Aproximación del modelo simulado mediante FIT3

1.4.1. Notch signalling regulates the patterning o f cell fates.

In many developing tissues, stocks of stem cells need to be retained at least until they have generated enough differentiated cells to complete the tissue. This depends on signalling between cells so that each cell knows what its neighbour is doing. Notch signalling is a mechanism for controlling cell fate through such local intercellular interactions.

Notch was first described in Drosophila as a mutation resulting in notches at the wing margin (Moohr, 1919). Stronger lethal loss-of-function mutations were discovered by Poulson (Poulson, 1937). The most obvious defect in Notch mutant flies is that they have a huge excess of neural cells, but abnormalities can also be found in many other tissues, including examples of tissues derived from all three germ layers (Hartenstein et a l, 1992). This multitude of defects led Poulson to describe the Notch mutant as “a kind of hopeless monster”. As well as being used many different tissues of the

Drosophila embryo. Notch signalling has also been conserved through evolution, being found in organisms ranging from sea urchins to humans. It is now clear that Notch is widely used as a tool for regulating differentiation decisions through local cell

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1.4.2. Notch pathway

The Notch gene encodes a single pass transmembrane receptor that becomes activated by transmembrane ligands Delta and Serrate (also called Jagged in vertebrates) on neighbouring cells (Artavanis-Tsakonas et a l, 1999). Upon activation. Notch

undergoes a set of proteolytic cleavages, ultimately releasing an intracellular fragment called NotchIC (Schroeter et a i, 1998). The Notch signalling pathway is unusual in that it involves no intermediate steps or cascades of secondary messengers: Notch IC moves directly to the nucleus where, by interaction with the DNA binding protein, Suppresser of Hairless (SuH: also called RBPJk in vertebrates) it activates transcription of target genes (Figure 1.11). (Tamura et al, 1995; Bailey and Posakony, 1995;

Lecourtois and Schweisguth, 1995). The target genes vary according to cell type and context. Common vertebrate targets are the bHLH transcription factors of the Hairy and Enhancer of Split (HES) family (Fisher and Gaudy, 1998). There is some evidence that Notch can signal independently of SuH in certain situations (Shawber et a i, 1996b; Matsuno et al., 1997; Wang et a!., 1997; Ordentlich et a!., 1998). However in most cases it is clear that SuH is the major activator of Notch signalling.

NOTCH

DELTA _NOTCH

SuH

Figure 1.11; The Notch signalling pathway

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1.4.3. Structure o f Notch and its ligands

Structure-function analysis has mainly focused on Drosphila Notch and its ligands. However, all of the structural elements described below can be recognised in their homologues in other species, including humans.

Figure 1.12 Notch N - t e r m .

EGF 1-12

EGF 24-29

Notch

Notch is a 300kd single pass transmembrane receptor (fig 1.12). Notch is cleaved in the trans-Golgi network by a furin-like convertase and appears on the cell surface as a heterodimer (Blaumueller et al, 1997; Logeât et al, 1998). The extracellular domain contains 36 tandem EGF like repeats. EGF repeats 11 and 12 are necessary and sufficient to physically bind ligand in trans (Rebay et al, 1991; Lieber et a l, 1992) whilst a region at around EGF repeats 24-29 mediates inhibitory interactions with ligand in cis, although it is not yet clear whether this region directly binds ligand (section 1.4.5) (De Cells and Bray, 2000). Close to the transmembrane domain lie three cysteine-containing repeats of unknown function termed the LNR (Lin 12 Notch Repeats: Lin 12 is worm Notch homologue).

LNR Transmembrane RAM Ankyrin Repeats C - t e r m .

The intracellular domain contains a region called the RAM domain that binds SuH. Binding of SuH is facilitated by an adjacent region containing six tandem ankyrin repeats (motifs associated with protein-protein interactions). Intracellular modulators of Notch activity such as Numb and Dishevelled interact with a region lying just C-terminal to the ankyrin repeats. The intracellular terminus is the least conserved region amongst the Notch family members, although they do all share a PEST sequence (proline-glutamate-serine- threonine rich region), a PDZ protein interaction domain, and a nuclear localisation signal.

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Four mammalian Notch receptors have been identified which share an overall identity of about 50% between themselves and the single Drosophila Noteh protein:

Notchl/TAN-1 (Ellisen <a/., 1991; del Amo et a l, 1993), Noteh2 (Weinmaster <3/.,

1992), Notch] (Lardelli et a l, 1994), and Notch4/int-3 (Uyttendaele et a l, 1996).

Delta

Delta is a 90 kDa transmembrane ligand for the Notch receptor. (Figure 1.13)

The extraeellular domain of Delta bears 8 EGF-like repeats and a 45 amino acid motif termed the DSL domain from Delta Serrate Lag2 (Lag2 is a worm Noteh ligand). The DSL domain ean be

described as a modified EGF-like repeat, lacking one of the six eharacteristic cysteine residues: this domain is required for funetion in invertebrates (Muskaviteh,

1994). The intracellular domain contains a hydrophobic motif at the C-terminus, whose sequenee suggests that it is recognised by PDZ domain proteins

N - t e r m