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Notch

Notch signaling is an evolutionary-conserved pathway in vertebrates and invertebrates which is involved many developmental processes, including cell-fate decisions, apoptosis, proliferation, and stem-cell self‑renewal. Aberrant expression of the Notch pathway is responsible for many forms of human disease, including breast cancer. Aberrant Notch signaling in the adult leads to tumor formation; Notch can act as an oncogene.

In mammals, the Notch genes encode large transmembane proteins that act as receptors for the DSL (Delta, Serrate, Lag-2) family of ligands. There are four different Notch receptors in mammals and five known DSL ligands.

Structure of Notch receptors in Drosophila and mammals. Diagrammatic representation of the Drosophila Notch (dNotch) receptor and the four known mammalian receptors. The Notch proteins are expressed on the cell surface as heterodimers composed of a large extracellular domain non-covalently linked to the intracellular domain. The extracellular domain of all Notch receptors contains epidermal growth-factor-like repeats (EGFLR) and three LIN Notch (LNR) repeats. The intracellular domain contains the RAM23 domain and seven Ankyrin/CDC10 repeats (ANK), necessary for protein- protein interactions. In addition, Notch receptors 1-3 contain two nuclear localization signals (NLS) compared to one NLS in Notch4. The NSL is necessary to target the intracellular domain to the nucleus where the transcriptional activation domain (TAD) activates downstream events. Note that Notch3 and Notch4 contain no TAD domain. All four Notch receptors contain a C-terminal Pro Glu Ser Thr (PEST) sequence for degradation.

The DSL ligands in Drosophila and mammals. The transmembrane DSL ligands (Delta, Serrate, LAG 2) of the Notch receptors in Drosophila and mammals. Mammals have five DSL family members. Delta-like 1, 3 and 4 are homologs of Drosophila Delta (dDelta), while Jagged1 and 2 are homologous to Drosophila Serrate (dSerrate). DSL ligands are transmembrane proteins of which the extracellular domain contains a characteristic number of EGF-like repeats and a cysteine rich N-terminal DSL domain. The DSL domain is a conserved motif found in all DSL ligands and required for their interaction with Notch. Serrate, Jagged1, and Jagged2 contain an additional cysteine rich domain (CRD).
Experiments in many different systems have provided a detailed model for DSL signaling via the Notch receptors. The signal is initiated by the interaction of DSL ligands with the extracellular domain of Notch on neighboring cells. The interaction between Notch and its DSL ligands involves EGF‑like repeats 11&12 in the extracellular portion of Notch. Ligand/receptor interaction induces two additional proteolytic cleavages within the Notch protein that release the Notch Intra-Cellular Domain (NICD, the activated form of Notch) from the cell membrane; the first cleavage occurs during the maturation of Notch at S1 site. In the absence of Notch ligand interaction, the cytoplasmic adaptor protein Numb  interacts directly with the cytoplasmic domain of Notch and inhibits Notch activation. On the cell surface, proteolytic cleavage (also referred as S2 cleavage) by the TNF-α-Converting Enzyme (TACE) and ADAM17 metalloprotease occurs extracellularly (residue 1711) to generate a membrane tethered fragment (NEXT). This is a transient intermediate that is cleaved again (S3 cleavage) by γ‑secretase. This occurs within the transmembrane domain (residue 1744) and leads to the release of the NICD into the cytoplasm. NICD subsequently translocates to the nucleus and binds to the CBF1 (also known as RBP-Jk) transcription factor. This is the primary nuclear effectors of the Notch signaling pathway which is bifunctional. In the absence of NICD, CBF1 binds to at least four co-repressors, the silencing mediator of retinoid and thyroid hormone receptor (SMRT), histone deacetylase‑1 (HDAC1), KyoT2, and Ski‑interacting protein (SKIP) and suppresses transcription. In contrast, the interaction CBF1 with the RAM23 and ANK repeats of the NICD displace these repressors to generate a transcriptional activator complex. The nuclear protein Mastermind‑like (MAML) also interacts with this complex to increase transcription further. However, the truncated version of MAML that maintains an association with the complex, behaves in a dominant-negative (DN-MAML) fashion and inhibits Notch activation.

The Notch signaling pathway. The Notch receptors are cleaved in the Golgi by the furin convertase enzyme, (a process known as S1 cleavage), which results in the expression of a non-covalently linked Notch heterodimer receptor on the cell surface. In the absence of a Notch-ligand interaction, Numb interacts directly with the cytoplasmic domain of Notch and inhibits its cleavage. Under these conditions, CBF1 binds to at least four co-repressors (SMRT, HDAC, SKIP and kyoT2) and suppresses transcription. Upon ligand binding, two sequential cleavages occur. One, known as S2 cleavage, mediated by the ADAM protease TACE (Tumour necrosis factor A-Converting Enzyme) occurs at aa 1771 near the transmembrane domain, while the other cleavage, known as S3 cleavage, is mediated by γ-secretase at aa 1744 within the transmembrane domain. This S3 cleavage liberates the intracellular domain of Notch (NICD), allowing its nuclear translocation and, where it binds to CBF1, converting it from a transcriptional repressor to a transcriptional activator by displacing the four co-repressors. Together with Mastermind‑like (MAML), NICD and CBF1 form a larger transcriptional activator complex and recruit coactivators such as p300.  The activated CBF1 can then transcribe its target genes which include members of the Hairy of enhancer of split (Hes) and the Hey family. Other targets, such as p21 (target in keratinocytes) are tissue specific. The stability of Numb is regulated by E3 ligases such as Mdm2, LNX and, Shia. In addition to this pathway, there is some evidence that points to the existence of a CBF1-independent Notch pathway which is not well characterized at present.

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