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Characterisation of G protein-coupled receptor 56-signalling and its potential role in tumour progression

Bauer, Lea Maria 2014. Characterisation of G protein-coupled receptor 56-signalling and its potential role in tumour progression. PhD Thesis, Cardiff University.
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The adhesion G protein-coupled receptor 56 (GPR56) plays a major role in early brain development. Mutations in Gpr56 cause the developmental brain disease bilateral frontoparietal polymicrogyria (BFPP), which is recapitulated in Gpr56-/- mice. GPR56 interacts with collagen III in the brain pial basement membrane and with tissue transglutaminase (TG2) in melanoma, where it potentially acts as a tumour suppressor by antagonising TG2-related functions. In glioblastoma, however, GPR56 is highly overexpressed and might play an important role for the invasive behaviour of these cells, which could be regulated by TG2 in the tumour stroma. The main aim of this thesis was to analyse GPR56 signalling in response to TG2, thus exploring a potential link to cancer development and progression. Identifying downstream signalling pathways activated by GPR56 in response to TG2 could provide valuable information regarding potential targets for future therapeutic intervention in the context of anti-cancer therapies. In order to investigate GPR56 signalling, a cell-based assay was established that measures GPR56 activation as metalloproteinase-dependent ectodomain shedding of alkaline phosphatase-tagged amphiregulin (AP-AR) in HEK293 cells. The assay was used to demonstrate for the first time activation of GPR56 by TG2. RhoA/Rho-associated protein kinase (ROCK)are activated by GPR56, which likely requires Gα12/13 coupling to GPR56. RhoA/ROCK activity is required for the activation of a disintegrin and metalloproteinase 17 (ADAM17), the main metalloproteinase responsible for GPR56-dependent AP-AR shedding. Shedding of EGF-like ligands such as amphiregulin leads to the activation of epidermal growth factor receptors, inducing cellular responses such as cell proliferation and migration. Further investigations using different GPR56 mutants revealed that the Nterminal domain of GPR56 is required for activation by TG2. The crosslinking activity of TG2 is dispensable for GPR56 activation and the C-terminal β-barrel domains of TG2 are sufficient to stimulate GPR56 signalling. Moreover, two novel potential GPR56 ligands, TG6 and TG7, were shown to stimulate GPR56-dependent AP-AR shedding. Using confocal microscopy, GPR56-dependent internalisation of TG2 via clathrin-coated pits was demonstrated, a mechanism that is well known for agonist-activated GPCRs. Finally, the potential role of GPR56 in glioblastoma was investigated by generating stable GPR56 knockdown glioma cells. Analysis of GPR56 knockdown cells indicated that GPR56 may play a role for glioblastoma migration and invasion. These results present a novel signalling pathway activated by GPR56 in response to TG2 that is involved in cell proliferation, growth and migration, potentially providing an explanation for the supposed tumour promoting functions of GPR56 in glioblastoma.

Item Type: Thesis (PhD)
Status: Unpublished
Schools: Dentistry
Subjects: R Medicine > RK Dentistry
Uncontrolled Keywords: GPR56; Adhesion G protein-coupled receptor; Deorphanisation; Transglutaminase 2; ADAM17; Shedding; Ligand-receptor pair
Funders: Tenovus (Grant number: PhD2009/L15)
Date of First Compliant Deposit: 30 March 2016
Last Modified: 29 May 2016 01:30

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