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Exploring the mechanisms of Fibroblast Growth Factor Receptor activation and regulation using structural tools

Farrell, Brendan (2019) Exploring the mechanisms of Fibroblast Growth Factor Receptor activation and regulation using structural tools. PhD thesis, University of Leeds.

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Abstract

Fibroblast growth factor receptors (FGFRs) are receptor tyrosine kinases (RTKs) that regulate developmental pathways in response to fibroblast growth factor binding. When dysregulated, FGFRs drive a number of oncogenic and developmental pathologies. While strides have been made in understanding RTK biology, many aspects are still poorly described. In this thesis, structural tools were used to explore the regulation processes of FGFRs and of kinases more generally, focusing on two key aspects: (a) activity regulation in the context of full-length FGFR receptors, and (b) the regulation of kinases by the cellular chaperone Hsp90. Currently, no full-length structure of FGFR nor any RTK is available. To address this lack of information, the expression of full-length FGFR3 and oncogenic variants thereof was established in insect cells. Purification of the receptor was optimised for preliminary negative-stain EM analyses of the receptor in apo and ligand-complexed forms. Though high-resolution structures of FGFR3 could not be determined during this project, the foundations were laid for future efforts. In exploring the chaperoning of kinases by Hsp90, using NMR spectroscopy, it was found that the Hsp90 co-chaperone Cdc37, which recognises client kinases, induces a remodelling of the kinase N-lobe that results in an apparent local unfolding at the kinase N-terminus. Moreover, analyses of wild-type, I538F-substituted and inhibitor-bound FGFR3 kinases suggested that differences in the client strength of kinases relates to a network of residues within their N-lobes. To explore the process of kinase transfer to Hsp90, FGFR3-bound ternary complexes of Hsp90 were reconstituted and characterised by complementary structural and biochemical techniques. These analyses suggested that novel complexes which may describe kinase transfer to Hsp90 were captured. Building on the low-resolution maps obtained of these complexes, efforts were focused on sample optimisation in aim to obtain higher resolution information regarding kinase chaperoning using cryo-electron microscopy.

Item Type: Thesis (PhD)
Related URLs:
Keywords: FGFR, receptor tyrosine kinase, NMR spectroscopy, cryo-EM, Hsp90, Cdc37, structural biology
Academic Units: The University of Leeds > Faculty of Biological Sciences (Leeds)
The University of Leeds > Faculty of Biological Sciences (Leeds) > Institute for Molecular and Cellular Biology (Leeds)
Depositing User: Mr Brendan Farrell
Date Deposited: 20 Feb 2020 13:33
Last Modified: 20 Feb 2020 13:33
URI: http://etheses.whiterose.ac.uk/id/eprint/25855

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