Characterisation of G protein-coupled receptor 56-signalling and its potential role in tumour progression by Lea Maria Bauer, Dipl. Biol. 2014 Extracellular Matrix in Repair & Remodelling Cardiff University School of Dentistry Cardiff, United Kingdom A thesis submitted to the Cardiff University for the degree of Doctor of Philosophy Acknowledgements I would like to thank my supervisors Dr Vera Knäuper and Prof Daniel Aeschlimann for their support and inspiration throughout my PhD. I would like to acknowledge Tenovus for funding this project. I would like to thank my colleagues Katarzyna Gaweł-Bęben, Magdalena Adamczyk, Ana Mafalda dos Reis Jegundo, Andreas Heil and Tim Wanger for their help and motivation. Finally, I would like to thank Andreas, my parents Irmgard and Peter and my sister Lisa for their love and support. iii Abstract 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 N- terminal 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. iv 1 Introduction ........................................................................................... 1 1.1 Why study GPR56? ......................................................................... 1 1.2 G protein-coupled receptors ............................................................ 3 1.2.1 Phylogenetic classification of GPCRs ........................................ 3 1.2.2 Structure of GPCRs .................................................................... 4 1.2.3 Activation of GPCRs ................................................................... 6 1.2.4 G protein-dependent signalling .................................................. 9 1.2.5 Desensitisation of G protein-dependent signalling ................... 12 1.2.6 Internalisation of GPCRs .......................................................... 13 1.2.6.1 Clathrin-dependent endocytosis ........................................ 14 1.2.6.2 Clathrin-independent endocytosis ...................................... 15 1.2.6.3 Endosomal sorting ............................................................. 17 1.2.7 G protein-independent signalling .............................................. 19 1.2.8 EGFR transactivation ............................................................... 21 1.2.8.1 A disintegrin and metalloproteinases (ADAMs) .................. 25 1.2.8.1.1 Structure of ADAMs ...................................................... 26 1.2.8.1.2 Biological functions of ADAMs ...................................... 27 1.2.8.1.3 ADAM17 ....................................................................... 28 1.2.8.1.4 ADAM inhibitors ............................................................ 29 1.3 Adhesion GPCRs .......................................................................... 30 1.3.1 Structure of adhesion GPCRs .................................................. 30 1.3.2 GPS cleavage and the GAIN domain ....................................... 32 1.3.3 Physiological functions of adhesion GPCRs ............................ 33 1.3.3.1 De-orphanised adhesion GPCRs ....................................... 33 1.3.3.2 Adhesion GPCRs in immunology ....................................... 34 1.3.3.3 Adhesion GPCRs in embryonic developmental ................. 34 1.3.3.4 Adhesion GPCRs in the CNS ............................................ 35 1.3.4 Signalling by adhesion GPCRs ................................................ 36 1.3.5 Adhesion GPCRs in tumorigenesis .......................................... 37 1.4 GPR56 ........................................................................................... 39 v 1.4.1 Expression of GPR56 ............................................................... 39 1.4.2 Structure of GPR56 .................................................................. 39 1.4.3 GPR56 interaction partners and potential ligands .................... 41 1.4.4 GPR56 in brain development ................................................... 41 1.4.4.1 GPR56 knockout mice ....................................................... 44 1.4.5 GPR56 and cancer ................................................................... 45 1.4.5.1 GPR56 as a tumour suppressor ........................................ 45 1.4.5.2 GPR56 as a tumour promoter ............................................ 46 1.4.6 GPR56 in immune cells ............................................................ 48 1.4.7 The role of N-GPR56 in GPR56 activation ............................... 48 1.4.8 Natural splice variants .............................................................. 49 1.5 Transglutaminases ........................................................................ 51 1.6 Tissue transglutaminase (TG2) ..................................................... 53 1.6.1 Expression of TG2 in the human body ..................................... 53 1.6.2 Enzymatic activities of TG2 ...................................................... 53 1.6.3 Biological functions of TG2-mediated transamidation .............. 57 1.6.4 Regulation of the transamidation activity .................................. 57 1.6.5 TG2 as a GTPase and G protein .............................................. 59 1.6.6 Non-enzymatic functions of extracellular TG2 and their physiological roles ................................................................................ 60 1.6.7 TG2 and cancer........................................................................ 61 1.6.8 TG2 knockout mice .................................................................. 62 1.7 Glioblastoma multiforme ................................................................ 64 1.8 Aims of the thesis .......................................................................... 67 2 Material and methods ......................................................................... 68 2.1 DNA manipulation .......................................................................... 68 2.1.1 Agarose gel electrophoresis ..................................................... 68 2.1.2 Restriction digest ...................................................................... 68 2.1.3 Ligation ..................................................................................... 69 2.1.4 Transformation ......................................................................... 69 2.1.5 Identifying positive clones ........................................................ 69 vi 2.1.6 Plasmid DNA midiprep ............................................................. 70 2.2 DNA mutagenesis .......................................................................... 70 2.2.1 PCR reactions .......................................................................... 70 2.2.2 Generating an intermediate GPR56 expression vector ............ 72 2.2.3 Restriction digest of PCR reactions .........................................