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Title: Investigating the mechanism of non-catalytic tyrosine-phosphorylated receptor triggering
Author: Denham, Eleanor Mary
ISNI:       0000 0004 6495 9704
Awarding Body: University of Oxford
Current Institution: University of Oxford
Date of Award: 2016
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Non-catalytic tyrosine-phosphorylated receptors (NTRs) are a large group of leukocyte receptors that bind to surface-associated ligands and include both activating and inhibitory members. They contain, or associate with adaptor molecules which contain, tyrosine residues within conserved cytoplasmic motifs that are phosphorylated and dephosphorylated by extrinsic kinases and phosphatases respectively. The mechanism by which NTR-ligand engagement leads to sustained phosphorylation of receptor tyrosinebased motifs and initiation of downstream signalling (termed "receptor triggering"), is as yet unknown. Our hypothesis is that the NTRs signal using the kinetic-segregation (KS) model. The model proposes that large inhibitory phosphatases, but not inner leaflet-bound activating kinases, are segregated in a size-dependent manner from engaged receptors upon ligand binding. This favours kinase activity and drives sustained receptor phosphorylation. To systematically test whether predictions of the KS model hold for all NTRs, we have developed an artificial generic ligand system in which biophysical and biochemical properties of NTR-ligand interactions can be manipulated. These include NTR-ligand dimensions, ligand densities and valency. Our system exploits the interaction between a Strep-Tag II, which is attached to the ectodomain of the NTR, and StrepTactin protein. We first demonstrate that representative NTRs, both activating and inhibitory, can be triggered through ligation of this Strep-Tag II. In agreement with a key KS model prediction, we next show that only ligation by surface-associated ligand leads to triggering, whilst ligation by soluble ligand does not. Additionally, by altering ligand valency and mobility, we provide evidence that receptor clustering can enhance activation. Also in agreement with the KS model, we show that elongation of the artificial ligand dramatically affects activation of a representative NTR. Finally, we provide evidence to suggest that increased receptor clustering may be able to compensate for ligand elongation-mediated defects in NTR signalling. The flexibility of this system will allow us to analyse other NTRs, test further predictions of the model, and investigate the role of other properties of NTR signalling.
Supervisor: Goyette, Jesse ; van der Merwe, P. Anton Sponsor: Wellcome Trust
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available