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Functional fluorescence imaging of receptor tyrosine kinase activity in cells

Reynolds, Andrew Robert; (2002) Functional fluorescence imaging of receptor tyrosine kinase activity in cells. Doctoral thesis (Ph.D), UCL (University College London). Green open access

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Abstract

Receptor tyrosine kinases (RTKs) are key players in the regulation of cell behaviour. These receptors share a common mode of action, i.e. ligand binding is coupled to downstream signalling pathways that co-ordinate diverse cellular activities. The ErbB family of RTKs consists of four receptors: ErbBl, ErbB2, ErbB3 and ErbB4. They are involved in mitogenic signalling and tissue development. Ligand binding initiates receptor dimerisation and receptor autophosphorylation at tyrosine residues, providing docking sites for the recruitment of downstream signalling molecules. Interactions between ErbB receptors, involving every possible combination of receptor hetero- and homodimer, have been proposed to mediate transphosphorylation between receptors. This receptor cross-talk may act to diversify the nature of the downstream signalling response. Receptor phosphorylation is additionally modulated by the action of protein tyrosine phosphatases (PTPs). PTPs have 10-1000 times greater catalytic activity than RTKs which entails that PTP activity must be switched off in order for RTKs to become stably phosphorylated in response to ligand. This thesis describes the application of functional fluorescence imaging methods to study the activation mechanisms of ErbB receptors in intact cells. Previous work from our laboratory has demonstrated the use of fluorescence lifetime imaging microscopy (FLIM) to measure fluorescence resonance energy transfer (FRET) between GFP-tagged ErbBl and Cy3 labelled anti-phosphotyrosine antibodies, thereby allowing ErbBl phosphorylation to be imaged in single cells. In this thesis, global analysis of FLIM data allows ErbBl phosphorylation levels to be quantified in single cells. The approach is used to show that the delivery of a highly localised threshold dose of EGF results in equivalent levels of ErbB receptor phosphorylation to a saturating dose of soluble EGF. In contrast, a locally delivered subthreshold stimulus only activates receptors locally. Evidence is presented which suggests that the extent of receptor phosphorylation induced by ligand stimulus is regulated by hydrogen peroxide mediated PTP inhibition.

Type: Thesis (Doctoral)
Qualification: Ph.D
Title: Functional fluorescence imaging of receptor tyrosine kinase activity in cells
Open access status: An open access version is available from UCL Discovery
Language: English
Additional information: Thesis digitised by ProQuest.
Keywords: Biological sciences; Receptor tyrosine kinases
URI: https://discovery.ucl.ac.uk/id/eprint/10098484
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