to activate Notch (see below). During exocytosis the Notch receptor is cleaved by PC5, a protein convertase, at Site 1 (S1). At the cell surface Notch is a heterodimer (HD) held together by non-covalent interactions within the HD domain which, together with three Lin-Notch repeats (LNR) forms the negative regulatory region (NRR). 2) The steady-state levels of the Notch receptor at the cell surface are regulated by several proteins, many of which are E3 ubiquitin ligases (Dtx, Nedd4). After recycling, inappropriate receptor activation within endosomes can occur in the absence of ligand binding (NICD*). The ESCRT complex proteins are involved in Notch down-regulation, and mutations in this complex may contribute to pathogenesis in different cellular contexts. 3) Ligand maturation. Notch ligands are also Type I transmembrane proteins. The two major classes of ligands are Delta and Jagged (Serrate in Drosophila), the latter containing a cysteine rich domain. To signal, ligands must undergo ubiquitinylation by E3 ubiquitin ligases Neur and Mib which trigger Epsin-mediated endocytosis, and an undefined modification produces an active ligand which recycles to the cell surface in a Rab11 dependent process. Current models explaining the nature of ligand modification include ligand clustering, post-translational modifications and/or recycling into specific membrane domains. 4–6) Activation. Productive receptor-ligand interactions occur between neighboring cells (in TRANS) whereas negative interactions occur between receptor-ligand proteins coexpressed in the same cell (in CIS). Fringe-modified Notch1 receptors favor Delta binding (shown). Ligand endocytosis is thought to generate sufficient force to unfold the NRR, exposing Notch to cleavage at site S2 by ADAM10 metalloproteases. The membrane-anchored NEXT (Notch extracellular truncation) fragment is recognized by γ-secretase, an enzymatic complex composed of PS, NCT, PEN2 and APH1. γ-secretase then cleaves the Notch transmembrane domain sequentially starting near the cytosolic surface (sites S3 and S4) to release the Notch intracellular domain (NICD) and Nβ peptides, respectively. 5) In the absence of NICD, the DNA-binding protein CSL associates with ubiquitous co-repressor (Co-R) proteins, HDACs and Sirt1 to repress transcription of target genes. NICD binding to CSL recruits the adaptor protein Mastermind (MAM), which recruits the mediator complex and assembles an active transcription complex on target promoters. 6) During the transcriptional activation process, NICD is phosphorylated on its PEST domain by kinases such as CDK8 and targeted for proteasomal degradation by E3 ubiquitin ligases such as Sel10/Fbw7. This terminates the Notch signal and resets the cell for the next round of signaling. This figure is reproduced from (Ilagan and Kopan, 2007) with permission from the publisher.