Combining structural, biophysical and in vivo techniques to probe structure/function of stem cell signalling receptor, Notch

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important challenge in biological research today. This is exemplified by studies of mutations in the Notch gene, an important signalling receptor regulating many aspects of development and stem cell activity. Notch is activated by binding of ligand to the extracellular domain which is comprised of numerous tandem repeats related to Epidermal Growth Factor (EGF). Ligand binding to the EGF region initiates proteolytic cleavages that release the Notch intracellular domain (NICD) to translocate to the nucleus, where NICD interacts with transcription factors and coactivator proteins to switch on target gene expression. The ankyrin domain region within the NICD plays an important role, providing binding sites for different components of the nuclear complex. The ankyrin region also forms a regulatory platform for binding components such as ubiquitin ligases and Adapter protein complexes involved in the endocytic trafficking and sorting which play an important part in modulating Notch signalling levels. Drosophila genetic/developmental analysis and cancer genome sequencing has shown that point mutations in different locations of the Notch protein including different EGF and Ankyrin repeats produce a diversity of phenotypic outcomes. The mechanistic basis, i.e. how the sequence change leads to phenotypic outcome through altered Notch function, is poorly understood for many of these mutations but the diversity of outcomes suggests different mutations probe distinct regulatory interactions.