Combining genetic and biophysical approaches to probe the structure and function relationships of the notch receptor

Abstract

Notch is a conserved cell signalling receptor regulating many aspects of development and tissue homeostasis. Notch is activated by ligand-induced proteolytic cleavages that release the Notch intracellular domain, which relocates to the nucleus to regulate gene transcription. Proteolytic activation first requires mechanical force to be applied to the Notch extracellular domain through an endocytic pulling mechanism transmitted through the ligand/receptor interface. This exposes the proteolytic cleavage site allowing the signal to be initiated following removal of the Notch extracellular domain. Ligands can also act, when expressed in the same cell, through non-productive cis-interactions to inhibit Notch activity. Furthermore, ligand selectivity and Notch activation are regulated by numerous post-translational modifications of the extracellular domain. Additional non-canonical trans and cis interactions with other regulatory proteins may modulate alternative mechanisms of Notch activation that depend on endocytic trafficking of the full-length receptor and proteolytic release of the intracellular domain from endo-lysosomal surface. Mutations of Notch, located in different regions of the protein, are associated with a spectrum of different loss and gain of function phenotypes and offer the possibility to dissect distinct regulatory interactions and mechanisms, particularly when combined with detailed structural analysis of Notch in complex with various regulatory partners.

Making sense out of missense mutations: Mechanistic dissection of Notch receptors through structure‐function studies inDrosophila

“…Furthermore, by analyzing a catalytically dead O-fut1 allele generated through a gene knock-in methodology in Drosophila, Notch receptors that lack O-fucosylation were shown to exhibit defects in endocytic trafficking at high temperature (30°C) (Ishio et al, 2015). In addition to studies based on genetics and biochemistry, recent crystallography and nuclear magnetic resonance (NMR) spectroscopy based structural biological approaches are beginning to unravel how Notch molecularly distinguishes Delta family ligands from Serrate/Jagged family ligands (Baron, 2017;Handford, Korona, Suckling, Redfield, & Lea, 2018), which will be discussed later in this article. Considering that the role of endocytic trafficking has been proposed to function as a buffering mechanism to provide robustness to Notch signaling under different temperature conditions (Shimizu et al, 2014) (Harvey et al, 2016;Xu et al, 2007).…”

“…Considering that the role of endocytic trafficking has been proposed to function as a buffering mechanism to provide robustness to Notch signaling under different temperature conditions (Shimizu et al, 2014) suggest that multiple EGFr that are modified by O-fut1/Fng contribute to ligand-receptor interactions and ligand selectivity (Harvey et al, 2016;Xu et al, 2007). In addition to studies based on genetics and biochemistry, recent crystallography and nuclear magnetic resonance (NMR) spectroscopy based structural biological approaches are beginning to unravel how Notch molecularly distinguishes Delta family ligands from Serrate/Jagged family ligands (Baron, 2017;Handford, Korona, Suckling, Redfield, & Lea, 2018), which will be discussed later in this article.…”

“…The N M1 allele was first reported in 1993 through an EMSmediated chemical mutagenesis screen and carries a p.E491V mutation in the Ca 2+ binding domain of EGFr-12(Baron, 2017;de Celis, Barrio, Arco, & Garcia-Bellido, 1993). Hemizygous and homozygous Abbreviations: ANK, ankyrin repeat; EGFr, epidermal growth factor-like repeat; HD, heterodimerization; LNR, LIN-12/Notch repeat; TAD, transactivation domain.…”

Neurovascular Alterations in Vascular Dementia: Emphasis on Risk Factors

“…Mutations in specific genes constitute the third most common cause of cSVD, among which the mutation of the neurogenic locus notch homolog protein (NOTCH)3 gene is the better characterized (Cannistraro et al, 2019;Marini et al, 2020). NOTCH3 is a member of the transmembrane receptor NOTCH family, which is critically involved in developmental patterning, cell fate decisions, regulation of cell survival, and proliferation (Kopan and Ilagan, 2009;Bray, 2016;Baron, 2017;Hosseini-Alghaderi and Baron, 2020). During adulthood, NOTCH3 regulates stem cells and their lineages to promote tissue maintenance and repair.…”

Notch3 in Development, Health and Disease

“…Notch is a transmembrane, developmental signalling receptor, which plays many crucial roles in developmental patterning, cell fate decisions, regulation of cell survival and proliferation [1][2][3]. As well as pleiotropic developmental roles, Notch receptors continue to play important roles in adult tissue maintenance and repair through regulation of stem cells and their lineages and other cellular processes.…”