Functional analysis of Notch ligand-binding mutations

Abstract

The Notch signalling pathway plays a key role in cell differentiation, proliferation and tissue homeostasis in all metazoans. The Notch receptor acts as both the signal receiving component of the pathway, via its extracellular region, and the signalling active molecule, through its intracellular region that is translocated to the nucleus upon activation. The pathway is tightly regulated through different processes that modulate the receptor’s plasma membrane interactions, endocytosis or transcriptional function. Dysfunction of the Notch pathway is associated with several types of cancer, in which it may act as a tumour suppressor or activator. This suggests that diverse regulatory processes may be perturbed in each case. The most studied aspect of the pathway is the receptors’ in-trans binding to ligands of neighbouring cells, which leads to pathway activation. Alternatively, interactions in-cis of the receptor with its ligands on the same cell execute an inhibitory effect on both the receptor and the ligands. In many cancer cases the receptor carries point mutations in the EGF repeats 8-12 of the Notch extracellular region, which mediates receptor-ligand interaction. A combined study of the structures of the human NOTCH1 receptor with its ligands (DLL4 and Jagged1) and functional studies of the homologous pathway components in Drosophila (dNotch, Delta and Serrate), indicated that residues within the ligand-binding region of the receptor may differentially contribute to its interactions in-trans and in-cis with the two ligands. In this thesis, the Drosophila model was used to investigate the outcome of Notch ligand-binding mutations on different aspects of receptor behaviour. It was found that different point mutations within the ligand-binding region of dNotch differentially affect the receptor’s ligand-binding and activation, and also that single dNotch residues could discriminate between its interaction with the two ligands. These Notch ligand-binding mutants did not affect the receptor’s self-association status. Additionally, this study demonstrated that Notch ligand-binding mutant alleles exhibit a different phenotypic behaviour to Notch null alleles in Drosophila, and their phenotype depends on the ligand profile of the tissue. This study suggests a functional classification of Drosophila Notch ligand-binding mutants that could serve in the functional interpretation of equivalent NOTCH1-4 residues mutated in cancer in future studies.

Date of Award	4 May 2021
Original language	English
Awarding Institution	The University of Manchester
Supervisor	Fiona Blackhall (Main Supervisor), Martin Lowe (Co Supervisor) & Dean Jackson (Co Supervisor)