REGULATION OF TIE-2 BY ANGIOPOIETIN-1 AND ANGIOPOIETIN-2 IN ENDOTHELIAL CELLS By Elena Bogdanovic A thesis submitted in conformity with the requirements for the degree of Doctor of Philosophy, Department of Medical Biophysics, in the University of Toronto © Copyright 2009 by Elena Bogdanovic Elena Bogdanovic Regulation of Tie-2 by Angiopoietin-1 and Angiopoietin-2 in Endothelial Cells (2009) Doctor of Philosophy Department of Medical Biophysics, University of Toronto Abstract The tyrosine kinase receptor Tie-2 is expressed on the surface of endothelial cells and is necessary for angiogenesis and vascular stability. To date, the best characterized ligands for Tie- 2 are Angiopoietin-1 (Ang-1) and Angiopoietin-2 (Ang-2). Ang-1 has been identified as the main activating ligand for Tie-2 while the role of Ang-2 has been controversial since its discovery; some studies reported Ang-2 as a Tie-2 antagonist while others described Ang-2 as a Tie-2 agonist. The purpose of this thesis was to understand: (1) how the receptor Tie-2 is regulated by Ang-1 and Ang-2 in endothelial cells, (2) to compare the effects of Ang-1 and Ang-2, and (3) to determine the arrangement and distribution of Tie-2 in endothelial cells. The research presented in this thesis indicates that Tie-2 is arranged in variably sized clusters on the endothelial cell surface. Clusters of Tie-2 were expressed on all surfaces of cells: on the apical plasma membrane, on the tips of microvilli, and on the basolateral plasma membrane. When endothelial cells were stimulated with Ang-1, Tie-2 was rapidly internalized and degraded. Upon Ang-1 stimulation, Tie-2 localized to clathrin-coated pits on all surfaces of endothelial cells indicating that one pathway mediating Tie-2 internalization is through clathrin-coated pits. After activation of Tie-2, Ang-1 dissociates from the endothelial cell surface and accumulates in the surrounding medium. When experiments were repeated with Ang-2, it was discovered that Ang-2 induced all of the same effects on Tie-2 as Ang-1 but at a much reduced level and rate, indicating that Ang-2 likely functions as a partial agonist for Tie-2 in endothelial cells. ii Dedication This thesis is dedicated to Dr. Howard Green from the University of Waterloo who first piqued my interest in cell surface receptors. His exciting lectures in physiology and passion for research have inspired me to pursue graduate studies. iii Acknowledgements I am grateful to my supervisor Dr. Dan Dumont for his support both personal and professional. The years I spent in his laboratory have been productive and a wonderful opportunity for growth and development. I would like to thank Dr. Neil Coombs for his collaboration on the electron microscopy studies and for his friendship. I also thank Steven Doyle, Lucy Andrighetti, Marianna Capurro, Vladimir Lhotak, Dominik Filipp, John Ebos, Christine Ichim, Giulio Francia, Yan Chen, Robert Temken, Angelika Burger, Ilya Gourevich and Daniel Voskas. I am indebted to Dr. Burton Yang for his guidance and support over the years. iv Table of Contents List of Tables viii List of Figures ix List of Appendices x List of Abbreviations xi Chapter 1: Tie-2 and the Angiopoietins 1.1 Introduction 1 1.2 The Receptor Tyrosine Kinase Tie-2/Tek 2 1.2.1 Expression and Function of Tie-2/Tek in the Cardiovascular System 2 1.2.2 Tie-2 Structure 4 1.3 The Angiopoietins 6 1.3.1 Angiopoietin-1 6 1.3.2 Angiopoietin-2 8 1.3.3 Angiopoietin-3/4 10 1.4 Tie-2 Signaling in Endothelial Cells 10 1.4.1 Downstream Signal Transduction 10 1.4.2 Tie-2 Heterodimerization with Other Cell Surface Proteins 14 1.5 Trafficking of Receptor Tyrosine Kinases 16 1.5.1 Endocytic Pathways Mediating Internalization of Cell Surface Proteins 18 1.6 Thesis Aim 20 Chapter 2: Regulation of Tie-2 by Ang-1 and Ang-2: A Biochemical Study 2.1 Abstract 21 2.2 Introduction 22 2.3 Materials and Methods 22 2.3.1 Cell Culture and Stimulations 22 v 2.3.2 Cell Lysis and Tie-2 Immunoprecipitation 23 2.3.3 Tie-2 Immunoblotting 23 2.3.4 Tie-2 Half-Life Determination and Synthesis 23 2.3.5 Tie-2 Biotinylation 25 2.3.6 Iodination of Ang-1 and Ang-2 25 2.3.7 Internalization of Ang-1 and Ang-2 26 2.3.8 Angiopoietin Immunoprecipitation and Immunoblotting 26 2.3.9 Soluble Tie-2 Determination 27 2.4 Results 27 2.4.1 Ang-1 and Ang-2 Activate Tie-2 in a Concentration Dependent Manner 27 2.4.2 Tie-2 Turnover in HUVECs 32 2.4.3 Tie-2 Internalization 36 2.4.4 Release of Bound Ang-1 and Ang-2 upon Tie-2 Activation 39 2.4.5 Potential Mechanisms Regulating Ligand Release 49 2.5 Discussion 53 Chapter 3: Oligomerized Tie-2 Localizes to Clathrin-Coated Pits in Response to Ang-1: An Electron Microscopy Study 3.1 Abstract 58 3.2 Introduction 59 3.3 Materials and Methods 59 3.3.1 Materials 59 3.3.2 Cell Culture and Stimulation 59 3.3.3 Scanning Electron Microscopy 60 3.3.4 Transmission Electron Microscopy 61 3.3.5 Double Label Transmission Electron Microscopy 62 3.3.6 Cell Surface Protein Cross-Linking 62 3.3.7 Tie-2 Internalization 63 3.3.8 Inhibition of Cellular Endocytosis 63 3.3.9 Knock-down of Clathrin Heavy Chain and Dynamin II Expression 63 3.4 Results 64 3.4.1 Tie-2 is Oligomerized on the Endothelial Cell Surface 64 3.4.2 Distribution of Tie-2 Clusters in Endothelial Cells 77 vi 3.4.3 Oligomerization of Ang-1 87 3.4.4 Tie-2 Internalization 91 3.4.5 Tie-2 Localization to Clathrin-Coated Pits in Response to Ang-1 94 3.4.6 Tie-2 Does Not Reside Within Endothelial Caveolae 106 3.5 Discussion 109 Chapter 4: Conclusion 117 Appendices APPENDIX A 125 APPENDIX B 126 APPENDIX C 127 APPENDIX D 128 APPENDIX E 129 APPENDIX F 130 APPENDIX G 131 APPENDIX H 132 APPENDIX I 133 APPENDIX J 134 References 135 vii List of Tables Chapter 2 Table 1: Cell and media distribution of 125I-Ang1 and 125I-Ang2 Table 2: Trypsin digestion of cell surface proteins Chapter 3 Table 3: Quantification of Tie-2 clusters on the HUVEC plasma membrane viii List of Figures Chapter 1 Figure 1: Domain structure of Tie-2 Figure 2: Domain structure of Ang-1 and Ang-2 Figure 3: Tie-2 Signaling in Endothelial Cells Chapter 2 Figure 4: Tie-2 tyrosine phosphorylation in the presence of Ang-1 and Ang-2 Figure 5: The metabolic half-life of Tie-2 Figure 6: Tie-2 synthesis Figure 7: Tie-2 degradation Figure 8: Examination of Tie-2 internalization by cell surface biotinylation Figure 9: Ang-1 and Ang-2 are released from the endothelial cell surface after activation of Tie-2 Figure 10: Potential mechanisms mediating Ang-1 and Ang-2 release Chapter 3 Figure 11: Specificity of anti-Tie2 antibodies Figure 12: Validation of the Tie-2 labeling strategy using antibody 33.1 Figure 13: Validation of the Tie-2 labeling strategy using goat polyclonal anti-Tie2 antibody Figure 14: Imaging Tie-2 on the endothelial cell surface Figure 15: Background control micrographs for Tie-2 labeling Figure 16: Various arrangements of Tie-2 on the endothelial cell surface Figure 17: Tie-2 is oligomerized on the endothelial cell surface Figure 18: Tie-2 clusters seen on the apical plasma membrane Figure 19: Tie-2 clusters seen on endothelial microvilli Figure 20: Tie-2 clusters seen on the basolateral plasma membrane Figure 21: Intracellular labeling of Rab5 and EEA1 Figure 22: Validation of the Ang-1 labeling strategy Figure 23: Ang-1 is oligomerized on the endothelial cell surface Figure 24: Tie-2 endocytosis Figure 25: Internalization of Tie-2 seen by scanning electron microscopy Figure 26: Inhibition of cellular endocytosis blocks Tie-2 internalization Figure 27: Tie-2 localizes to clathrin- coated pits in response to Ang-1 Figure 28: Knockdown of CHC expression and Tie-2 internalization Figure 29: Knockdown of dynamin II and Tie-2 internalization Figure 30: Tie-2 does not reside in endothelial caveolae ix List of Appendices APPENDIX A: Cross-linking Ang-2 does not enhance Tie-2 activation. APPENDIX B: Conventional scanning electron micrographs of the 293F cell surface. APPENDIX C: Conventional scanning electron micrographs of the 293F cell surface. APPENDIX D: Conventional scanning electron micrographs of the HUVEC cell surface. APPENDIX E: Conventional scanning electron micrographs of the HUVEC cell surface. APPENDIX F: Conventional scanning electron micrographs of the 293F cell surface. APPENDIX G: Conventional scanning electron micrographs of the HUVEC cell surface. APPENDIX H: Control Western blot for experiments using trypsin. APPENDIX I: Tie-2 internalization in confluent HUVEC cell cultures in response to Ang-1. APPENDIX J: Transmission electron micrographs showing Tie-2 at the cell edges in HUVECs. x List of Abbreviations Akt acutely transforming retrovirus BCA bicinchoninic acid BS3 Bis(sulphosuccinimidyl) suberate BSA bovine serum albumin c-Fes feline sarcoma oncogene c-Fyn proto-oncogene related to src CHO Chinese hamster ovary DAB 3,3’-Diaminobenzidine DMEM Dulbecco’s Modified Eagle Medium Dok-R Downstream of kinase – related EEA1 early endosomal antigen 1 ERK extracellular signal regulated kinase Grb growth-factor-receptor bound GTP guanosine triphosphate IL-8 interleukin-8 MAP kinase mitogen activated protein kinase MEK MAP kinase-ERK kinase Nck non-catalytic region of tyrosine kinase adaptor protein NFκB nuclear factor of kappa light polypeptide gene PMA phorbol 12-myristate 13-acetate PAGE polyacrylamide gel electrophoresis PECAM platelet/endothelial cell adhesion molecule PI-3 kinase phosphatidylinositol-3 kinase PKB protein kinase B Rab5 Ras associated binding protein 5 Rac1 Ras-related C3 botulism toxin substrate 1 Raf rat fibrosarcoma Ras rat sarcoma RhoA Ras homolog gene family member A RIPA radioimmunoprecipitation assay buffer SDS sodium dodecyl sulphate Shc Src homology collagen-like SH-PTP2 SH2-domain-containing inositol phosphatase 2 Src sarcoma STAT Signal transducer and activator of transcription Tek Tunica intima endothelial kinase VE-cadherin vascular endothelial cadherin VEGF vascular endothelial growth factor xi CHAPTER 1 Tie-2 and the Angiopoietins 1.1 Introduction The ability of a cell to communicate with another cell is essential for the existence of multi-cellular organisms.