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Alternative Signaling Pathways Triggered by Different Mechanisms of Serpin Endocytosis

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Alternative Signaling Pathways Triggered by Different Mechanisms of Serpin Endocytosis ALTERNATIVE SIGNALING PATHWAYS TRIGGERED BY DIFFERENT MECHANISMS OF SERPIN ENDOCYTOSIS INAUGURALDISSERTATION zur Erlangung der Würde Doktors der Philosophie Vorgelegt der Philosophisch-Naturwissenschaftlichen Fakultät Der Universität Basel von Xiaobiao Li aus Nanning, China Friedrich Miescher Institute, Basel, 2006 Genehmigt von der Philosophisch-Naturwissenschaftlichen Fakultät auf Antrag der Herren Prof. Dr. Denis Monard, PD Dr. Jan Hofsteenge und Frau Dr. Ariane de Agostini. Basel, den 18 March 2006. Prof. Dr. Hans-Jakob Wirz 2 Acknowledgments: It is a pleasure for me to express my gratitude to those who contributed to this work. In the first place, I would like to thank Prof. Dr. Denis Monard, who gave me the opportunity to work in his laboratory and the freedom to develop my projects. His support was essential for completion of my projects and my scientific development. I would also like to thank the members of my PhD thesis committee, PD Dr. Jan Hofsteenge and Dr. Ariane de Agostini, for their comments and help during the course of the thesis. I am grateful to my colleagues at FMI, especially in the lab, for their helpful discussions, suggestions, technical assistance, and their kindly company over all these years. In particular, I would like to thank Ulrich Hengst and Mirna Kvajo for helping me to get started, Catherine Vaillant for some valuable suggestions, Anne-Catherine Feutz and Sabrina Taieb for their continuous encouragement. I am especially grateful to all my friends for their friendship, which was a great support to me, especially at the time when I experienced difficulty during these years. The most special thank goes to my family, who has always encouraged me in pursuing my professional interest, supported me with their love. My gratitude to them is already beyond the words. 3 Table of contents Acknowledgements………………………………………………………………………………… 3 Abbreviations……………………………………………………………………………………….. 6 Summary……………………………………………………………………………………………. 8 1 Introduction ...................................................................................................................10 1.1 Clathrin-mediated endocytosis and signal transduction........................................11 1.2 Caveolae-mediated endocytosis and signal transduction .....................................12 1.3 Low-density lipoprotein receptor family.................................................................12 1.3.1 Structure of LDLR family...............................................................................13 1.3.2 Functions of LDLR family..............................................................................14 1.3.2.1 LDLR family-dependent endocytosis........................................................15 1.3.2.1.1 Lipoprotein and lipid metabolism .........................................................16 1.3.2.1.2 LDLR family-mediated lipoprotein and lipid metabolism......................18 1.3.2.2 LDLR family-independent endocytosis .....................................................19 1.3.2.3 LDLR family-mediated signal transduction ...............................................19 1.3.2.3.1 VLDLR and ApoER2 function in Reelin signaling pathway..................19 1.3.2.3.2 LRP function in Wnt signaling pathway ...............................................20 1.3.2.3.3 LRP2 (Megalin) function in signaling transduction...............................20 1.3.2.4 LRP1-mediated endocytosis and signal transduction...............................21 1.3.2.4.1 LRP1-mediated endocytosis: ligand families and their binding sites ...22 1.3.2.4.2 Interactions between LRP1 and intracellular adaptor proteins ............23 1.3.2.4.3 Potential role of LRP1 in Alzheimer disease........................................24 1.3.2.4.4 LRP1 function in neurotransmission....................................................25 1.3.2.4.5 Role of LRP1 in cell adhesion and migration.......................................26 1.3.2.4.6 Phosphorylation of LRP1: regulation of endocytosis and signal transduction ..........................................................................................................27 1.4 Heparan sulfate proteoglycan...............................................................................29 1.4.1 Syndecan family ...............................................................................................32 1.4.1.1 HS synthesis: generating specific HS binding sites..................................33 1.4.1.2 Syndecan core proteins............................................................................34 1.4.1.3 HSPGs-mediated internalization...............................................................35 1.4.1.3.1 HSPGs function in lipoprotein metabolism as co-receptor...................35 1.4.1.3.2 HSPGs-mediated ligand internalization...............................................36 1.4.1.4 Regulation of cytoskeletal organization by syndecans .............................36 1.4.1.4.1 Syndecans in signaling transduction and cytoskeleton organization ...37 1.4.1.4.2 Syndecans in tumor metastasis...........................................................39 1.5 Serine protease inhibitors .....................................................................................40 1.5.1 Serine protease ............................................................................................40 1.5.2 Extracellular serine proteases and their receptors in cell migration..............41 1.5.3 Serine protease inhibtor-Serpin family..........................................................42 1.5.3.1 Serpin inhibitory mechanism and activity regulation.................................43 1.5.3.2 Clearance of serpin-protease complexes .................................................44 1.5.3.3 Protease Nexin-1......................................................................................45 4 1.5.3.4 Serpins in cell adhesion and tumor invasion ............................................46 2 Aim and course of this work..........................................................................................49 3 Materials and Methods..................................................................................................51 3.1 Materials ...............................................................................................................51 3.2 Methods................................................................................................................51 4 Results..........................................................................................................................59 4.1 PN-1 uptake in cortical primary neuronal culture ..................................................59 4.2 Both active and complexed PN-1 is internalized in both LRP1-dependent and independent pathways ......................................................................................................61 4.3 Properties of PN-1 uptake in LRP1-/- and wild type MEF cells.............................65 4.4 HSPGs are involved in PN-1 uptake in LRP1-/- MEF cells ...................................67 4.5 Syndecan-1 plays a predominant role in PN-1 uptake in LRP1-/- MEF cells ........70 4.6 PN-1 activates the ERK signaling pathway in LRP1-/- MEF cells.........................73 4.7 PN-1 increases LRP1-/- MEF cell migration by activating ERK and its downstream effector Rac1.....................................................................................................................76 4.8 Enhanced interaction between PN-1 and syndecan-1 promotes LRP1-/- MEF cell migration. ..........................................................................................................................79 4.9 PN-1 and syndecan-1 are coimmunoprecipitated with integrin β3........................81 5 Discussion and Outlook ................................................................................................84 References………………………………………………………………………………..………… 97 Curriulum Vitae……………………………………………………………………………………..120 5 Abbreviations: apoER2: apolipoprotein E Receptor2 APP: Amyloid Precursor Protein CCP: Clathrin-Coated Pit CCV: Clathrin-Coated Vesicle CS: Chondroitin Sulfate DMEM: Dulbecco’s Modified Eagle’s Medium ECM: Extracellular Matrix EGF: Epidermal Growth Factor ERK: Extracellular signal-Regulated Kinase FCS: Fetal Calf Serum GAG: Glycosaminoglycan GPCR: G-Protein Couples Receptor GPI: Glycosylphosphatidylionsitol HS: Heparan Sulfate HSPG: Heparan Sulfate Proteoglycan KPI: Kunitz proteinase inhibitor LDL: Low-Density Lipoprotein LDLR: LDL receptor LpL: Lipoprotein Lipase LRP1: Low-density lipoprotein receptor Related Protein1 MAPK: Mitogen-Activated Protein Kinase MEF: Mouse Embryonic Fibroblast MLCK: Myosin Light Chain Kinase NMDA: N-Methy-D-Aspartate PAI: Plasminogen Activator Inhibitor PDGF: Platelet Derived Growth Factor PN-1: Protease Nexin-1 RAP: Receptor Associating Protein RTK: Receptor Tyrosine Kinase SFM: Serum Free Medium Shh: Sonic hedgehog SMC: Smooth Muscle Cell tPA: Tissue-type Plasminogen Activator 6 uPA: Urokinase-type Plasminogen Activator uPAR: uPA Receptor VLDL: Very Low-Density Lipoprotein VLDLR: VLDL receptor 7 Summary Summary: Protease Nexin-1 (PN-1), a 43 KDa glycoprotein, is known as a serpin (serine protease inhibitor) regulating extracellular proteolytic activity. It strongly inhibits the activity of several serine proteases such as thrombin, tissue plasminogen activator (tPA), urokinase-type plasminogen activator (uPA), trypsin and plasmin. Consequently it contributes to tissue homeostasis
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