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Characterization of the Tetraspanin Protein Tspan18

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Characterization of the Tetraspanin Protein Tspan18 CHARACTERIZATION OF THE TETRASPANIN PROTEIN TSPAN18 by DARIO COLOMBO A thesis submitted to The University of Birmingham for the degree of DOCTOR OF PHILOSOPHY Department of Cardiovascular Sciences School of Clinical and Experimental Medicine The University of Birmingham September 2010 - University of Birmingham Research Archive e-theses repository This unpublished thesis/dissertation is copyright of the author and/or third parties. The intellectual property rights of the author or third parties in respect of this work are as defined by The Copyright Designs and Patents Act 1988 or as modified by any successor legislation. Any use made of information contained in this thesis/dissertation must be in accordance with that legislation and must be properly acknowledged. Further distribution or reproduction in any format is prohibited without the permission of the copyright holder. ABSTRACT Tetraspanins are a superfamily of four-transmembrane proteins. They associate laterally with one another and with other transmembrane proteins to form membrane microdomains, in which associated proteins are regulated. Tetraspanins regulate diverse processes such as cell signalling and fusion, intracellular trafficking, viral infection and cancer. The primary aim of this thesis was to study a previously uncharacterized tetraspanin, Tspan18. Unlike other tetraspanins, over-expression of Tspan18 in lymphocyte cell lines activated an NFAT/AP-1 reporter, which responds to calcium and MAPK signalling. In DT40 B cell lines, Tspan18 signalling was independent of Lyn, Syk, PLC2 and IP3 receptors, which are essential for B cell receptor signalling. However, Tspan18 signalling did require extracellular calcium and the calcium-activated phosphatase calcineurin, which activates NFAT. Additional studies that included the Jurkat T cell line showed that Tspan18 could not activate the MAPK-responsive AP-1 promoter, but instead mimicked the effect of the calcium ionophore ionomycin. For the secondary thesis aim, over-expression of Tspan18 or other tetraspanins did not affect signalling by the platelet collagen receptor GPVI. Finally, Tspan18 mRNA was found to be relatively highly expressed in endothelial cells and peripheral blood leukocytes. Together these data suggest a role for Tspan18 in calcium signalling on these cell types. - ACKNOWLEDGEMENTS Thanks to Mike Tomlinson, for his immense patience, time, enthusiasm, optimism and day-by day supervision, which have been always great and much appreciated; thanks also for being an inspirational team leader. Thanks to Steve Watson, for having given me the opportunity to be part of the Watson lab for three years. Thanks also for his guidance, supervision and encouragements. Thanks to all the present and past members of the Watson lab, who made these years special. In sparse order: Majd, for being an amazing friend, technology buddy (one day, the systems will converge…), and a fantastic lab technician. Neil, for being a great tetraspaner, Ban, for always having looked properly after me, Jun, for the incredible friendship, the long hours spent talking about music, photos, and antibodies on the New Street-University-New Street train, Chris and Karis (CKK), for being my “adoptive family” and for pushing me well behind what I believed were my limits, Andy and Jocelyin, for their friendship, support, encouragements, feedbacks, for answering all my questions, and for offering their help during difficult times, Simon, for having been always positive, Gemma, for the Medici and bunny advices, Alex and Sonia, for their French touch, Craig, for his friendship and for his jokes, Stevie T, for all his microscope-related advices, Jen, for her several cups of tea and for all the molecular biology help, Stuart, for being a great office mate, Yotis, for being an endless source of encouragements, Galye, for keeping the lab together with fun and enthusiasm, Beata, for looking after all the lab equipments and being so nice. Thanks also to Ilaria, for the huge support, and Piera, for having been a good friend. Thanks to Alison and Andrew at The Open University, for they encouragements, patience and support. Finally, thanks to my family, Magda, Primo and Corinna, to whom this thesis is dedicated to, without the immense day-by-day support and encouragements this adventure would not have been possible. Thanks to the British Heart Foundation, which financially supported this work, and to The University of Birmingam. - To my Family… TABLE OF CONTENTS CHAPTER 1: GENERAL INTRODUCTION...........................................................................1 1.1 TETRASPANINS.................................................................................................................2 1.1.1 Overview .......................................................................................................................2 1.1.2 Structure of tetraspanin proteins....................................................................................2 1.1.3 Tetraspanin web hypothesis and classification of tetraspanin interactions ...................7 1.1.4 Specific tetraspanin functions......................................................................................10 1.2 CALCIUM SIGNALLING ...............................................................................................21 1.3 ITAM RECEPTOR SIGNALLING ..................................................................................27 1.3.1 Overview .....................................................................................................................27 1.3.2 ITAM motif .................................................................................................................27 1.3.3 Proximal B and T cell receptor signalling...................................................................28 1.3.4 Activation of PLC ....................................................................................................30 1.3.5 Calcium mobilization leads to NFAT activation.........................................................31 1.3.6 The MAP kinase pathway leads to AP-1 activation....................................................35 1.3.7 NFAT/AP-1 promoter activation.................................................................................35 1.4 Platelet collagen receptor GPVI and platelet tetraspanins..................................................37 1.4.1 Platelets........................................................................................................................37 1.4.2 Platelet collagen receptor GPVI ..................................................................................38 1.4.3 Regulation of platelet function by tetraspanins ...........................................................38 1.5 THESIS AIM......................................................................................................................41 CHAPTER 2: MATERIALS AND METHODS......................................................................42 2.1 MATERIALS .....................................................................................................................43 2.1.1 Antibodies and reagents ..............................................................................................43 2.1.2 Plasmids and constructs...............................................................................................44 2.1.3 Inhibitors and agonists used in luciferase assays.........................................................44 2.1.4 Cells and tissues ..........................................................................................................46 2.2 CELL CULTURE...............................................................................................................48 2.3 FUNCTIONAL STUDIES .................................................................................................49 2.3.1 Transient cell transfection ...........................................................................................49 2.3.2 Luciferase assay...........................................................................................................50 2.3.3 -galactosidase assay...................................................................................................51 2.3.4 Flow cytometry............................................................................................................51 2.4 PROTEIN STUDIES..........................................................................................................52 2.4.1 SDS polyacrylamide gel electrophoresis, western blotting and immunoblotting .......52 2.4.2 Lipid raft separations...................................................................................................53 2.4.3 Dot-blot analysis..........................................................................................................54 2.5 MOLECULAR BIOLOGY ................................................................................................54 2.5.1 Generation of tetraspanin constructs ...........................................................................54 2.5.2 CD9/Tspan18 chimera and Tspan18 isoform 1 constructs..........................................54 2.5.3 DNA amplification by PCR.........................................................................................55 2.5.4 Restriction enzyme digest............................................................................................56 2.5.5 Ligation of DNA into vectors......................................................................................56
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