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FIG 22.2  Domain architecture of Notch, its ligands and nuclear proteins associated with its intracellular segment.

Both Notch and its ligands contain numerous EGF-like domains. This renders them long and flexible, but the flexibility is restrained at domains that bind Ca2 . The DSL domains interact with Notch at different sites. The mature Notch protein is cleaved in the heterodimerization domain (S1). The intracellular segment contains a RAM domain, approximately 100 residues and loosely defined as a region that commences at the -secretase cleavage site (see Figure 22.9) and ends at the first ankyrin repeat. The C-terminal region contains TAD and PEST motifs, both subject to phosphorylation by CDK8. The nuclear protein CSL binds the intracellular fragment of Notch, in which both the BT and C-terminal Rel-like domains take part. Mastermind-1, which binds CSL and the intracellular Notch segment (Nicd) with its N-terminal regions, has no recognizable domains.

fragments are reassembled as a heterodimer. This is essential for Notch activity in mammals, although Drosophila appears to get away with uncleaved receptors.14 The extracellular segment contains numerous (26–36) EGF-like domains and three cysteine-rich repeats (Lin repeats) that mask an essential second extracellular cleavage site (S2) (Figure 22.3). As an indication of its importance, the region covering the Lin repeats is altered in 26% of activating mutations that are associated with T cell acute lymphoblastic leukaemia.15 The DSL domain of the ligand interacts with EGF-like repeats number 11

and 12, but other regions are not excluded (Figure 22.2). Some of the EGF-like repeats contain Ca2 binding sites, which stiffen the molecule (as in the cadherin adhesion molecules), whereas others are modified by fucosylation on serine or threonine (for instance T466 on EGF-like repeat 12).16 Mammals have four genes, Notch-1–4, while Drosophila carries only one copy and

C. elegans has two (see Table 22.1).

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Table 22.1  Components of the Notch signalling pathway

Receptor associated proteins (involved in activity and endocytosis)

Continued

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Table 22.1  Continued

Notch E3-ligases acting in the cytoplasm (regulation of trafficking)

Adapted from Fiuza and Arias17 and Nichols et al.18

Functional Notch requires plasma membrane-expression of the tetraspan membrane protein Sanpodo.19 Little is known, about this protein, which was originally discovered as the homologue of the actin-associated tropomodulin. Sanpodo acts downstream of Delta and upstream of Notch to which it binds. Importantly, loss of its expression at the plasma membrane with subsequent accumulation in endocytic vesicles, renders cells unresponsive to Delta. We return to Sanpodo below (see page 720).

Glycosylation of ligands and receptor

The fucosylation sites on the EGF-like repeats of Notch and its ligands are further modified by addition of N-acetylglucosamines (so-called glycosylation elongation). Lack of the initial fucosylation enzyme, OFUT1, results in a phenotype similar to loss of Notch.20 It may possibly also act as a chaperone in the rough endoplasmic reticulum, stimulating Notch signalling simply by

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