The role of HRS phosphorylation in endosomal trafficking and signalling.



Grimes, DJ
(2019) The role of HRS phosphorylation in endosomal trafficking and signalling. PhD thesis, University of Liverpool.

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Abstract

Hepatocyte growth factor receptor substrate (HRS) is a component of the ESCRT-0 complex. It is involved in the sorting of ubiquitylated receptors into the lumen of multivesicular bodies (MVBs), thus targeting them for lysosomal degradation. HRS is highly phosphorylated in response to stimulation by various growth factors, but despite this, the functional role of its phosphorylation is still contentious. Preliminary data from our laboratory suggested that HRS may also act as a signalling adaptor, involved in the crosstalk between receptor tyrosine kinases (RTKs) and G protein couple receptors (GPCRs). In order to investigate this hypothesis, I first examined the EGF dependent interactors of HRS by mass spectrometry. I then developed tools for the measurement of endosomal cAMP production and PKA signalling. My data suggests that HRS interacts with the E3 ligase deltex3-like (DTX3L) in an EGF dependent manner. Although an association between these proteins has previously been made, its EGF dependence is novel. DTX3L has previously been shown to modulate the recruitment of HRS to endosomes in response to stimulation of the G-protein coupled receptor CXCR4. This new finding offers a theoretical means through which different receptor classes could crosstalk and elicit an integrated signalling response. I further show that GFP-HRS is recruited directly to newly formed vesicles containing EGF and its receptor. Recruitment of HRS to endosomes/vesicles in response to TGFα, but not EGF, is partly dependent on phospho-tyrosine residues Y329 and Y334. Furthermore, HRS has recently been implicated in the recruitment of the recycling complex WASH to endosomes. This, combined with the previous observation, suggests that phosphorylation of these two sites may be involved in receptor recycling. Overall, my thesis generates new tools for the study of HRS function and for the analysis of endosomal signalling. I provide evidence for the role of HRS in RTK and GPCR crosstalk alongside providing insights into the mechanisms governing recruitment of HRS.

Item Type: Thesis (PhD)
Divisions: Fac of Health & Life Sciences > Institute of Translational Medicine
Depositing User: Symplectic Admin
Date Deposited: 07 Jan 2020 16:44
Last Modified: 08 May 2020 21:22
DOI: 10.17638/03061365
Supervisors:
URI: http://livrepository.liverpool.ac.uk/id/eprint/3061365