A Notch updated

Abstract

Cell-cell signaling mediated by the Notch receptor is iteratively involved in numerous developmental contexts, and its dysregulation has been associated with inherited genetic disorders and cancers. The core components of the signaling pathway have been identified for some time, but the study of the modulation of the pathway in different cellular contexts has revealed many layers of regulation. These include complex sugar modifications in the extracellular domain as well as transit of Notch through defined cellular compartments, including specific endosomes.

Figure 1.

Schematic illustration of Notch and the pathway. (A) The N-terminal part of the Notch ectodomain consists of 36 EGF-like repeats (gray ovals) and three LNRs (lin-12 Notch repeat; orange ovals). EGF repeats 11 and 12 interact with the ligands (pink ovals). NICD consists of an N-terminal RAM (recombination binding protein-J -associated molecule) domain, an ankyrin (ANK) domain (gray rectangles), and less-conserved regions, including a variable transactivation domain and a C-terminal PEST sequence (gray star). The red arrows indicate the cleavage sites: S1 (Furin), S2 (ADAM metalloprotease), and S3/S4 (γ-secretase). (B, 1) Secretory pathway: the modifications during the secretion of Notch to the membrane in the ER (purple) and the Golgi (orange). Notch is translated inside ER, where it is glycosylated by an O-fucosyltransferase O-Fut1 (light purple) and O-glucosyltransferase Rumi (yellow). Note the black circle on the Notch molecule in the ER; because Notch is not cleaved, the extracellular and intracellular domains are physically linked. Notch is then translocated into Golgi, where it is cleaved by Furin protease (scissors) at the S1 site and further modified by the N-acetylglucosaminyltransferase, Fringe. Note the red circle on the Notch molecule in the Golgi after S1 cleavage; the extracellular and intracellular domains are not physically linked. (2) Ligand-mediated activation: Notch (gray) interacts with the DSL ligands, Delta (blue) and Serrate (green), resulting in a series of proteolytic cleavage events induced by ligand binding. The S2 cleavage is mediated by ADAM protease (purple), whereas the S3/4 cleavage event is mediated by γ-secretase (g-secretase, in orange). Several studies also suggest that γ-secretase–mediated cleavage can occur inside endocytic compartment (shown in the light blue circle). (3) The endocytic regulation of the Notch receptor: full-length Notch can undergo endocytosis, leading to translocation of Notch into EEs, MVB, and lysosomes. From genetic data, several proteins have been identified to modulate this process, including Hrs and Bib, possibly during the EE-to-MVB transition of Notch, Lgd, and ESCRT complex, or during the MVB-to-lysosomes transition. These proteins further modulate Notch activity as described in the text. The dotted red arrow shows that in mutants that affect trafficking from the MVB to the lysosome, or if Notch is not trafficked to the lumen of the lysosome, Notch can undergo γ-secretase cleavage, resulting in a Notch GOF phenotypes.

Figure 2.

X-ray structure of the human NOTCH2 negative regulatory region (NRR) in its autoinhibited conformation, and models for signal activation. The top panel shows a ribbon representation of the NOTCH2 NRR. The LNR modules are colored in different shades of pink and purple, and the HD is in white and green. The three bound Ca²⁺ ions are shown in green, the bound Zn²⁺ ion is purple, and the disulfide bonds are red. The positions of S1 and S2 cleavage are indicated with red arrows. The bottom panel shows the model for activation by mechanical force driven by the DSL endocytosis. For unbound Notch, the LNRs (pink structure in 1–4) protect the S2 cleavage site in HD (white structure in 1–4). When Notch binds to DSL, which then undergoes endocytosis (1 and 2), this generates a mechanical force for disengaging LNRs from the HD (shown in 1–3). This relaxation between LNRs–HD interaction allows the metalloprotease to access the S2 cleavage site (shown in 4), leading to Notch activation. The figure is adapted from Gordon et al. (2007, 2008).