Notch signaling

The Notch signaling pathway is a highly conserved cell signaling system present in most multicellular organisms. Notch is present in all metazoans, and vertebrates possess four different notch receptors, referred to as Notch1 to Notch4. The Notch receptor is a single-pass (i.e. it crosses the membrane once, in contrast to many other transmembrane proteins which loop back and forth between the extracellular and intracellular spaces) transmembrane receptor protein. It is a hetero-oligomer composed of a large extracellular portion which associates in a calcium dependent, non-covalent interaction with a smaller piece of the Notch protein composed of a short extracellular region, a single transmembrane-pass, and a small intracellular region.

Discovery
The Notch gene was discovered in 1917 by Thomas Hunt Morgan when it was first noticed in a strain of the fruit fly Drosophila melanogaster with notches apparent in their wingblades. Its molecular analysis and sequencing was undertaken in the 1980s.

Action
The Notch protein sits like a trigger spanning the cell membrane, with part of it inside and part outside. Ligand proteins binding to the extracellular domain induce proteolytic cleavage and release of the intracellular domain, which enters the cell nucleus to alter gene expression.

Functions
The Notch signaling pathway is important for cell-cell communication, which involve gene regulation mechanisms that control multiple cell differentiation processes during embryonic and adult life. Notch signaling also has a role in the following processes:
 * neuronal function and development
 * stabilizing arterial endothelial fate and angiogenesis
 * regulating crucial cell communication events between endocardium and myocardium during both the formation of the valve primordial and ventricular development and differentiation
 * cardiac valve homeostasis and has implications in other human disorders involving the cardiovascular system
 * timely cell lineage specification of both endocrine and exocrine pancreas
 * influencing binary fate decisions of cells that must choose between the secretory and absorptive lineages in the gut
 * expanding the HSC compartment during bone development and participate in commitment to the osteoblastic lineage suggesting a potential therapeutic role for Notch in bone regeneration and osteoporosis
 * regulating cell-fate decision in mammary gland at several distinct development stage
 * possibly some non-nuclear mechanisms, such as controlling the actin cytoskeleton through the tyrosine kinase Abl

Notch signaling is often repressed in many cancers, and faulty Notch signaling is implicated in many diseases including T-ALL (T-cell acute lymphoblastic leukemia), CADASIL (Cerebral Autosomal Dominant Arteriopathy with Sub-cortical Infarcts and Leukoencephalopathy), MS (Multiple Sclerosis), Tetralogy of Fallot, Alagille syndrome, and myriad other disease states.

Details of the pathway
Maturation of the Notch receptor involves cleavage at the prospective extracellular side during intracellular trafficking in the Golgi complex. This results in a bipartite protein, composed of a large extracellular domain linked to the smaller transmembrane and intracellular domain. Binding of ligand promotes two proteolytic processing events; as a result of proteolysis, the intracellular domain is liberated and can enter the nucleus to engage other DNA-binding proteins and regulate gene expression.

Notch and most of its ligands are transmembrane proteins, so the cells expressing the ligands typically need to be adjacent to the Notch expressing cell for signaling to occur. The Notch ligands are also single-pass transmembrane proteins and are members of the DSL (Delta/Serrate/LAG-2) family of proteins. In Drosophila melanogaster (the fruit fly) there are two ligands named Delta and Serrate. In mammals, the corresponding names are Delta-like and Jagged. In mammals there are multiple Delta-like and Jagged ligands, as well as possibly a variety of other ligands, such as F3/contactin. There has been at least one report that suggests that some cells can send out processes which allow signaling to occur between cells which are as much as four or five cell diameters apart.

The Notch extracellular domain is composed primarily of small cysteine knot motifs called EGF-like repeats. Notch 1 for example has 36 of these repeats. Each EGF-like repeat is approximately 40 amino acids, and its structure is defined largely by six conserved cysteine residues that form three conserved disulfide bonds. Each EGF-like repeat can be modified by O-linked glycans at specific sites. An O-glucose sugar may be added between the first and second conserved cysteine, and an O-fucose may be added between the second and third conserved cysteine. These sugars are added by an as yet unidentified O-glucosyltransferase, and GDP-fucose Protein O-fucosyltransferase 1 (POFUT1) respectively. The addition of O-fucose by POFUT1 is absolutely necessary for Notch function, and without the enzyme to add O-fucose, all Notch proteins fail to function properly. As yet, the manner in which the glycosylation of Notch affects function is not completely understood.

The O-glucose on Notch can be further elongated to a trisaccharide with the addition of two xylose sugars by xylosyltransferases, and the O-fucose can be elongated to a tetrasaccharide by the ordered addition of an N-acetylglucosamine (GlcNAc) sugar by an N-Acetylglucosaminyltransferase called Fringe, the addition of a galactose by a galactosyltransferase, and the addition of a sialic acid by a sialyltransferase.

To add another level of complexity, in mammals there are three Fringe GlcNAc-transferases, named Lunatic Fringe, Manic Fringe, and Radical Fringe. These enzymes are responsible for something called a "Fringe Effect" on Notch signaling. If Fringe adds a GlcNAc to the O-fucose sugar, then the subsequent addition of a galactose and sialic acid will occur. In the presence of this tetrasaccharide, Notch signals strongly when it interacts with the Delta ligand, but has markedly inhibited signaling when interacting with the Jagged ligand. The means by which this addition of sugar inhibits signaling through one ligand, and potentiates signaling through another is not clearly understood.



Once the Notch extracellular domain interacts with a ligand, an ADAM-family metalloprotease called TACE (Tumor Necrosis Factor Alpha Converting Enzyme) cleaves the Notch protein just outside the membrane. This releases the extracellular portion of Notch, which continues to interact with the ligand. The ligand plus the Notch extracellular domain is then endocytosed by the ligand-expressing cell. There may be signaling effects in the ligand-expressing cell after endocytosis; this part of Notch signaling is a topic of active research. After this first cleavage, an enzyme called γ-secretase (which is implicated in Alzheimer's disease) cleaves the remaining part of the Notch protein just inside the inner leaflet of the cell membrane of the Notch-expressing cell. This releases the intracellular domain of the Notch protein, which then moves to the nucleus where it can regulate gene expression by activating the transcription factor CSL. Other proteins also participate in the intracellular portion of the Notch signaling cascade.

Triggering
Because most ligands are also transmembrane proteins, the receptor is normally only triggered from direct cell-to-cell contact. In this way, groups of cells can organise themselves, such that if one cell expresses a given trait, this may be switched off in neighbour cells by the inter-cellular Notch signal. In this way groups of cells influence one another to make large structures.

The Notch cascade consists of Notch and Notch ligands, as well as intracellular proteins transmitting the Notch signal to the cell's nucleus. The Notch/Lin-12/Glp-1 receptor family was found to be involved in the specification of cell fates during development in Drosophila and C. elegans. The Notch signaling pathway begins to inhibit new cell growth when adolescence is reached, and keeps neural networks stable in adulthood.