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. One major mechanism mediating communication
between cells is through cell surface receptors and their ligands. Receptors are protein
molecules located mainly at the plasma membrane while ligands are molecules secreted
by cells which bind to and activate the receptors. Ligands can be secreted by neighboring cells, by distal cells where the ligands travel via the blood stream, or even by the same cell that contains the receptor (Srivastava and Wood, 1997). To date, a large number of cell surface receptors have been identified and have been grouped into various receptor families based on their overall structure and the manner in which they initiate signal transduction (Srivastava and Wood, 1997). Some examples include the G-protein coupled receptor superfamily, the ion channel receptors, cytokine receptors, and the tyrosine kinase receptor superfamily (Srivastava and Wood, 1997).
The receptor tyrosine kinases (RTKs) comprise a large family of receptors
exclusively expressed in multi-cellular organisms (Robinson et al., 2000; Manning et al.,
2002; Grassot et al., 2003). In humans, the RTK superfamily contains 58 members that
have been further grouped into 20 subfamilies based on the sequence of their kinase
domains (Robinson et al., 2000). RTKs are transmembrane proteins composed of an extracellular ligand binding domain, a single transmembrane domain, and a cytoplasmic
domain containing tyrosine kinase catalytic activity (Hubbard and Till, 2000). Ligand
binding to the extracellular domain activates the tyrosine kinase contained within the
1 cytoplasmic domain. Tyrosine kinases catalyze the transfer of γ-phosphate from ATP to
tyrosine residues on target proteins (Hunter, 1998). Once activated by ligand binding,
RTKs initiate intracellular signaling cascades that lead to a variety of biological responses involved in development and growth such as cell proliferation, differentiation, migration, metabolism, survival, attachment, shape, polarity, and fate determination
(Hubbard and Till, 1998; Schlessinger 2000).
1.2 The Receptor Tyrosine Kinase Tie-2/Tek
1.2.1 Expression and Function of Tie-2/Tek in the Cardiovascular System
The receptor Tie-2, originally named Tek (Dumont et al., 1992; Dumont et al.,
1993), belongs to the Tie (tyrosine kinase with immunoglobulin and epidermal growth
factor homology domains) subfamily of RTKs together with the closely related receptor
Tie-1 (Partanen et al., 1992; Schnurch and Risau, 1993). Tie-2 is expressed on the
surface of endothelial cells, which line the entire cardiovascular system (Dumont et al.,
1992; Schnurch and Risau, 1993). Endothelial cells assemble into a monolayer and form
the innermost lining of the heart, arteries, veins, capillaries, and lymphatic vessels (Aird,
2007).
The expression of Tie-2 can be detected early in embryonic development at day
8.0-8.5 in mouse embryos (Dumont et al., 1992). At this time, Tie-2 is expressed in the
endocardium, in the vessels of the head, and the dorsal aorta (Dumont et al., 1992). By
day 12.5, Tie-2 expression can be detected within the inner lining of all major blood
vessels (Dumont et al., 1992). In the embryo, Tie-2 activity is crucial for the
development of the cardiovascular system (Dumont et al., 1994; Sato et al., 1995).
2 The first blood vessels begin to form early in embryonic development by a process called vasculogenesis (day 7.0-7.5 in mouse embryos) (reviewed by Patan, 2000).
Endothelial cells differentiate in situ from endothelial cell precursors, proliferate, and coalesce to form the primary capillary plexus containing vessels that are primitive and of equal size (Patan, 2000; Yancopoulos et al., 2000). The primary capillary plexus is rapidly expanded and remodeled by a process called angiogenesis where vessels begin to sprout and form a hierarchy of large and small vessels (Yancopoulos et al., 2000).
Angiogenesis is the principal mechanism of blood vessel formation in the embryo and is important after birth to support the growth and development of the organism (Carmeliet,
2003).
In the absence of Tie-2, mice die during development at day 9.5 due to
hemorrhaging, reduced heart and head size, the endocardium poorly associated with the
underlying myocardium, fewer endothelial cells, lack of capillary sprouts, and an absence
of large and small vessels (Dumont et al., 1994; Sato et al., 1995). The genetic studies indicated that Tie-2 activity is important for angiogenesis and for the integrity and survival of the endothelium (Dumont et al., 1994; Sato et al., 1995).
In the adult, Tie-2 expression is maintained throughout the cardiovascular system
although the levels of Tie-2 are lower compared to the levels observed during embryonic
development (Dumont et al., 1992; Schnurch and Risau, 1993; Wong et al., 1997). The
endothelium in the adult is essentially quiescent, with angiogenesis occurring only in the
female reproductive system and in pathological situations such as wound healing and in
disease states such as cancer (Carmeliet, 2003; Risau, 1995). Tie-2 expression in the
quiescent adult vasculature appears to continue to maintain the integrity and survival of
3 the endothelium (Dumont et al., 1994). Tie-2 has been shown to be upregulated in
healing skin wounds (Wong et al., 1997) and in the ovary and placenta (Wong et al.,
1997; Seval et al., 2008) indicating that Tie-2, although necessary to maintain the
quiescent endothelium, is also involved at sites of active angiogenesis in the adult.
In addition to being expressed in endothelial cells, Tie-2 is also expressed
in hematopoietic stem cells (Suda et al., 1997), in certain monocytes (De Palma et al.,
2005), neutrophils (Sturn et al., 2005), eosinophils (Feistritzer et al., 2004), thyroid
follicular cells (Ramsden et al., 2001), and in dorsal root ganglion cells (Kosacka et al.,
2005).
1.2.2 Tie-2 Structure
Tie-2 is a 140 kDa glycoprotein composed of an extracellular ligand binding
domain, a transmembrane domain which anchors the receptor to the plasma membrane, and an intracellular tyrosine kinase domain (Dumont et al., 1993; Schnurch and Risau,
1993). The extracellular domain of Tie-2 contains 3 immunoglobulin (Ig) domains, 3
epidermal growth factor repeats (EGF), and 3 fibronectin type three repeats (FNIII) (Fig.
1) (Dumont et al., 1993; Schnurch and Risau, 1993; Barton et al., 2006; Macdonald et al.,
2006). The Ig and EGF domains tightly pack into a globular structure resembling an
arrowhead with Ig2 at the tip and the FNIII domains forming the stalk (Fig. 1) (Barton et
al., 2006; Macdonald et al., 2006). The intracellular portion of Tie-2 contains the
juxtamembrane region, the catalytic domain divided by a kinase insert, and a carboxyl tail containing tyrosine residues important for the initiation of downstream signaling
(Dumont et al., 1993; Schnurch and Risau, 1993).
4 Tie-2 and the closely related receptor Tie-1 share 76% sequence identity between their intracellular domains and 33% and 37% between the extracellular and transmembrane regions respectively (Schnurch and Risau, 1993).
Ig2
Ig1 EGF
Ig3
FNIII
Plasma Membrane
TK1
KI
TK2
CT
Figure 1: Domain structure of Tie-2. This diagram of Tie-2 illustrates the various domains within the receptor compiled from schematics of Tie-2 depicted in published literature (Dumont et al. 1993; Barton et al. 2006). The extracellular domain of Tie-2 contains three immunoglobulin domains (Ig1, Ig2, and Ig3) where Ig2 and Ig3 are separated by three epidermal growth factor repeats (EGF) followed by three fibronectin type III repeats (FNIII). The intracellular domain of Tie-2 contains the tyrosine kinase domain (TK1 and TK2) that is divided by the kinase insert (KI). Following the tyrosine kinase domain is the C- terminal tail (CT).
1.3 The Angiopoietins
The ligands for Tie-2 are the family of angiopoietins: angiopoietin-1
5 (Ang-1), Ang-2, and Ang-3/4 (Davis et al., 1996; Maisonpierre et al., 1997; Valenzuela et al., 1999). All angiopoietins are similarly structured but are secreted by different cells and appear to have different functional roles within the vasculature (Davis et al., 1996;
Maisonpierre et al., 1997; Valenzuela et al., 1999; Davis et al., 2003).
1.3.1 Angiopoietin-1
Ang-1 was the first ligand for Tie-2 to be identified and is so far the main activating ligand for the receptor (Davis et al., 1996; Suri et al., 1998). Most of the functions of Tie-2 in the vasculature have been attributed to the binding of Ang-1 (Suri et al., 1996). Mice deficient in Ang-1 die during embryonic development by day 12.5 and display vascular abnormalities similar to mice lacking Tie-2 (Suri et al., 1996). In the absence of Ang-1, mouse embryos display defects in heart development with poor association of the endocardium with the underlying myocardium, a less complex vascular network, and poor association of endothelial cells with surrounding cells and the extracellular matrix (Suri et al., 1996). During embryonic development Ang-1 is expressed in the myocardium and in the mesenchyme surrounding developing vessels
(Suri et al., 1996). The expression pattern of Ang-1 within the embryo indicated that
Ang-1 is synthesized and secreted by surrounding cells and acts in a paracrine manner on
Tie-2 on endothelial cells (Davis et al., 1996; Suri et al., 1996).
In the adult, Ang-1 is synthesized and secreted by smooth muscle cells (SMC) and pericytes that surround and support endothelial cells (Davis et al., 1996; Sundberg et al.,
2002; Jain, 2003). Although Ang-1 production by SMC/pericytes has gained the most attention, Ang-1 is also secreted by osteoblasts (Horner et al., 2001), platelets (Li et al.,
6 2001), podocytes (Satchell et al., 2002), fibroblasts (Stacker et al., 2000), and hepatic stellate cells (Taura et al., 2008).
Ang-1 is a 70 kDa glycoprotein that binds to Tie-2 with high affinity (KD
~ 3nM) (Davis et al., 1996; Maisonpierre et al., 1997). Structurally, Ang-1 is composed of various domains that are critical for the activation of Tie-2 (Fig. 2) (Davis et al., 1996;
Davis et al., 2003). At the C terminus, Ang-1 contains a fibrinogen-like domain that is responsible for binding to the Ig2 domain at the extracellular tip of Tie-2 (Barton et al.,
2006). Towards the N-terminus, next to the fibrinogen-like domain, is the coiled-coil domain which mediates the multimerization of Ang-1 monomers (Davis et al., 2003). At the N-terminus, Ang-1 contains a superclustering domain that further oligomerizes the
Ang-1 molecules and a secretion signal peptide (Fig. 2) (Davis et al., 2003; Kim et al.,
2005). In solution, Ang-1 forms homotrimers, tetramers, pentamers, and higher order clusters composed of basic trimeric, tetrameric, and pentameric structures (Procopio et al., 1999; Davis et al., 2003; Kim et al., 2005). Multimerization of Ang-1 is critical for the activation of Tie-2 (Procopio et al., 1999; Davis et al., 2003; Kim et al., 2005). The minimal cluster size of Ang-1 required to activate Tie-2 was reported to be a tetramer
(Kim et al., 2005).
The binding of Ang-1 to Tie-2 on the surface of endothelial cells induces tyrosine phosphorylation of the receptor and activates several intracellular signaling pathways leading to endothelial cell migration (Witzenbichler et al., 1998; Jones et al.,
1999), sprouting (Koblizek et al., 1998; Kim et al., 2000), tube formation (Hayes et al.,
1999), survival (Jones et al., 1999; Kwak et al., 1999; Papapetropolous et al., 1999) and a
7 strengthening/maintenance of endothelial cell junctions thereby preventing endothelial
permeability (Gamble et al., 2000).
Ang-1
1 19 79 263 284 498
Ang-2
1 18 71 257 282 496
S SCD CC FLD
N C
Figure 2: Domain structure of human Angiopoietin-1 (Ang-1) and Angiopoietin-2 (Ang-2). Schematic diagram illustrating the various domains contained within Ang-1 and Ang-2 reproduced from a publication by Kim et al. (2005). At the N-terminus both Ang-1 and Ang-2 contain a signal sequence (S) that directs secretion from cells, a superclustering domain (SCD), a coiled-coil domain (CC), and the fibrinogen like domain (FLD). The numbers refer to the linear sequences of amino acid residues within the proteins.
1.3.2 Angiopoietin-2
Ang-2 was the second angiopoietin family member to be identified (Maisonpierre
et al., 1997). Ang-2 is synthesized by endothelial cells and stored within Weibel-Palade
bodies (Fiedler et al., 2004) and is secreted from endothelial cells in response to certain
stimuli such as phorbol esters (PMA), histamine, or thrombin (Fiedler et al., 2004). Like
Ang-1, Ang-2 is a 75 kDa glycoprotein that binds to Tie-2 with similar affinity (KD ~
3nM) and shares 60% sequence homology with Ang-1 (Maisonpierre et al., 1997)). Ang-
2 contains an N-terminal signal peptide, followed by a superclustering domain, a coiled-
8 coil domain, and a C-terminal fibrinogen-like domain that binds to the Ig2 domain of Tie-
2 (Fig. 2) (Maisonpierre et al., 1997; Davis et al., 2003; Kim et al., 2005; Barton et al.,
2006). In solution, Ang-2 forms dimers, trimers, tetramers, and pentamers with few
higher order clusters (Procopio et al., 1999; Davis et al., 2003; Kim et al., 2005). Ang-2 was initially described as an antagonistic ligand where the binding of Ang-2 to Tie-2 did not induce receptor activation and competed with Ang-1 for binding to Tie-2
(Maisonpierre et al., 1997). Transgenic overexpression of Ang-2 in mouse embryos resulted in death as a result of severe cardiovascular defects similar to those seen in Tie-2 and Ang-1 knockout mice (Maisonpierre et al., 1997). It was concluded that Ang-2 was a naturally occurring inhibitor of Tie-2 (Maisonpierre et al., 1997). After the initial characterization of Ang-2, papers began appearing in the literature indicating that Ang-2 was able to activate Tie-2 and promoted many of the same biological outcomes as Ang-1
(Kim et al., 2000; Teichert-Kuliszewska et al., 2001; Mochizuki et al., 2002). The published literature on Ang-2 contained some papers describing Ang-2 as a Tie-2 antagonist while others described Ang-2 as an agonist. This led to considerable confusion. It was then concluded that Ang-2 behaved as an agonist/antagonist depending on the cell and tissue type examined although a specific cellular context was never defined. It seems likely, as described in Chapter 2, that Ang-2 behaves as a partial agonist for Tie-2. As a partial agonist, Ang-2 is capable of eliciting the same biological outcomes as Ang-1 but at a much reduced level, and as a weaker agonist, could diminish the activity of Tie-2 in the presence of Ang-1 by blocking the binding of Ang-1.
The precise function of Ang-2 in the vasculature is not completely understood but
Ang-2 appears to be involved in post-natal vascular remodeling and lymphatic
9 development (Gale et al., 2002; Shimoda, 2008) and may act as a pro-inflammatory agent
(reviewed by Fiedler and Augustin, 2006).
1.3.3 Angiopoietin 3/4
Compared to Ang-1 and Ang-2, relatively little is known about Ang-3/4.
Ang-3 and Ang-4 are mouse and human counterparts respectively of the same gene loci
that have diverged (Valenzuela et al., 1999). Like Ang-1 and Ang-2, Ang-3/4 contain at
the N-terminus a signal peptide, followed by a superclustering domain, a coiled coil
domain, and a C-terminal fibrinogen like domain (Valenzuela et al., 1999). In the mouse,
Ang-3 is highly expressed in the heart, kidney, and testis, whereas in the human, Ang-4 is
strongly expressed in the lung (Valenzuela et al., 1999). In solution, Ang-3 and Ang-4
form dimers (Lee et al., 2004) and most likely form higher order clusters. Ang-3 and
Ang-4 activate Tie-2 in a species specific manner where Ang-3 activates mouse Tie-2
and Ang-4 activates human Tie-2 (Lee et al., 2004). In vitro, Ang-3/4 induced survival
and migration of endothelial cells and stimulated angiogenesis in a corneal micropocket
angiogenesis assay (Lee et al., 2004).
1.4 Tie-2 Signaling in Endothelial Cells
1.4.1 Downstream Signal Transduction
All angiopoietin family members induce Tie-2 activation to various
extents and consequently activate similar signaling pathways. To date, many downstream signaling molecules have been identified (Fig. 3) although some are controversial and even proteomic screens have been published identifying proteins that
10 become tyrosine phosphorylated in response to Ang-1 in endothelial cells (Kim et al.,
2007).
Ligand binding to the extracellular domain of Tie-2 activates the intrinsic
tyrosine kinase which leads to the phosphorylation of specific tyrosine residues contained within the kinase domain and the C terminal tail (Jones et al., 1999). Phosphorylated tyrosine residues at the C terminus serve as high affinity binding sites for downstream
signaling molecules (Jones et al., 1999). Upon activation by Ang-1, the intracellular
domain of Tie-2 was shown to engage several proteins. The adapter protein Dok-R (also called Dok-2) binds to phosphorylated Y1106 (mouse Tie-2) (Jones and Dumont, 1998;
Jones et al., 2003) and itself becomes phosphorylated on specific tyrosine residues
(Master et al., 2001). Dok-R then associates with the proteins Nck and the p21 activating kinase (PAK) (Jones and Dumont, 1998; Master et al., 2001). The formation of the Dok-
R-Nck-PAK complex forms part of the signaling pathway leading to endothelial cell migration (Master et al., 2001).
The A20 binding inhibitor of NFκB -2 (ABIN-2) also binds to the intracellular
domain of Tie-2 in a phosphotyrosine dependent manner (Hughes et al., 2003). In
response to Ang-1, ABIN-2 inhibits endothelial cell apoptosis induced by serum
deprivation and promotes cell survival (Tadros et al., 2003). ABIN-2 also inhibits NF-κB
activity which in turn suppresses the expression of genes involved in inflammation
(Hughes et al., 2003).
Phosphorylation of Y1101 (human Tie-2) in the C-terminal tail was shown
to induce the binding of the p85 subunit of PI 3-kinase to subsequently induce PI 3-
kinase activity (Kontos et al., 1998). Activation of PI 3-kinase and its downstream
11 signaling molecules protects endothelial cells from apoptosis, promotes cell survival, and
promotes Ang-1 induced endothelial cell migration and sprouting (Kontos et al., 1998;
Jones et al., 1999; Kim et al., 2000; Kim et al., 2000). In response to Ang-1, PI 3-kinase
activates the serine/threonine kinase Akt/PKB (Kontos et al., 1998; Kim et al., 2000), p70
S6 kinase (Moon et al., 2005), focal adhesion kinase (FAK) (Kim at al., 2000), and the
GTPase Rac1 (Cascone et al., 2003; Mammoto et al., 2007). Akt/PKB upregulates the
apoptosis inhibitor survivin (Papapetropolous et al., 2000) and inhibits the forkhead
transcription factor FKHR (FOXO1) (Daly et al., 2004). FOXO1 increases the
expression of genes involved in endothelial cell apoptosis and vascular destabilization.
In one report, the activation of the GTPase Rac1 by Ang-1 led to the inhibition of
RhoA (Mammoto et al., 2007). Inhibition of RhoA prevented cytoskeletal
rearrangements that led to vascular permeability (Mammoto et al., 2007). In another report, Ang-1 activated RhoA (Cascone et al., 2003; Gavard et al., 2008) and that led to
the activation of mDia (Gavard et al., 2008). mDia then associated with src and prevented
the activation of src by VEGF (Gavard et al., 2008). Sequestering src away from the
VEGF signaling pathway maintained VE-cadherin at cell-cell junctions and prevented
paracellular permeability (Gavard et al., 2008). Another report has shown that Ang-1
activates both Rac1 and RhoA and induces cytoskeletal rearrangements that lead to
endothelial cell migration downstream of PI 3-kinase (Cascone et al., 2003).
In response to Ang-1, the adapter protein Shc (p52) also binds to tyrosine 1101 of
Tie-2. This association appears to be important for the stimulation of endothelial cell
migration and survival (Audero et al., 2004).
12 Ang-1 was also shown to activate the Ras-Raf-MEK-ERK1/2 pathway ( Kim et al.,
2002) that leads to the activation of sphingosine kinase-1 (SK-1) resulting in reduced
vascular permeability by decreasing PECAM-1 phosphorylation and re-localizing VE-
cadherin to endothelial cell-cell contacts (Li et al., 2008).
Ang-1 and Ang-2 were shown to activate src, c-Fes, and c-Fyn to
promote endothelial cell migration, tube formation, and increases in capillary length
(Mochizuki et al., 2002; Kanda et al., 2007). Ang-1 was also shown to inhibit the activation of caspase-9,-7, and-3 (Harfouche et al., 2003)
Tie-2 was also shown to activate the p38 MAP kinase (Harfouche et al., 2006) and
the STAT proteins (1, 3, and 5) (Korpelainen et al., 1999). In vitro, activated forms of
Tie-2 were shown to associate with Grb2, SH-PTP2, Grb7 and Grb14 (Huang et al.,
1995; Jones et al., 1999).
13 Ang
Tie-2 Tie-2
Akt/PKB
p70 S6 kinase p85 Shc Y1100 Y1100 FAK
ABIN-2 Y1106 RAC1 RhoA Ras Dok-R
Nck PAK Raf NFκB
MEK
ERK1/2
SK-1
Figure 3: Tie-2 signaling pathways in endothelial cells modified slightly from Brindle et al. (2006).
1.4.2 Tie-2 Heterodimerization with Other Cell Surface Proteins
Tie-2 heterodimerizes with several cell surface proteins that appear to either attenuate or enhance Tie-2 activity. Tie-2 has been shown to heterodimerize with: (1) the closely related receptor Tie-1 (Marron et al., 2000; Saharinen et al., 2005), (2) the vascular endothelial protein tyrosine phosphatase (VE-PTP) (Fachinger et al., 1999), and
(3) the integrin α5β1 (Cascone et al., 2005).
Studies examining the heterodimerization of Tie-2 with Tie-1 have suggested that Tie-1 downregulates Tie-2 activity and downstream signaling.
Stimulation of endothelial cells with Ang-1 induces tyrosine phosphorylation of Tie-1
14 that appears to be enhanced in the presence of Tie-2, indicating that Tie-2 may
phosphorylate Tie-1 (Saharinen et al., 2005). Studies where the expression of Tie-1 was
knocked down using short interfering RNA (siRNA) showed that under these conditions
the Ang-1 induced tyrosine phosphorylation of Tie-2 and the activation of downstream
signal transduction were markedly enhanced suggesting that the presence of Tie-1
downregulates Tie-2 signaling (Marron et al., 2007; Yuan et al., 2007). Tie-1 may
compete for Ang-1 since Tie-1 becomes tyrosine phosphorylated in response to Ang-1 in the absence of Tie-2 (Saharinen et al., 2005; Kim et al., 2006; Marron et al., 2007).
Tie-2 has been shown to associate with the vascular endothelial protein
tyrosine phosphatase (VE-PTP) (Fachinger et al., 1999). VE-PTP is the mouse
homologue of the human receptor type protein tyrosine phosphatase β (HPTPβ)
(Fachinger et al., 1999). Association of Tie-2 with VE-PTP resulted in the
dephosphorylation of Tie-2 when Tie-2 and VE-PTP were co-expressed in COS-1 cells
(Fachinger et al., 1999).
Tie-2 has also been shown to be constitutively associated with the integrin
α5β1 in endothelial cells (Cascone et al., 2005). Association with α5β1 integrin
sensitizes Tie-2 to Ang-1 where lower concentrations of Ang-1 were able to activate Tie-
2 and this association between Tie-2 and α5β1 integrin was important for angiogenesis in
vivo (Cascone et al., 2005). Ang-1 was also shown to bind to integrins in the absence of
Tie-2 in fibroblasts and cardiac myocytes to enhance cell adhesion, spreading, and
migration (Carlson et al., 2001; Dallabrida et al., 2005).
15 1.5 Trafficking of Receptor Tyrosine Kinases
It is now well recognized that cell surface receptors are dynamic within cells.
Cell surface receptors are continually in a cycle of synthesis, transport to the plasma membrane, followed by internalization and eventual degradation (Limbird 2004). Once synthesized, receptors are transported to specific locations within the cell depending on the signaling properties and function of a particular receptor. In polarized epithelial cells, some receptors are exclusively targeted to either the apical or basolateral plasma membrane (Beer Stolz and Jacobson 1992; Ojakian and Schwimmer 1988; Prydz et al.
1990). For example, in the collecting tubules of the kidney, the epidermal growth factor receptor (EGFR) is exclusively expressed on the basolateral plasma membrane (Orellana et al. 1995). Mislocalization of the EGFR to the apical surface has been defined as a contributing factor in polycystic kidney disease (Orellana et al. 1995).
In response to ligand binding, many members of the tyrosine kinase family of
receptors markedly increase their rates of internalization resulting in the rapid removal of
receptors from the cell surface (Sorkin and Waters 1993; Wiley 2003). For many
tyrosine kinase receptors, internalization in response to ligand activation leads to
degradation of the receptor (Sorkin and Waters 1993). This phenomenon, known as
receptor downregulation, has been observed for numerous RTKs such as the EGFR
(Beguinot et al., 1984; Stoschek and Carpenter, 1984), platelet derived growth factor
receptor (PDGFR) (Nilsson et al., 1983; Keating and Williams, 1987), insulin receptor
(Kasuga et al., 1981), insulin like growth factor 1 receptor (IGF1R) (Michelangelo et al.,
2004), fibroblast growth factor receptor (FGFR) (Cha et al., 2008), Met (Parachoniak and
Park, 2008), and the vascular endothelial growth factor receptor 2 (VEGFR2) (Ebos et
16 al., 2008). In the case of the EGFR; one of the earliest and best characterized RTKs
(Carpenter and Cohen, 1976), binding of the epidermal growth factor (EGF) to the EGFR
results in receptor autophosphorylation and the internalization of the EGF:EGFR
complex into the cell (Beguinot et al., 1984; Stoschek and Carpenter, 1984). In the
presence of EGF, the rate of EGFR internalization increases ~ 5-10 fold compared to the
unstimulated state (Wiley, 2003). Upon binding EGF, the EGF:EGFR complex
concentrates within clathrin-coated pits at the cell surface which then pinch off into the
cell and deliver the EGF:EGFR complex to early endosomes (Beguinot et al., 1984).
From early endosomes the EGF:EGFR complex is sorted to late endosomes
(multivesicular bodies) and then to the lysosome where the receptor and ligand are degraded (Beguinot et al., 1984).
Two main hypotheses exist to explain why ligand activated receptors are rapidly
internalized and targeted for degradation. The first hypothesis suggests that
internalization and degradation of activated receptors together with their ligands is likely
a necessary mechanism to regulate the duration of signaling since degradation of
activated receptors would terminate signal transduction (Sorkin and Waters 1993; Wells
et al. 1990; Wiley 2003). The removal of receptors from the plasma membrane results in
an overall loss of receptors from the cell surface diminishing the ability of the cell to
respond to further stimulation (Sorkin and Waters, 1993). Receptor downregulation
would serve a regulatory role controlling the duration of signaling and consequently the
biological response. Wells et al. (1990) have shown that an EGFR mutant capable of ligand activation but impaired for internalization transformed cells in culture. Regulating receptor signaling is critical to maintain normal cellular homeostasis. Unregulated
17 receptor signaling contributes to the development of diseases such as cancer (Yarden and
Sliwkowski, 2001). For example, a mutant EGFR with a deletion in the extracellular
domain renders the receptor constitutively active (Moscatello et al., 1995). This mutant
receptor is frequently expressed in gliomas and in lung, ovary, and breast carcinomas
(Moscatello et al., 1995; Yarden and Sliwkowski, 2001).
In addition to serving a prominent regulatory role, some reports have suggested
that rapid receptor internalization after ligand binding actually facilitates downstream
signaling and that some signals are initiated from endosomal compartments (Bevan et al.,
1996; Wiley, 2003). The rate of internalization of activated receptors is considerably faster than the rate of receptor degradation resulting in the accumulation of large pools of activated receptors within the cell (Wiley, 2003). Receptors such as the EGFR and
PDGFR remain tyrosine phosphorylated within intracellular compartments and some receptor substrates are highly enriched within endosomes (Burke et al., 2001; Wiley,
2003). Blocking the internalization of the insulin receptor impaired the activation of the
MAP kinase pathway while Akt/PKB phosphorylation and activation were unaffected
(Caresa et al., 1998).
1.5.1 Endocytic Pathways Mediating Internalization of Cell Surface Proteins
The best characterized endocytic pathway mediating the internalization of cell
surface proteins is through clathrin-coated pits and vesicles first identified in 1964
(reviewed by Conner and Schmid, 2003; Young, 2007). In clathrin-mediated
endocytosis, proteins destined for internalization are recruited to specialized regions of
the plasma membrane through their intracellular domains by binding to adaptor proteins
18 such as AP-2 and β-arrestin (Kirchhausen, 1999). In addition to recruiting protein cargo,
AP-2 and β-arrestin also bind to membrane lipids and the protein clathrin (Young, 2007).
These specialized regions of the plasma membrane invaginate and form pits with the protein clathrin surrounding the pit on the intracellular side (Mousavi et al., 2004). These clathrin-coated pits then pinch off from the cell surface and enter the cell as clathrin- coated vesicles (Mousavi et al., 2004). Scission of clathrin-coated pits from the plasma membrane is mediated by the GTPase dynamin (Mousavi et al., 2004). Upon entering the cell, clathrin and the adaptor proteins are removed from the vesicle and the vesicle, containing membrane cargo, then fuses with early endosomes where the cargo is sorted to various subcellular compartments (Stahl and Schwartz, 1986; Mousavi et al., 2004).
A clathrin-independent mechanism of receptor internalization that has received
attention has been endocytosis through caveolae (Conner and Schmid, 2003). Caveolae
are flask-shaped invaginations of the plasma membrane that are found in many cell types
particularly endothelial cells (Cohen et al., 2004). The membrane within caveolae is rich
in cholesterol, sphingolipids and the protein caveolin thereby creating a more rigid
structure compared to the surrounding membrane (Cohen et al., 2004). Caveolae are
thought to be important in the transcellular transport of material across cells particularly
endothelial cells where proteins from the blood stream are transported across endothelial
cells to the tissues (Conner and Schmid 2003). Numerous cell surface receptors and their
downstream effector proteins have been shown to localize to caveolae suggesting the
possibility that caveolae may also serve as platforms to organize receptor signaling
(Cohen et al., 2004).
19 Internalization via clathrin- and caveolar- independent mechanisms also exist indicated by the observation that the internalization of some viruses and toxins still occurs in cells where both clathrin- and caveolar- mediated internalization have been inhibited (Conner and Schmid 2003). The molecular mechanisms involved in these pathways are now beginning to emerge (reviewed by Sandvig et al., 2008).
1.6 Thesis Aim
The purpose of this study was to understand how the receptor tyrosine kinase Tie-
2 is regulated by the angiopoietins, the ligands for Tie-2, in endothelial cells. The specific research questions addressed were: (1) to determine the arrangement and distribution of Tie-2 within endothelial cells, (2) to determine and compare the effect of
Ang-1 and Ang-2 on Tie-2 phosphorylation, synthesis, internalization, and degradation,
(3) to examine the internalization of Ang-1 and Ang-2 in endothelial cells and (4) to identify the endocytic pathway mediating the internalization of Tie-2 in endothelial cells.
Research aims (2) and (3) are addressed in Chapter 2. Research aims (1) and (4) are addressed in Chapter 3. An overall discussion is presented in Chapter 4.
20 CHAPTER 2
Regulation of Tie-2 by Ang-1 and Ang-2: A Biochemical Study
2.1 Abstract
The receptor tyrosine kinase Tie-2 is expressed in endothelial cells and is critical for angiogenesis and vascular maintenance. The ligands for Tie-2 are the angiopoietins of which Ang-1 and Ang-2 have been the most studied. Ang-1 has been characterized as the primary activating ligand for Tie-2 while the role of Ang-2 remains controversial with
Ang-2 activating Tie-2 in some studies and inhibiting Tie-2 in others. Our studies were aimed at understanding the regulation of Tie-2 in endothelial cells by Ang-1 and Ang-2 and revealed that both ligands activated Tie-2 in a concentration dependent manner.
Ang-2 was considerably weaker at activating Tie-2 compared to Ang-1 suggesting that
Ang-2 may be a partial agonist. Activation of Tie-2 by these ligands resulted in differential turnover of the receptor where binding of Ang-1, and to a lesser extent Ang-2, induced rapid internalization and degradation of Tie-2. Furthermore, binding studies demonstrate that both ligands are differentially released from the endothelial cell surface after receptor activation and accumulate in the surrounding medium. Altogether, these data begin our understanding of the regulation of Tie-2 and the activity of the
Angiopoietins after engaging the endothelial cell surface. The data presented in this chapter was published in the Journal of Cell Science (2006, vol 119, pages 3551-3560).
21 2.2 Introduction
In this chapter the regulation of Tie-2 by Ang-1 and Ang-2 in endothelial cells
was evaluated and compared using biochemical techniques. The work presented in this
chapter examined Ang-1 and Ang-2 induced tyrosine phosphorylation of Tie-2, the metabolic half-life of the receptor, the internalization of Tie-2 from the cell surface, and the internalization of Ang-1 and Ang-2.
2.3 Materials and Methods
2.3.1 Cell Culture and Stimulation
Human umbilical vein endothelial cells (HUVECs) (GlycoTech Corp., Rockville,
Maryland) were grown on gelatinized (2% gelatin, Sigma) tissue culture plates in F12K medium (ATCC) supplemented with 10% fetal bovine serum, penicillin-streptomycin,
0.1 mg/ml heparin sodium, 10 ng/ml EGF, 10 ng/ml VEGF, 5 ng/ml bFGF, and 2 mM L- glutamine, and were used up to passage 10. EA.hy926 endothelial cells were cultured as described previously (Jones et al., 1999). Human recombinant Ang1 and Ang2
containing a polyhistidine tag were obtained from R&D Systems Inc. All cell stimulations were performed in supplemented F12K medium containing varying
concentrations of Ang-1 or Ang-2. For some stimulations with Ang-1, recombinant Ang-
1 was pre-incubated together with an anti-polyhistidine monoclonal antibody (10 µg/ml,
R&D Systems) to facilitate cross-linking of Ang-1 multimers.
22 2.3.2 Cell Lysis and Tie-2 Immunoprecipitation
HUVECs were washed two times with ice-cold phosphate buffered saline (PBS) and
lysed with RIPA lysis buffer (50 mM Tris⋅Cl pH 7.5, 150 mM NaCl, 1% Igepal, 0.5%
sodium deoxycholate, 0.1% SDS, 2 mM sodium orthovanadate, and protease inhibitors).
The cells were incubated on ice for 20 minutes followed by centrifugation at 14 000 g for
5 minutes at 4°C. Tie-2 was immunoprecipitated from the supernatant using 2-4 μg of anti-Tie2 antibody C20 (Santa Cruz Biotechnology) that had been pre-coupled to 25 μl protein A-sepharose (Amersham Biosciences).
2.3.3 Tie-2 Immunoblotting
Proteins were resolved on a 7.5% polyacrylamide gel and transferred to PVDF (Perkin
Elmer) membranes. The membranes were blocked for 1 hour at room temperature in 5% bovine serum albumin (BSA) prior to detecting phosphotyrosine, or in 5% non-fat dry milk for detecting total Tie-2. The membranes were incubated with either 1 μg/ml anti- phosphotyrosine antibody 4G10 (Upstate), anti-phospho Tie-2 Tyr1102/1108 antibody
(1:5000 dilution, Calbiochem), or 0.5 μg/ml anti-Tie2 monoclonal antibody (BD
Biosciences Pharmingen). Proteins were visualized using secondary antibodies conjugated to HRP (Biorad) followed by enhanced chemiluminescence.
2.3.4 Tie-2 Half-Life Determination and Synthesis
The half-life of Tie-2 in un-stimulated cells and in cells treated with either Ang-1 or Ang-
2 was determined by 35S labeling of proteins as described (Burke and Wiley, 1999).
HUVECs were incubated for 16-18 hours at 37°C in DMEM lacking methionine and
23 cysteine (Gibco) supplemented with 10% dialyzed fetal bovine serum (Gibco), penicillin-
streptomycin, 2 mM L-glutamine, and 100 μCi/ml [35S]methionine/cysteine (Tran35S-
Label, MP Biomedicals). Cells were washed six times with HUVEC growth medium and incubated in the same medium either alone, or containing 800 ng/ml Ang-1 (cross-linked with the anti-polyhistidine monoclonal antibody) or 800 ng/ml Ang-2 for various times at
37°C. The cells were lysed and Tie-2 immunoprecipitated and resolved on a 7.5% polyacrylamide gel. The gel was dried and exposed to a Molecular Dynamics phosphorimager screen. The intensities of the Tie-2 bands were quantified using the
Biorad Molecular Imager FX. The intensity of the band at time = 0 was designated
100%. The intensities of all other bands were expressed as a percentage of the initial (t =
0) value. The natural logarithm of the percentage values was taken and plotted against time. The slope of the line was used to determine the time required for the initial value
(100%) to decrease to 50%.
To examine Tie-2 synthesis in the presence of Ang-1 or Ang-2 , cells were incubated in
DMEM lacking methionine and cysteine as described above containing 100 μCi/ml
[35S]methionine/cysteine and either 800 ng/ml Ang-1 (cross-linked) or 800 ng/ml Ang-2
for various times at 37°C. The cells were lysed and Tie-2 immunoprecipitated and
resolved on a 7.5% polyacrylamide gel. The gel was dried and exposed to a Molecular
Dynamics phosphorimager screen and the bands quantified using the Biorad Molecular
Imager FX.
24 2.3.5 Tie-2 Biotinylation
HUVECs were incubated for various times with either Ang-1 (800 ng/ml, cross-linked) or Ang-2 (800 ng/ml) at 37°C. The cells were placed on ice, washed two times with ice- cold PBS and incubated for 15 minutes at 4°C with 0.5 mg/ml biotin (Sulfo-NHS-SS-
Biotin, Pierce) in PBS containing Ca2+ and Mg2+. The biotin solution was removed and the cells were washed once with 25 mM Tris pH 8.0 to quench non-reacted biotin followed by three washes with cold PBS. The cells were lysed with RIPA lysis buffer and cell lysates containing equal amounts of protein were incubated with 25 μl streptavidin conjugated to agarose (Pierce). The beads were washed 3 times with 1 ml
RIPA lysis buffer, resuspended in 85 μl sample buffer, and boiled for 5 minutes.
Supernatants (60μl) were immunoblotted for Tie-2 as described. For control purposes, surface biotin was removed by incubating cells three times in cold stripping solution (50 mM reduced glutathione, 75 mM NaCl, 75 mM NaOH, 1 mM EDTA, and 1% BSA) for
10 minutes each at 4°C followed by two washes with cold PBS as described previously
(Burke et al., 2001).
2.3.6 Iodination of Ang-1 and Ang-2
Carrier free human recombinant Ang-1 and Ang-2 (25 μg each, R&D Systems) were iodinated using IODO-GEN pre-coated iodination tubes (Pierce) and 0.2-0.5 mCi Na[125I]
(MP Biomedicals). All steps were performed according to the manufacturer’s instructions. Free iodine was separated using 10 ml Dextran desalting columns (Pierce).
25 2.3.7 Internalization of Ang-1 and Ang-2
HUVECs grown to confluency were washed once with PBS and incubated in ice-cold
binding medium (F12K medium containing 0.1% BSA) containing either 125I-Ang1 or
125I-Ang2 for 90 minutes on ice at 4°C to allow ligand binding. Cell monolayers were washed six times (4 ml per wash) with cold binding medium, fresh binding medium was
added, and the cells transferred to 37°C. At various times, the cells were removed from
the incubator and the medium collected and counted. The cells were lysed with
solubilization buffer (0.1 M NaOH, 0.1% SDS) as described previously (Waterman et al.,
1998). Radioactivity contained within the media and the cells was counted in a
Compugamma CS gamma counter. To differentiate between surface bound or
internalized ligands, surface ligands were removed by trypsin. Cell monolayers were placed on ice, washed three times with cold PBS, and incubated with trypsin (1 mg/ml) for 55 minutes on ice at 4°C followed by 5 minute incubation at 37°C. The cells were collected and centrifuged at 1000 g for 5 minutes. The supernatant which contained digested extracellular proteins including cleaved surface ligands was removed and counted. The cell pellet which contained internalized ligand was resuspended in solubilization buffer and counted.
2.3.8 Angiopoietin Immunoprecipitation and Immunoblotting
Ang-1 and Ang-2 were immunoprecipitated from cell culture media using 2 μg anti-
polyhistidine monoclonal antibody (R&D Systems) that was pre-coupled to 25 μl protein
G sepharose (Amersham Biosciences). The immunoprecipitates were immunoblotted using 1 μg/ml anti-polyhistidine monoclonal antibody (R&D Systems). Proteins were
26 visualized using goat anti-mouse secondary antibodies conjugated to HRP (Biorad) followed by enhanced chemiluminescence.
2.3.9 Soluble Tie-2 Determination
HUVECs were grown to 25% confluency in 24-well dishes for 24 hours prior to
treatment with Ang-1 (cross-linked) or Ang-2. Each treatment was done in triplicate.
Following cell stimulations, the culture media was collected and cleared by brief centrifugation. Soluble Tie-2 present in the culture media was quantified by the
Quantikine Human Tie-2 Immunoassay (R&D Systems) according to the manufacturer’s instructions.
2.4 Results
2.4.1 Ang-1 and Ang-2 activate Tie-2 in a concentration dependent manner
The regulation of many tyrosine kinase receptors has shown to be dependent on ligand concentration (Kasuga et al., 1981) and consequently, on receptor activation where higher levels of receptor activation stimulate faster rates of receptor internalization and
degradation. To understand the relationship between the concentration of Ang-1 and
Ang-2, and Tie-2 activation, we examined Tie-2 tyrosine phosphorylation in response to
increasing concentrations of each ligand (Fig. 4). Pre-incubating recombinant Ang1
where a polyhistidine tag has been added at the C-terminus together with an anti- polyhistidine monoclonal antibody enhanced Tie-2 tyrosine phosphorylation compared to
Ang-1 alone presumably by cross-linking Ang-1 multimers (Fig. 4A, C). Under these conditions, Ang-1 activated Tie-2 in a concentration dependent manner with maximal
27 levels of Tie-2 activation observed when the cells were stimulated with 800 ng/ml Ang-1
(Fig. 4A). The anti-polyhistidine antibody alone did not induce Tie-2 activation (Fig. 4C) and was included in Ang1 stimulations to achieve higher levels of Tie-2 activation. Ang-
2 also activated Tie-2 in a concentration dependent manner (Fig. 4B). Similar to Ang1, maximal activation of Tie-2 was observed when the cells were stimulated with 800 ng/ml
Ang-2 (Fig. 4B). In contrast to Ang-1, pre-incubating Ang-2 with the anti-polyhistidine antibody did not enhance Tie-2 phosphorylation (Appendix A). To study the regulation of Tie-2 by Ang-1 and Ang-2, 800 ng/ml of each ligand was used in subsequent experiments since these concentrations produced maximal activation of Tie-2.
When tyrosine phosphorylation of Tie-2 induced by Ang-1 and Ang-2 was compared, Tie-2 phosphorylation induced by Ang-1 was considerably higher compared to Ang-2 (Fig. 4C). We examined the phosphorylation of tyrosine residues 1102/1108
(mouse 1100/1106) on Tie-2 in response to Ang-1 and Ang-2. Phosphorylation on tyrosine residues 1102 and 1108 is important for the initiation of downstream signaling pathways (Jones et al., 1999; Jones et al., 2003). Both Ang-1 and Ang-2 stimulated phosphorylation of Tyr1102/1108 on Tie-2 although phosphorylation induced by Ang-2 was weaker compared to Ang-1 (Fig. 4D).
28 A
IP: Tie2 BLOT: anti-pY Tie2
116-
IP: Tie2 BLOT: Tie2 116- Ang1 0 100 400 800 1000 (ng/ml)
120 100 80 60 40 20
Tie2 Phosphorylation 0 0 200 400 600 800 1000 1200 Ang1 (ng/ml)
Figure 4: Ang-1 activates Tie-2 in a concentration dependent manner. (A) HUVECs were stimulated with the indicated concentrations of Ang-1 pre-clustered with 10 μg/ml anti- polyhistidine antibody for 15 minutes at 37°C. Top Panel Representative Immunoblot. Lower Panel Intensities of Tie-2 tyrosine phosphorylation were quantified by densitometry and plotted versus Ang-1 concentration. The value at 800 ng/ml was designated 100%. Data shown are a representative experiment repeated three times. The arrow indicates mature Tie-2 (~140kDa). A molecular mass standard is shown on the left.
29 B
IP: Tie2 BLOT:anti-pY Tie2 110-
IP: Tie2 BLOT: Tie2
110- Ang2 0 100 200 400 800 1000 (ng/ml)
120 100 80 60 40 20 0 Tie2 PhosphorylationTie2 0 200 400 600 800 1000 1200 Ang2 (ng/ml)
Figure 4: Ang-2 activates Tie-2 in a concentration dependent manner. (B) HUVECs were stimulated with the indicated concentrations of Ang-2 for 15 minutes at 37°C. Top Panel Representative Immunoblot. Lower Panel Intensities of Tie-2 tyrosine phosphorylation were quantified by densitometry and plotted versus Ang-2 concentration. The value at 800 ng/ml was designated 100%. Data shown are a representative experiment repeated three times. The arrow indicates mature Tie-2 (~140kDa). A molecular mass standard is shown on the left.
30 C
IP: Tie2 Tie2 BLOT: anti-pY 116-
IP: Tie2 BLOT: Tie2 116-
Non Ang1 Ang1 Ang2 anti-his + anti-his D
IP: Tie2 BLOT: pTyr1102/1108 116-
IP: Tie2 BLOT: Tie2
116- Non Ang1 Ang2
Figure 4: (C) Comparison of Tie2 tyrosine phosphorylation when HUVECs are stimulated with Ang-1 (800 ng/ml), Ang-1 (800 ng/ml) pre-clustered with the anti-polyhistidine antibody (10 μg/ml), Ang-2 (800 ng/ml), or the anti-polyhistidine antibody (10 μg/ml) alone. (D) EA.hy926 cells were stimulated with Ang- 1 (800 ng/ml, pre-clustered) or Ang-2 (800 ng/ml) for 15 minutes at 37°C. Tie-2 was immunoprecipitated and immunoblotted with an anti-phospho Tyr1102/1108 antibody (Top Panel) or an anti-Tie2 antibody (Lower Panel). Data shown are a representative experiment repeated three times. The arrow indicates mature Tie-2 (~140kDa). A molecular mass standard is shown on the left.
31 2.4.2 Tie-2 Turnover in HUVECs
To examine the effect of Ang-1 and Ang-2 on the turnover of Tie-2 in HUVECs,
we determined the metabolic half-life of Tie-2 by pulse-chase in un-stimulated cells, and
in cells stimulated with either Ang-1 or Ang-2. HUVECs were first incubated in media
containing [35S]-methionine/cysteine to label newly synthesized Tie-2. The cells were
then washed and incubated in normal growth medium (with or without ligands) for
various times. Upon transfer of the cells to normal growth medium, the initial
radioactively labeled Tie-2 population disappeared over time as part of the regular
turnover of membrane proteins and lipids. This approach allowed us to measure the rate
of Tie-2 degradation and how this may be modulated by Angiopoietin stimulation. Under
these conditions, Tie-2 degraded exponentially over time (Fig. 5A). Semi-logarithmic plots of Tie-2 levels versus time were generated and the slopes of the lines were used to calculate the time required for half of the initial receptor mass to decrease by 50%. The half-life of Tie-2 in un-stimulated cells was ∼9 hours (range 7.5 to 12 hours), in the
presence of Ang-1 the half-life was determined to be ∼3 hours (range 1.8 to 4.0 hours),
and in the presence of Ang-2 the half-life was ∼7 hours (range 4.9 to 8.2 hours) (Fig. 5B).
Using a one way analysis of variance (ANOVA) followed by the Tukey’s HSD Test
revealed that the half-life values of Tie-2 differ significantly between un-stimulated cells
and in cells treated with either Ang-1 and Ang-2 (p<0.05). In the absence of the cross-
linking antibody, the half-life of Tie-2 in the presence of Ang-1 was still shorter
compared to un-stimulated cells or in the presence of Ang-2 (t1/2 = 2.6 h). The rate of Tie-
2 synthesis was unchanged in the presence of either ligand compared to un-stimulated
cells (Fig. 6).
32 A
Tie2 -116 Time 0 5 10 15 0 5 10 15 0 5 10 15 (Hours)
Non (•) Ang1 (◊) Ang2 (■)
B
5
4.5
4
3.5
3 Ln (Optical Density)
2.5
2 0481216 Time (Hours)
Figure 5: Turnover of Tie-2 in HUVECs. [35S]methionine/cysteine labeled HUVECs were washed and incubated at 37°C for the indicated times in either HUVEC media alone, or containing pre-clustered Ang-1 (800 ng/ml), or Ang-2 (800 ng/ml). The cells were lysed, Tie-2 immunoprecipitated with an anti-Tie2 antibody, and immunoprecipitates resolved by SDS-PAGE. (A) Representative autoradiograph. (B) Semi-logarithmic plot of the relative Tie-2 intensities versus time in un-stimulated cells (•), in cells stimulated with Ang-1 (◊), and Ang-2 (■). Each point on the graph is an average value from three to nine independent experiments. The arrow indicates mature Tie-2 (~140kDa). A molecular mass standard is shown on the right.
33 A -116 Time 3 6 9 12 3 6 9 12 3 6 9 12 (Hours)
Non (•) Ang1 (◊) Ang2 (■)
B
160 140 120 100 80 60
(% of Maximum) of (% 40 Relative Tie2 Levels Levels Tie2 Relative 20 0 03691215 Time (Hours)
Figure 6: Ang-1 and Ang-2 do not influence Tie-2 synthesis. [35S]methionine/cysteine labeled HUVECs were incubated for various times together with pre-clustered Ang-1 (800 ng/ml), or Ang-2 (800 ng/ml). Tie-2 immunoprecipitates were resolved by SDS-PAGE and the dried gel exposed to a phosphorimager screen. (A) Representative autoradiograph. (B) The intensities of the bands were quantified and plotted against time. Time = 12 hours was designated 100%. All other values were expressed as a percentage of the 12 hours time point. Un-stimulated cells (•), Ang-1 (◊), Ang- 2 (■). Results are mean ± s.e.m. from three independent experiments. The arrow indicates mature Tie-2 (~140kDa). A molecular mass standard is shown on the right.
The increased rate of degradation of Tie2 in the presence of Ang-1 and Ang-2 could also be observed in cell lysates by Western blot analysis (Fig. 7) further suggesting
34 that the rate of Tie2 synthesis was unable to compensate for the ligand-induced degradation of the receptor and that activation of the Tie-2 pathway does not up-regulate its own synthesis.
182- A 116- Tie2
49- β-Actin
Time 0 15min 2h 6h 15min 2h 6h
Ang1 Ang2
B
120
100
80
60
40
20
0 Relative Tie2 Levels (% of Initial) of Relative Tie2 Levels (% 02468 Time (Hours)
Figure 7: (A) HUVECs were stimulated with pre-clustered Ang-1 (800 ng/ml), or Ang-2 (800 ng/ml) for the indicated times. Total cell lysates were analyzed by Western immunoblotting using a monoclonal Tie-2 antibody to detect total receptor levels and an anti-β-actin antibody to verify that equal amounts of protein were analyzed. Time (0) represents cells stimulated with media alone. (B) The intensities of the bands in (A) were quantified and expressed as a percentage of the t = 0 value and plotted against time, Ang-1 (◊) Ang-2 (■). Results are mean ± s.e.m. from three independent experiments. The arrows indicates mature Tie-2 (~140kDa) and β-actin (~40kDa). Molecular mass standards are shown on the left.
35 2.4.3 Tie-2 Internalization
Upon ligand binding, many tyrosine kinase receptors are rapidly internalized into intracellular compartments (Sorkin and Waters 1993; Gaborik and Hunyady 2004). The internalization of Tie-2 upon binding Ang-1 or Ang-2 was indirectly assessed by tagging
Tie-2 at cell surface with biotin. Biotinylated Tie-2 was isolated with streptavidin conjugated to agarose beads and detected by immunoblotting (Fig. 8). To ensure that only Tie-2 at the plasma membrane was being isolated, biotin was stripped from the cell surface and proteins pulled-down with streptavidin agarose. After stripping the cell surface most of the Tie2-specific signal in the immunoblot was lost confirming that Tie-2 receptors isolated with streptavidin agarose represented the receptors at the plasma membrane (Fig. 8A).
Internalization of Tie-2 in the presence of Ang-1 and Ang-2 was determined by examining the amount of receptor remaining at the cell surface at various times after the addition of either ligand. HUVECs were incubated with either Ang-1 or Ang-2 for various times at 37°C followed by biotinylation of cell surface proteins. Cell lysates containing equal amounts of protein were incubated with streptavidin agarose and the amount of Tie-2 remaining at the cell surface was determined by immunoblotting for Tie-
2. In the presence of Ang-1, Tie-2 was rapidly internalized into the cell as indicated by the reduced amount of Tie-2 isolated with streptavidin agarose (Fig. 8B). In contrast, in the presence of Ang-2, Tie-2 internalization was considerably slower compared to Ang-1 with almost all of the receptor still remaining at the cell surface after 90 minutes of stimulation (Fig. 8B). When cell stimulations with Ang-1 were performed at 0°C, a temperature where receptor endocytosis is suppressed, the disappearance of Tie-2 from
36 the cell surface was inhibited indicating that the reduced amount of Tie-2 isolated with streptavidin agarose was a consequence of receptor internalization (Fig. 8D). Ang-1 increased the rate of Tie-2 internalization even in the absence of the cross-linking antibody compared to un-stimulated cells and cells stimulated with Ang-2 (Fig. 8E and data not shown).
A
182- IP: Streptavidin 116- BLOT: Tie2 Non Biotin Biotin Cell lysate + Stripping
B
182- IP: Streptavidin BLOT: Tie2 116- Time 0 30 60 90 0 30 60 90 (min) Ang1 Ang2 C 120
100
80
60
40
20 Cell Surface Tie2 of(% Initial) 0 0306090 Time (minutes)
Figure 8: Surface biotinylation of HUVECs was used to assess the amount of Tie-2 remaining at the cell surface upon Ang-1 and Ang-2 stimulation. (A) Surface biotinylated HUVECs were lysed and incubated with streptavidin conjugated to agarose beads. Biotinylated proteins were resolved by SDS- PAGE and immunoblotted using a monoclonal anti-Tie2 antibody. Non-biotinylated cells (Non), biotinylated (Biotin), biotinylated followed by stripping (Biotin + Stripped), and total cell lysate (Cell Lysate). (B) HUVECs were incubated with pre-clustered Ang-1 (800 ng/ml), or Ang-2 (800 ng/ml) for the indicated times at 37°C. Cell surface proteins were biotinylated and isolated with streptavidin agarose followed by immunoblotting as described in (A), t = 0 represents cells stimulated with media alone. (C) The intensities of the bands in (B) were quantified and plotted against time, Ang-1 (◊) Ang-2 (■). Results are mean ± s.e.m. from four independent experiments. Western blots show mature Tie-2 (~140kDa). Molecular mass standards are shown on the left.
37 D
185- Tie-2 112- 85- 64-
Non 0° 4° 37° E Ang-1
182-
116- Tie-2
Time (min) 0 120 0 30 120 0 30 120
Non Ang-1 Ang-1 + Xab
Figure 8: (D) HUVECS were incubated with Ang-1 (800ng/ml) at the indicated temperatures (0, 4, or 37°C) for 90 min. The cells were washed and the cell surface was biotinylated as described in Section 2.3.5. Cell surface proteins were isolated using streptavidin conjugated to agarose beads and resolved by SDS-PAGE and immunoblotted using anti-Tie2 antibody 33.1. Non represents non-stimulated cells at 37°C. The Western blot showing Tie-2 at the cell surface indicates that Ang-1 induced endocytosis of Tie- 2 is temperature dependent. Numbers indicate molecular mass in kDa shown on the left. (E) HUVECs were stimulated for the indicated times at 37°C with Ang-1 or Ang-1 that was cross-linked using the anti- his antibody (Ang-1 + Xab). The cell surface was then biotinylated, cell surface proteins isolated, resolved by SDS-PAGE and immunoblotted using anti-Tie2 antibody 33.1. Non represents non-stimulated cells at 37°C for the indicated times. Molecular mass standards are shown on the left.
38 2.4.4 Release of Bound Ang-1 and Ang-2 upon Tie-2 Activation
Most peptide growth factors that bind to their cognate receptors are co-
internalized upon activation and internalization of the receptor. In order to determine
whether Ang-1 or Ang-2 would also internalize together with Tie-2, HUVECs were
stimulated with iodinated Ang-1 or Ang-2. Iodination of Ang-1 and Ang-2 did not
disrupt the multimerization state of either ligand as shown by gel electrophoresis and
Western blot analysis. Under reducing conditions, both native Ang-1 and 125I-Ang1
migrated with a molecular mass of ∼70 kDa, and under non-reducing conditions migrated
with a minimum molecular mass of ∼ 200 kDa suggesting that the minimal multimeric
state of Ang-1 and 125I-Ang1 is a trimer as reported previously for Ang-1 (Procopio et al.,
1999) (Fig. 9A). Ang-2 and 125I-Ang2 migrated, under reducing conditions, with a molecular mass of ∼70 kDa, and under non-reducing conditions migrated with a minimum molecular mass of ∼150 kDa, suggesting that the minimal multimeric state of
Ang-2 and 125I-Ang2 is a dimer as reported previously for Ang-2 (Procopio et al., 1999)
(Fig. 9B).
39 A 125I-Ang1 Ang1
182- 182- 116- 116- 82- 82- 64- 64- R NR R NR Autoradiography Blot: anti-his
B 125I-Ang2 Ang2
182- 182-
116- 116- 82- 85- 64- 64- R NR R NR Autoradiography Blot: anti-his
Figure 9: (A) Electrophoretic mobility of 125I-Ang1 and Ang-1. Aliquots of 125I-Ang1 and 50 ng Ang-1 were resolved by SDS-PAGE under reduced (R) and non-reduced (NR) conditions. To visualize 125I-Ang1, the gel was dried and exposed to X-ray film. To visualize Ang-1, proteins were transferred to PVDF membranes and immunoblotted using an anti-polyhistidine antibody. Numbers indicate relative molecular masses. (B) Electrophoretic mobility of 125I-Ang2 and Ang2 was evaluated as described in (A).
40 The binding of 125I-Ang1 and 125I-Ang2 to HUVECs was performed at 0°C. At
this temperature receptor endocytosis is inhibited thereby allowing ligand binding with
minimal receptor internalization. Following ligand binding at 0°C, the cells were washed
to remove unbound ligands and then transferred to 37°C to initiate receptor
internalization. Immediately upon transfer of the cells to 37°C there was a progressive
loss of radioactivity from the cells evident within 5 minutes of incubation (Fig. 9C). This
was observed for both 125I-Ang1 and 125I-Ang2 with the loss of radioactivity from the
cells faster when 125I-Ang2 was bound (Fig. 9C). When 125I-Ang2 was bound, more than half of the initial radioactivity was lost from the cells after 30 minutes of incubation at
37°C, whereas when 125I-Ang1 was bound, half of the initial radioactivity was lost after
90 minutes of incubation. Cross-linking 125I-Ang1 with the anti-polyhistidine antibody
did not significantly influence the rate of release of the ligand from the cell surface and
was therefore omitted from the binding studies.
41 C
120 100 80 60
(% of Initial) 40 20
Cell Associated Radioactivity 0 0 30 60 90 120 Time (minutes)
Figure 9: (C) 125I-Ang1 and 125I-Ang2 were allowed to bind to HUVECs at 0°C. The cells were washed to remove unbound ligands and then transferred to 37°C. At the indicated times, the cells were solubilized and cell associated radioactivity counted and plotted against time. Radioactivity at t = 0 was designated 100%, 125I-Ang1 (◊) 125I-Ang2 (■).
42 When the culture medium was analyzed for the presence of Ang-1 and Ang-2 it
was found that there was a progressive accumulation of radioactivity in the media. This
release into the medium occurred more rapidly with 125I-Ang2 compared to 125I-Ang1.
Radioactivity was detected in the media within 5 minutes at 37°C and continued to
increase up to 2 hours of incubation for both ligands (Fig. 9D).
D
100
80
60
40 (% of Total)
20 Radioactivity in Media
0 0 306090120 Time (minutes)
Figure 9: (D) Time dependent appearance of radioactivity in the media collected from cells bound with 125I-Ang1 (◊) and 125I-Ang2 (■). Radioactivity was expressed as a percentage of the total radioactivity contained within the sample.
43 When the cells were maintained at 0°C the release of 125I-Ang1 was significantly
reduced. In contrast, 125I-Ang2 had a tendency to be released at 0°C (Table 1).
Table 1: Cell and Media Distribution of 125I-Ang1 and 125I-Ang2 at 0ºC and 37ºC.
0ºC 37ºC
% Cell Associated % Media % Cell Associated % Media
125 I-Ang1 82±5.4 24±3.8 28±3.0 76±2.8
125 I-Ang2 53±7.7 56±5.9 14±2.2 86±2.8
HUVECs were incubated with 125I-Ang1 and 125I-Ang2 at 0°C to allow ligand binding as described. The cells were washed to remove unbound ligand and incubated at 0°C or 37°C in fresh binding medium for 2 hours. The medium was collected and radioactivity quantified in a gamma counter. The cells were washed, solubilized, and counted in a gamma counter. Values are expressed as a percentage of the total radioactivity contained within the sample (cell associated radioactivity + media radioactivity = 100%). Results are mean ± s.e.m. of four independent experiments.
To determine whether the released radioactivity represented intact Ang-1 or Ang-
2, or possibly a proteolytic fragment of the ligands, the media was collected and incubated with anti-polyhistidine antibodies pre-coupled to protein G-sepharose.
Following SDS-PAGE under reducing conditions and autoradiography, a band with a
molecular mass of ∼70 kDa was immunoprecipitated from the media collected from cells
previously incubated with 125I-Ang1 or 125I-Ang2 (Fig. 9E and 9F). The ∼70 kDa
proteins isolated from the media collected from 125I-Ang1 and 125I-Ang2 bound cells
migrated at the same molecular mass as full length 125I-Ang1 and 125I-Ang2 respectively,
44 suggesting that the 125I-Ang1 and 125I-Ang2 released from the cells upon Tie-2 internalization were not proteolytically modified upon release from the receptor. As predicted from our earlier studies, there was significantly less 125I-Ang1 in the medium from cells incubated at 0°C (Fig. 9E), whereas 125I-Ang2 was released into the medium even when the cells were maintained at 0°C (Fig. 9F).
45
E
82- IP: anti-his Autoradiography 64-
125I-Ang1 30 60 120 cold
Time (min)
F
182- IP: anti-his 116- Autoradiography 82- 64- 49-
125I-Ang2 0°C 37°C 60min
Figure 9: (E) Media was collected at the indicated times from HUVECs bound by 125I-Ang1 and immunoprecipitated with an anti-polyhistidine antibody. Immunoprecipitates were resolved by SDS-PAGE and exposed to X-ray film. Cold refers to cells incubated at 0°C for 120 minutes. Numbers refer to relative molecular masses. (F) Media was collected at the indicated times from cells bound with 125I-Ang2 and processed as in (E).White line indicates that intervening lanes have been spliced out.
46 To further investigate the observation that 125I-Ang1 and 125I-Ang2 remain at the
cell surface and may not be internalized into endothelial cells, surface bound ligands were
differentiated from internalized ligands by cleaving surface proteins with trypsin.
Surface bound 125I-Ang1 and 125I-Ang2 were not readily removed by washing the cells
with high salt and acid buffers which are commonly used to remove surface ligands from
their receptors. By treating HUVECs with trypsin at 0°C, ∼90% of surface bound 125I-
Ang1 and 125I-Ang2 were removed by this procedure.
To determine whether Ang-1 and Ang-2 remained at the cell surface, HUVECs
were incubated with 125I-Ang1 or 125I-Ang2 for 30 minutes at 37°C. The cells were
washed to remove unbound ligands and the proteins at the cell surface were then cleaved
with trypsin. Following digestion of surface proteins by trypsin, 90% of 125I-Ang1 and
86% of 125I-Ang2 were removed from the cells as evidenced by the loss of radioactivity associated with the cells and the concomitant increase of radioactivity in the supernatant fraction (Table 2). These findings suggest that 125I-Ang1 and 125I-Ang2 were not
internalized into endothelial cells but remained at the cell surface where they were accessible to trypsin. Similar results were obtained when the cells were incubated with
125I-Ang1 or 125I-Ang2 for 15 minutes, 1 hour, or 2 hours (data not shown).
47 Table 2: Trypsin Digestion of Cell Surface Proteins
Trypsin Treatment
Total cell radioactivity Pellet Supernatant
125I-Ang1 44 570 4681 33 599
125I-Ang2 29 400 4780 23 664
HUVECs were incubated in binding medium containing 125I-Ang1 or 125I-Ang2 for 30 minutes at 37°C. The cells were washed and incubated at 0°C with 1 mg/ml trypsin for 55 minutes followed by 5 minutes at 37°C. The cells were collected and centrifuged. The supernatant containing digested extracellular proteins was removed and the radioactivity quantified in a gamma counter. The cell pellet was solubilized and counted in a gamma counter. Numbers refer to radioactive counts per minute (cpm).
To insure that the surface release of the ligands was not a consequence of the
iodination, we incubated HUVECs with non-iodinated Ang1 or Ang2 and repeated our
release experiment. Western blot analysis using the anti-polyhistidine antibody revealed that Ang-1 and Ang-2 were released into the media with similar kinetics as that observed with 125I-Ang1 and 125I-Ang2 (Fig. 9G).
48 G
82- IP: anti-his BLOT: anti-his 64- IgG
Ang1 cold 0.5h 3.0h Ang2 cold 0.5h 3.0h
Ang1 Ang2
Figure 9: (G) Ang1 and Ang2 were allowed to bind to HUVECs. Cells were washed and incubated at 37°C in fresh binding medium. At the indicated times, the media was immunoprecipitated and immunoblotted using an anti-polyhistidine antibody. As controls for the position of Ang1 and Ang2, 50 ng of each ligand were resolved alongside the media samples. Cold refers to media collected from cells incubated at 0°C for 3 hours.
2.4.5 Potential Mechanisms Regulating Ligand Release
To determine whether Ang-1 and Ang-2 release was due to shedding of the
extracellular domain of Tie-2, we measured the amount of Tie-2 present in the culture medium of cells after stimulation with either Ang-1 or Ang-2 (Fig. 10A). Our results reveal that although soluble Tie2 is present in the culture medium of HUVECs as
described previously (Reusch et al., 2001), the amount of soluble Tie-2 released to the
media was not increased by Ang-1 or Ang-2 stimulation indicating that the release of
Ang-1 and Ang-2 does not involve cleavage of the extracellular domain of Tie-2. This
observation is consistent with our preliminary experiments suggesting that released Ang-
49 1 and Ang-2 are biologically active and are capable of rebinding to fresh cells (data not
shown). A
0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 sTie-2 Concentration (ng/mL)sTie-2 Concentration 0 224 Time (Hours)
Figure 10: (A) HUVECs were treated with either Ang1 (800 ng/ml cross-linked, white bars), Ang2 (800 ng/ml) (black bars), Mock (cross-linking antibody alone, hatched bars), or left untreated (grey bars). At the indicated times, the media was collected and the amount of soluble Tie2 (sTie2) quantified using a Human Tie2 Immunoassay Kit. Figure contributed by Vicky PKH Nguyen.
Blocking Tie-2 internalization using chemical inhibitors such as phenylarsine
oxide (PAO) or hypertonic sucrose (0.5 M), agents shown to block internalization of a
variety of cell surface receptors (Hertel et al., 1985; Faussner et al., 2004) had no effect
on the release of Ang-1 although treatment of cells with 0.5 M sucrose only partially
blocked Tie-2 internalization in response to Ang-1 (data not shown). Receptor
internalization is likely not sufficient for ligand release since Ang-2 did not significantly
promote Tie-2 internalization but was released faster compared to Ang-1.
50 To stimulate intracellular signaling within endothelial cells, we exposed
endothelial cells to pervanadate, a powerful protein tyrosine phosphatase inhibitor known
to significantly enhance phosphorylation on tyrosine, serine, and threonine residues of
proteins. Exposure of HUVECs to pervanadate led to marked increases in tyrosine
phosphorylation of cellular proteins (data not shown). When endothelial cells were exposed to pervanadate Ang-1 was released faster compared to endothelial cells not treated with pervanadate (Fig. 10B). Examination of Tie-2 internalization in the presence of Ang-1 and pervanadate revealed that although Ang-1 induced Tie-2 internalization, the addition of pervanadate did not further stimulate Tie-2 internalization (Fig. 10C) indicating that receptor internalization alone is not sufficient for Ang-1 release.
Pervanadate also enhanced Ang-2 release but more consistent results were obtained with
Ang-1 (data not shown). In addition, stimulation of endothelial cells with pervanadate led to increased levels of Tie-2 tyrosine phosphorylation (data not shown).
51 B Ang1
Ang1 Ang1 + pervanadate C
Tie2
Non Ang1 Ang1 + pervanadate
Figure 10: (B) HUVECs were incubated with Ang1 (800 ng/ml) for 90 minutes at 0°C in the presence or absence of 100 µM pervanadate. The cells were washed to remove unbound Ang1 and incubated for 30 minutes at 37°C in fresh media alone or containing 100 µM pervanadate. Ang1 was immunoprecipitated from the media and immunoblotted using an anti-polyhistidine antibody. (C) The cells from (B) were washed, surface proteins were biotinylated and isolated with streptavidin agarose, followed by immunoblotting for Tie2. As a control for Tie2 internalization, cells incubated in media alone in the absence of Ang1 or pervanadate were included (Non). White line indicates that lanes have been spliced out.
52 2.5 Discussion
In this study, we demonstrate that in response to Ang-1, Tie-2 is rapidly
internalized and targeted for degradation. In contrast, Ang-2 only weakly activated Tie-2
and did not significantly stimulate receptor internalization compared to Ang-1 while
mildly inducing Tie-2 degradation. More importantly, we show that the Angiopoietins are released from the endothelial cell surface after binding and accumulate in the surrounding medium.
The observation that Ang-1 induced rapid internalization and degradation of Tie-2
is consistent with studies of other tyrosine kinase receptors, such as the EGFR (Stoscheck
and Carpenter, 1984), where degradation of the receptor may serve to turn off signal
transduction, a process critical for maintaining cellular homeostasis. Evidence that Tie-2
signaling may be regulated in the vasculature comes from studies that have identified
constitutively active mutants of Tie-2 causing vascular abnormalities known as venous
malformations in humans (Vikkula et al., 1996; Calvert et al., 1999). The degradation of
Tie-2 upon binding Ang-1 may serve to regulate the magnitude and duration of Ang-1
signal transduction. Consistent with this possibility, Ang-2 only weakly activated Tie-2
and resulted in a correspondingly reduced rate of Tie-2 degradation.
Similar to our study, Hashimoto et al. (2004) have shown that Tie-2 is
downregulated in cell lysates in the presence of Ang-1 but not Ang-2. In our study, the
range of half-life values obtained for Tie-2 in the presence of Ang-2 overlapped with the
range of half-life values in un-stimulated cells indicating that in some experiments the
downregulation of Tie-2 by Ang-2 may not be as pronounced.
53 The differences in the rate of Tie-2 internalization stimulated by Ang-1 and Ang-2
may be related to the level of Tie-2 activation induced by these ligands where higher
levels of Tie-2 activation promoted a more rapid rate of Tie-2 internalization. It is
possible that due to the higher multimerization state of Ang-1, a larger population of Tie-
2 receptors were activated and subsequently targeted for degradation. In line with this
possibility, rapid downregulation of the EGFR has been shown to be dependent on the tyrosine kinase activity of the receptor (Wiley et al., 1991). Recently, receptor
dimerization has also been shown to be sufficient to induce rapid internalization of the
EGFR (Wang et al., 2005). This could also be applied to Tie-2 where Ang1, due to its
higher multimerization state, would induce correspondingly higher multimerization states
of Tie-2 compared to Ang-2. To date, the mechanism(s) regulating Tie2 internalization
are not known.
Our study has characterized Ang-2 as a partial agonist for Tie-2 rather than a
complete antagonist since both Ang-1 and Ang-2 exerted similar effects on Tie-2 but
with varying potency and kinetics. In addition to inducing phosphorylation of tyrosine
1102/1108 on Tie-2, Ang-1 and Ang-2 have been shown to activate similar signaling pathways such as the activation of Akt (Kim et al., 2000a; Papapetropoulos et al., 2000;
Kim et al., 2000b) although a higher concentration of Ang-2 was required (Kim et al.,
2000b). As a partial agonist, Ang-2 could still antagonize the Ang-1 signal as has been shown previously (Maisonpierre et al., 1997).
The role of Ang-2 in Tie-2 signaling has been controversial in the literature with
some studies describing Ang-2 as a Tie-2 antagonist (Maisonpierre et al., 1997) while
other studies reported the agonist properties of Ang-2 (Kim et al., 2000; Teichert-
54 Kuliszewska et al., 2001). The reason for the controversy among the various studies is not clear. In our study, the activation of Tie-2 by Ang-2 is weak and concentration dependent. Perhaps the weak induction of Tie-2 by Ang-2 may be interpreted as background phosphorylation. Another Angiopoietin family member, Ang-4, elicits similar levels of Tie2 activation as Ang-2 (unpublished observations) but has been described in the literature as a Tie-2 agonist (Valenzuela et al., 1999).
The observation that Ang-1 and Ang-2 were not internalized into endothelial cells but instead were released from the cell surface into the surrounding medium was surprising since many ligands have been shown to internalize together with their receptors. Similar to our results, Wang et al. (2002) have shown that VEGF is released into the surrounding medium after binding to human colonic vascular endothelial cells and that released VEGF can be reutilized. Ang-1 and Ang-2 which are released into the medium after binding to endothelial cells are capable of rebinding to fresh cells (data not shown), suggesting that these ligands may also be recycled or reutilized by endothelial cells.
Our data have indicated that shedding of the extracellular domain of Tie2, and receptor internalization are not responsible for the release of the Angiopoietins but suggests that the mechanism(s) may be dependent in part or can be modulated by intracellular signaling. The observation that pervanadate appears to enhance
Angiopoietin release suggests that activation of Tie-2 together with the activation or inhibition of particular signaling pathways or molecules is responsible for the release of
Ang-1 and Ang-2 from the endothelial cell surface. Although the data obtained with pervanadate may suggest the involvement of a signaling pathway(s), the mechanism(s)
55 involved may be more complex. The mechanism(s) regulating Ang-1 and Ang-2 release
may be different. For example, Ang-2 only weakly induces tyrosine phosphorylation of
Tie-2 but was released faster from endothelial cells compared to Ang-1. One possibility
is that Angiopoietin release may be related more to changes in receptor affinity after
binding to these ligands. Further work will be required to completely elucidate the mechanism(s) regulating ligand release.
The fact that Ang-1 binds to other receptors such as Tie-1 and α5β1 integrin in
addition to Tie-2, poses the question whether Ang-1 dissociates from all receptors equally
or whether Ang-1 specifically bound to Tie-2 internalizes while Ang-1 bound to the other
receptors is released to the media. This question is difficult to answer because, of the
total Ang-1 bound to the cell surface, it is not known what proportion is specifically
bound to Tie-2 vs Tie-1 and α5β1. In addition, the relative affinities of Ang-1 for Tie-1
and α5β1 are not known. Experiments using radioactive Ang-1 revealed that at 2 h ~85%
of bound Ang-1 is released to the media. Longer release times (longer than 2 h) were not
evaluated, so the possibility exists that with longer release times, >85% of the bound
Ang-1 would be released. A full 100% release will not likely occur since the plasma membrane still contains the receptor Tie-2 (at a much reduced level) and the background constitutes ~10% of the radioactive counts. Given that Ang-1 has a relatively high affinity for Tie-2, it is likely that a significant portion of exogenously added Ang-1 binds to Tie-2 and is released after receptor activation. Further evidence indicating that Ang-1 is released specifically from Tie-2 came from microscopy experiments (Chapter 3). The
goat polyclonal anti-Tie2 antibody used to visualize internalized Tie-2, binds to Tie-2 in the Ang-1 binding site where Ang-1 and the antibody compete for the same site on Tie-2.
56 Therefore, the ability to visualize Tie-2 inside endothelial cells after Ang-1 activation
using this antibody indicates that the site on the receptor is available for binding.
The affinities of Ang-1 and Ang-2 for Tie-2 were reported to be the same when
binding of either ligand to the extracellular domain of Tie-2 was examined in vitro
(Maisonpierre et al., 1997). The interaction of Ang-1 and Ang-2 with the endothelial cell
surface may be more complex as several reports have shown that Ang-1 can bind to other
surface receptors such as Tie-1 and integrins (Carlson et al., 2001; Saharinen et al.,
2005). A recent report has shown that Ang-2 can also bind to integrins and activate
intracellular signaling pathways (Hu et al., 2006). The fact that Ang-2 was released faster after binding compared to Ang-1 suggests that the interaction between these
ligands and the endothelial cell surface may be different. One possibility is that Ang-1
may bind with higher affinity to other receptors such as Tie-1 and/or integrins and therefore remain on the cell surface longer compared to Ang-2. The release of the
Angiopoietins after binding and activation of Tie-2 may make them available to bind to other receptors on endothelial cells and on non-endothelial cells.
The Angiopoietins together with VEGF are an emerging class of ligands that
instead of being co-internalized together with their cognate receptors as described for
numerous other receptor-ligand systems are released. We believe that the ability of
ligands to be released after receptor activation will be a consideration when studying
endothelial cell biology.
57 CHAPTER 3
Oligomerized Tie-2 Localizes to Clathrin-Coated Pits in Response to
Ang-1: An Electron Microscopy Study.
3.1 Abstract
The tyrosine kinase receptor Tie-2 is expressed on endothelial cells, and
together with its ligand angiopoietin-1 (Ang-1), are important for angiogenesis and
vascular stability. Upon activation by Ang-1, Tie-2 is rapidly internalized and degraded, a mechanism most likely necessary to attenuate receptor activity. Using immunogold
electron microscopy, we show that on the surface of endothelial cells, Tie-2 is arranged in
variably sized clusters containing dimers and higher order oligomers. Clusters of Tie-2
were expressed on the apical and basolateral plasma membranes, and on the tips of
microvilli. Upon activation by Ang-1, Tie-2 co-localized with the clathrin heavy chain at
the apical and basolateral plasma membranes and within endothelial cells indicating that
Tie-2 internalizes through clathrin coated pits. Inhibiting cellular endocytosis by
depleting cellular potassium or by acidifying the cytosol blocked the internalization of
Tie-2 in response to Ang-1. Our results suggest that one pathway mediating the
internalization of Tie-2 in response to Ang-1 is through clathrin coated pits. The data
presented in this chapter was published in Histochemistry and Cell Biology (2009, DOI:
10.1007/s00418-009-0603-3).
58 3.2 Introduction
In this chapter, the arrangement and distribution of Tie-2 within endothelial
cells was examined using immuno-gold electron microscopy. In addition, the research
presented in this chapter was aimed at identifying possible pathways mediating the
internalization of Tie-2 in response to Ang-1 with a focus on clathrin coated pits. Ang-1
was selected since the internalization of Tie-2 was significantly enhanced in the presence
of this ligand while Ang-2 had little effect on the internalization of Tie-2.
3.3 Materials and Methods
3.3.1 Materials
Human recombinant Ang-1 was purchased from R&D Systems. Mouse monoclonal anti-
clathrin heavy chain antibody (TD.1) was obtained from Santa Cruz Biotechnology.
Mouse monoclonal anti-β actin antibodies were from Sigma.
3.3.2 Cell Culture and Stimulation
Human umbilical vein endothelial cells (HUVECs) (BD Biosciences and Cambrex) were grown on gelatinized (gelatin from porcine or bovine skin) tissue culture plates, and cultured up to passage 10 in F-12K media (ATCC) supplemented with fetal bovine serum
(10%), 2mM L-glutamine, and 100μg/ml each of epidermal growth factor, vascular
endothelial growth factor, and basic fibroblast growth factor. For cell stimulations, Ang-
1 was resuspended (50µg/ml) in phosphate buffered saline (PBS) and diluted further in F-
12K media without serum to a final concentration of 800ng/ml.
59 3.3.3 Scanning Electron Microscopy
HUVECs were grown on gelatinized (0.05% gelatin) Thermanox plastic coverslips
(Nunc) and fixed using 4% paraformaldehyde and 0.2% glutaraldehyde in PBS (20mM sodium phosphate, 150mM NaCl, pH 7.5) for 25 min at room temperature. Cells were washed three times with PBS (5 min per wash) followed by a 10 min wash with 50mM glycine in PBS (pH 7.5). Cells were blocked in PBS containing 7% bovine serum albumin (BSA) and 3% normal serum (goat, donkey, or rabbit) for 1 h at room temperature. Primary antibodies were added to the blocking buffer and the cells incubated for 1 h at room temperature. The primary antibodies used were: mouse monoclonal anti-Tie2 antibody 33 (10μg/ml, BD Biosciences), goat polyclonal anti- human Tie-2 (2μg/ml, R&D Systems), or mouse monoclonal anti-Ang1 antibodies
(1μg/ml, R&D Systems). The cells were washed three times with PBS (5 min per wash) and re-blocked for 5 min followed by incubation for 1 h with secondary antibodies (goat anti-mouse, donkey anti-mouse, or rabbit anti-goat antibodies) conjugated to 0.8nm gold particles (Electron Microscopy Sciences) at a 1:10 dilution. The cells were washed six times with PBS (5 min per wash) and post-fixed with 1% glutaraldehyde in PBS for 10 min at room temperature. The cells were washed with PBS followed by six washes with distilled water (Gibco). The sizes of the gold particles were enhanced by coating the particles with silver using the Aurion R-Gent SE-EM kit (Electron Microscopy Sciences) according to the manufacturer’s instructions. The cells were washed again six times (5 min per wash) with distilled water and then dehydrated using a series of ascending ethanol solutions (50%, 70%, 90%, 95%, 100%, 100%, 2 min each). The samples were
60 then critical point dried and imaged using the Hitachi S-5200 scanning electron microscope.
3.3.4 Transmission Electron Microscopy
To image Tie-2 within cells using transmission electron microscopy, cells were processed as described for scanning electron microscopy. After the final dehydration step in 100% ethanol, the samples were embedded in Poly/Bed 812 (Polysciences) for 16-18 h at 65°C.
The blocks were peeled off from the culture dish, turned upside down (cell side up), and re-embedded for 24 h with fresh resin allowing cells to be contained between two layers of resin. The blocks were sectioned (~90nm thick) and stained with uranyl acetate and imaged using the Hitachi S-5200 scanning electron microscope.
3.3.5 Double Label Transmission Electron Microscopy
Double immunogold labeling was performed as described by Yi et al. (2001). HUVECs were grown on gelatinized (0.05% gelatin) tissue culture plates and processed as described for scanning electron microscopy with some modifications. Following fixation, cells were permeabilized with 0.01% Triton X-100 in PBS for 5 min at room temperature. The cells were blocked for 1 h and incubated with goat polyclonal anti-Tie2 antibodies (2μg/ml, R&D Systems) for 1 h at room temperature. The cells were washed and incubated with rabbit anti-goat antibodies conjugated to 0.8nm gold particles at a dilution of 1:10. The gold particles were then silver enhanced to enlarge the particle size.
The cells were washed with 0.03M sodium thiosulphate followed by distilled water, re- blocked, incubated with goat anti-clathrin heavy chain antibodies (5μg/ml, Santa Cruz
61 Biotechnology) followed by rabbit anti-goat secondary antibodies conjugated to 0.8nm
gold particles (1:10 dilution). The use of the same secondary antibody for double
labeling experiments was described by Bienz et al. (1986). The cells were post-fixed
with 1% glutaraldehyde and the particles silver enhanced. The cells were washed
extensively with distilled water and dehydrated using ascending ethanol solutions (20%,
30%, 50%, 70%, 90%, 95%, 100%, 100%, 2 min each) and embedded as described
above. The blocks were sectioned into ~90nm thick sections and stained with uranyl
acetate and imaged using the Hitachi S-5200 scanning electron microscope or the Hitachi
H-7000 transmission electron microscope.
3.3.6 Cell Surface Protein Cross-Linking
HUVECs were grown on gelatinized (0.2% gelatin) tissue culture plates. The cells were
placed on ice and washed with ice-cold PBS and incubated on ice in PBS containing
5mM BS3 (Pierce) for 30 min. The cells were washed and incubated with 20mM Tris, pH 7.5 for 15 min on ice followed by 2 washes with ice-cold PBS. The cells were lysed in RIPA lysis buffer (50mM Tris pH 7.5, 150mM NaCl, 0.5% Sodium deoxycholate, 1%
Igepal, 0.1% SDS, and protease inhibitors) and cellular protein quantified by the BCA protein assay kit (Pierce). Tie-2 was immunoprecipitated using antibody C20 (4µg, Santa
Cruz Biotechnology) conjugated to Protein A-sepharose beads (25μl, Amersham).
Immunoprecipitates were resolved by SDS-polyacrylamide gel electrophoresis (SDS-
PAGE, 5% gel) and Western blotted using monoclonal anti-Tie2 antibody 33 (0.5µg/ml) and goat anti-mouse secondary antibodies (Biorad, 1:5000) followed by enhanced chemiluminescence.
62 3.3.7 Tie-2 Internalization
Tie-2 internalization was determined by cell surface biotinylation as described previously
(Bogdanovic et al. 2006). Internalized Tie-2 was also assessed by trypsinizing cell
surface proteins. HUVECs were stimulated with Ang-1 for various times at 37°C. The
cells were placed on ice, washed twice with ice-cold PBS, and incubated on ice in PBS
containing 1 mg/ml trypsin for 90 min. Cells were collected in ice-cold HUVEC media
and centrifuged. Cell pellets were lysed in RIPA lysis buffer and cellular proteins quantified using the BCA protein assay (Pierce). Total cell lysates were resolved by
SDS-PAGE and Western blots were performed using anti-Tie2 antibody 33 (0.5µg/ml).
3.3.8 Inhibition of Cellular Endocytosis
Endocytosis was inhibited in HUVECs by depleting cellular potassium as described by
Larkin et al. (1983) or by acidifying the cytosol as described by Sandvig et al. (1987).
3.3.9 Knockdown of Clathrin Heavy Chain and Dynamin II Expression
To knockdown the expression of the clathrin heavy chain, cells were seeded at 30-50%
confluency on gelatinized (0.2% gelatin) tissue culture plates. Cells were transfected
with 50nM siRNA using Lipofectamine RNAiMAX (Invitrogen) according to the
manufacturer’s instructions. HUVECs were assayed 48 h post transfection. The siRNA
sequences used to knockdown the expression of the clathrin heavy chain and dynamin II
were chc-2 (Motley et al. 2003) designated siRNA 1, CHC duplex 2 (Huang et al. 2004)
designated siRNA 2 and DYN2 siRNA Duplex 2 (Huang et al. 2004). All siRNAs were
63 synthesized by Dharmacon Inc. Non-targeting siRNA #3 (Dharmacon Inc.) was used as a negative control.
3.4 Results
3.4.1 Tie-2 is Oligomerized on the Endothelial Cell Surface
To visualize Tie-2 on the surface of HUVECs we employed indirect immunogold electron microscopy using antibodies directed against the extracellular domain of Tie-2.
Two different primary antibodies were used; a mouse monoclonal anti-Tie2 antibody
(clone 33), and a goat polyclonal anti-Tie2 antibody. Both antibodies displayed high specificity for Tie-2 and detected a prominent ~140 kDa band in the membrane fraction in Western blots using HUVEC total membrane and cytosolic preparations (Fig. 11A and
B), and neither anti-Tie2 antibody cross-reacted with the closely related receptor Tie-1 to any detectable level.
64 A B
180- 180- 115-* 115-* 82- 82- 64-
64- 49-
49- 37- 37-
26- 26- Mem Cyt Mem Cyt
Figure 11: Specificity of anti-Tie2 antibodies. HUVECs were homogenized and centrifuged at 400 000g to obtain total membrane (Mem) and cytosolic (Cyt) fractions. Samples from each fraction were resolved by SDS-PAGE and Western blots were performed using either (A) mouse monoclonal anti-Tie2 antibodies, or (B) goat polyclonal anti-Tie2 antibodies. * Indicates the position of mature Tie-2. Numbers indicate molecular mass in kDa shown on the left.
Secondary antibodies conjugated to ultra-small (0.8nm) gold particles were used to label the primary antibodies. Due to their small size, ultra-small gold antibody conjugates allow high labeling efficiency likely due to reduced steric hindrance compared to when larger gold particles are used (reviewed by Robinson et al. 2000). To render the
0.8nm gold particles easily visible in the electron microscope, the sizes of the particles
65 were enhanced by coating the particles with metallic silver, a process referred to as silver enhancement (reviewed by Lackie 1996).
To validate this labeling strategy, 293F cells stably expressing Tie-2 were used as positive controls and parental 293F cells that did not express endogenous Tie-2 were used as negative controls. The cells were chemically fixed as described in Methods and labeled with primary and secondary antibodies, followed by silver enhancement. When the cell surface was imaged by scanning electron microscopy, numerous silver particles were seen on the surfaces of 293F cells expressing Tie-2 with relatively few particles seen on the surfaces of the parental 293F cells. This was observed for both anti-Tie2 antibodies (Fig.12 and 13). To visualize silver particles on the cell surface, backscatter images were collected where contrast between the silver particles and underlying plasma membrane was maximized. Conventional scanning electron micrographs showing the structure of the plasma membrane for the corresponding fields of view are shown in
Appendix B and Appendix C.
66 A
B
Figure 12: Validation of the Tie-2 labeling strategy. 293F cells and 293F cells stably overexpressing Tie- 2 were grown on glass coverslips that were pre-coated with poly-l-lysine. The cells were fixed as described for HUVECs in Methods. Both 293F cells and 293F cells overexpressing Tie-2 were treated with mouse monoclonal anti-Tie2 antibodies (clone 33, 10μg/ml) followed by goat anti-mouse secondary antibodies conjugated to 0.8nm gold particles. The sizes of the gold particles were enhanced with silver. Cells were viewed using the Hitachi S-5200 scanning electron microscope. Backscatter images showing the surface of (A) 293F cells, and (B) 293F cells overexpressing Tie-2.
67 A
B
Figure 13: Validation of the Tie-2 labeling strategy. 293F cells and 293F cells stably overexpressing Tie- 2 were grown on glass coverslips that were pre-coated with poly-l-lysine. The cells were fixed as described for HUVECs in Methods. Both 293F cells and 29F cells overexpressing Tie-2 were treated with goat polyclonal anti-Tie2 antibodies (2μg/ml) followed by rabbit anti-goat secondary antibodies conjugated to 0.8nm gold particles. The sizes of the gold particles were enhanced with silver. Cells were viewed using the Hitachi S-5200 scanning electron microscope. Backscatter images showing the surface of (A) 293F cells, and (B) 293F cells overexpressing Tie-2.
68 When the surface of HUVECs was examined using this labeling strategy, the expression of Tie-2 appeared to be distributed evenly over the endothelial cell surface seen as scattered silver particles in the scanning electron micrographs obtained by randomly imaging portions of the HUVEC plasma membrane (Fig. 14). This was observed using both anti-Tie2 antibodies (Fig. 14A, B). Few silver particles were seen in the background control cells where the primary antibodies were omitted (Fig. 15 A, B).
Conventional scanning electron micrographs showing the structure of the HUVEC plasma membrane for the corresponding fields of view are shown in Appendix D and
Appendix E.
69 A
B
Figure 14: Tie-2 was visualized on the HUVEC plasma membrane by indirect immunogold electron microscopy. Cells were fixed as described in Methods and treated with primary anti-Tie2 antibodies followed by secondary antibodies conjugated to 0.8nm gold particles. The sizes of the gold particles were enhanced with metallic silver. Cells were imaged using the Hitachi S-5200 scanning electron microscope. Backscatter images showing Tie-2 labeling using (A) goat polyclonal anti-Tie2 antibodies followed by rabbit anti-goat secondary antibodies, and (B) mouse monoclonal anti-Tie2 antibodies followed by goat anti-mouse secondary antibodies.
70 A
B
Figure 15: Background control images for the Tie-2 labeling strategy. HUVECs were fixed as described in Methods and treated with secondary antibodies conjugated to 0.8nm gold particles followed by silver enhancement. Cells were imaged using the Hitachi S-5200 scanning electron microscope. Backscatter images showing background labeling using (A) rabbit anti-goat secondary antibodies, and (B) goat anti- mouse secondary antibodies.
71 Upon closer examination of the micrographs shown in Fig. 14A and B, Tie-2 appeared to be arranged in discrete but variably sized clusters seen as aggregates of silver particles. After examination of ~70µm2 surface area of the HUVEC plasma membrane using each anti-Tie2 antibody, a variety of cluster sizes were observed ranging from a single silver particle to higher order clusters containing ~10 particles suggesting that within the plasma membrane Tie-2 is arranged in variably sized clusters containing various numbers of receptors. The various clusters of Tie-2 observed are shown in Fig.
16 and Table 3 shows the frequency at which the various clusters of Tie-2 occurred.
Although both anti-Tie2 antibodies produced similar images, the goat polyclonal anti-
Tie2 antibody appeared to be more efficient at labeling Tie-2 than the mouse monoclonal antibody judged by the higher labeling density observed in micrographs where the goat polyclonal antibody was used to label Tie-2. The goat polyclonal antibody was raised against human Tie-2 and acts as a neutralizing antibody; having the ability to block receptor-ligand interactions and therefore may be more efficient at binding to Tie-2 in the native state. In all further experiments, the goat polyclonal anti-Tie2 antibody was used to label Tie-2.
72 i ii iii
iv v vi
vii viii
Figure 16: Tie-2 is oligomerized on the endothelial cell surface. The various cluster sizes of Tie-2 observed on the endothelial cell surface using both antibodies were enlarged and are shown (i-viii).
73 Table 3: Quantification of Tie-2 Clusters on the HUVEC Plasma Membrane
Cluster 1 2 3 4 5 6 7 8 9 10 Size Mouse 49 17 7 2 3 1 1 - - - monoclonal Goat 99 48 18 5 4 2 2 1 1 3 polyclonal
After examining 70μm2 of the HUVEC plasma membrane, the number of times a cluster size appeared was counted for each anti-Tie2 antibody. Cluster size is defined as the number of aggregated silver particles. Bold numbers refer to the number of silver particles in the cluster; a cluster size of 1 refers to a single isolated silver particle, 2 refers to a cluster containing two silver particles, 10 refers to a cluster containing 10 silver particles.
Tie-2 oligomerization could also be detected by Western blotting. HUVECs were
incubated with the cell impermeable chemical cross-linker BS3 which cross-links proteins
on the extracellular surface within 1.14nm (11.4Å). Tie-2 was immunoprecipitated and
Western blots were performed (Fig. 17). In the presence of BS3, only a small fraction of
Tie-2 was observed as a monomer compared to control cells not treated with BS3 (Fig.
17A). Double bands are regularly observed in Tie-2 Western blots (Fig 17A). The lower band likely represents an immature or less glycosylated form of the receptor that is recognized by the anti-Tie2 antibodies. After a longer exposure of the Western blot shown in Fig. 17A, high molecular mass species became clearly visible in cells treated with BS3 (Fig. 17B). A protein band with a molecular mass >250 kDa is seen in Fig.17B
suggesting that the minimal oligomeric state of Tie-2 at the cell surface is a dimer
(monomeric Tie-2 is ~140kDa). Some immunoreactivity was also detected at the top of the gel suggesting the presence of higher molecular mass forms of Tie-2 that were too large to be resolved on a 5% polyacrylamide gel. Fig. 17C shows a Western blot of β-
74 actin levels taken from samples shown in Fig. 17B. The β-actin levels were similar
between conditions indicating that the cross-linker BS3 did not cross-link intracellular
proteins. Cross-linking Tie-2 at the cell surface with BS3 also suggests that within
HUVECs, the vast majority of Tie-2 is located at the plasma membrane since the majority of Tie-2 within HUVECs was available for cross-linking (Fig. 17A).
75 A
250-
150- Tie-2
BS3 - +
B Tie-2
Tie-2
250-
150- Tie-2
BS3 - +
Figure 17: Tie-2 is oligomerized on the endothelial cell surface. HUVECs were incubated on ice with the cell impermeable protein cross-linker BS3 (5mM). Cells were lysed, and (A) Tie-2 was immunoprecipitated and resolved by SDS-PAGE (5% gel) followed by Western blotting using mouse monoclonal anti-Tie2 antibodies. (B) Longer exposure of the Tie-2 Western blot shown in (A). Numbers indicate molecular mass in kDa.
76
C
β-actin
BS3 - +
Figure 17: Tie-2 is oligomerized on the endothelial cell surface. HUVECs were incubated on ice with the cell impermeable protein cross-linker BS3 (5mM). Cells were lysed, and (C) Western blot of β-actin levels in cell lysates obtained from samples shown in (A).
3.4.2 Distribution of Tie-2 Clusters in Endothelial Cells
The distribution of Tie-2 within HUVECs was determined by transmission
electron microscopy. Cells were grown as a monolayer to confluence, fixed, labeled with
anti-Tie2 antibodies as described in Methods, and embedded as a monolayer to preserve cell orientation. To preserve the silver coating around the gold particles, post-fixation
with osmium tetroxide was omitted since the addition of even small amounts (0.1%) of
osmium tetroxide completely dissolved the silver coating around the gold particles and,
in the presence of PBS, produced insoluble silver chloride precipitates. Cell
ultrastructure was thus compromised in order to preserve Tie-2 labeling.
Images were obtained from sections collected by sectioning through the cell
monolayer perpendicular to the culture dish enabling large portions of the cell length to
be imaged. Under these conditions, clusters of Tie-2 were found throughout the cell
length on all surfaces of the cell (Figs. 18 - 20). Tie-2 oligomers were expressed on the
77 apical plasma membrane (defined as the plasma membrane surface facing the media)
(Fig. 18 A, B), on the tips of microvilli (Fig. 19 A, B), and on the basolateral plasma membrane (defined as the plasma membrane surface in contact with the culture dish)
(Fig. 20 A, B). Similar results were observed using the mouse monoclonal anti-Tie2 antibody (data not shown). The expression of Tie-2 appeared to be relatively equal between the apical and basolateral plasma membranes.
78 A
B
Figure 18: Tie-2 clusters on the apical plasma membrane. HUVECs were grown in a monolayer to confluence. Following fixation, Tie-2 was labeled using goat polyclonal anti-Tie2 antibodies followed by rabbit anti-goat secondary antibodies conjugated to 0.8nm gold particles. The sizes of the gold particles were enhanced with silver. The cells were embedded, sectioned (90nm thick) and stained with uranyl acetate. Sections were viewed using the Hitachi S-5200 scanning electron microscope.
79 A
Figure 19: (A) Tie-2 clusters are expressed on endothelial microvilli. Following fixation, Tie-2 was labeled using goat polyclonal anti-Tie2 antibodies followed by rabbit anti-goat secondary antibodies conjugated to 0.8nm gold particles. The sizes of the gold particles were enhanced with silver. The cells were embedded, sectioned (90nm thick) and stained with uranyl acetate. Sections were viewed using the Hitachi S-5200 scanning electron microscope.
80 B
Figure 19: (B) Tie-2 clusters are expressed on endothelial microvilli. Image obtained on the Hitachi H- 7000 transmission electron microscope.
81 A
Figure 20: (A) Tie-2 clusters are distributed in the basolateral plasma membrane. HUVECs were grown in a monolayer to confluence. Following fixation, Tie-2 was labeled using goat polyclonal anti-Tie2 antibodies followed by rabbit anti-goat secondary antibodies conjugated to 0.8nm gold particles. The sizes of the gold particles were enhanced with silver. The cells were embedded, sectioned (90nm thick) and stained with uranyl acetate. Sections were viewed using the Hitachi S-5200 scanning electron microscope.
82 B
Culture media
Culture dish
Figure 20: (B) Tie-2 clusters are expressed on the basolateral plasma membrane. Image obtained using the Hitachi H-7000 transmission electron microscope.
83 The number of silver particles within a cluster likely reflects the oligomerization
state of Tie-2 within the plasma membrane and not non-specific aggregation of antibodies
or gold particles since not all antigens appeared as clusters. For example, the endosomal markers Rab5 and EEA1 were observed as single spheres within the cell (Fig. 21 A, B)
while proteins known to form oligomers such as the clathrin heavy chain and Ang-1
(described below) appeared as larger aggregates of silver particles. In addition, primary
and secondary antibodies derived from different species produced similar images (data
not shown).
84 A
Figure 21: (A) Intracellular labeling of Rab5. HUVECs were grown as a monolayer to confluence, fixed, permeabilized, and treated with anti-Rab5 antibodies followed by donkey anti-mouse secondary antibodies conjugated to 0.8nm gold particles. The sizes of the particles were enhanced with silver. The cells were embedded and sectioned (90nm thick). Sections were stained with uranyl acetate and imaged using the Hitachi H-7000 transmission electron microscope.
85 B
Figure 21: (B) Intracellular labeling of EEA1. HUVECs were grown as a monolayer to confluence, fixed, permeabilized, and treated with anti-EEA1 antibodies followed by goat anti-mouse secondary antibodies conjugated to 0.8nm gold particles. The sizes of the particles were enhanced with silver. The cells were embedded and sectioned (90nm thick). Sections were stained with uranyl acetate and imaged using the Hitachi H-7000 transmission electron microscope.
86 3.4.3 Oligomerization of Ang-1
As mentioned in Chapters 1 and 2, protein electrophoresis and structural studies
have shown that Ang-1, the ligand for Tie-2, exists in variably sized homo-oligomeric
complexes (trimers, tetramers, pentamers, and higher order clusters) (Kim et al. 2005;
Procopio et al. 1999). To visualize Ang-1 on the surface of cells by scanning electron
microscopy, a similar labeling strategy was employed as that used for Tie-2. HUVECs
were incubated with Ang-1 on ice at 4°C to allow ligand binding. At this temperature
subsequent receptor internalization is inhibited (Fig. 8D). The cells were washed
extensively to remove unbound ligand, fixed, treated with anti-Ang1 antibodies followed
by secondary antibodies and silver enhancement. This labeling strategy for Ang-1 was
validated using 293F cells expressing Tie-2. As negative controls, cells were incubated
in the absence of Ang-1 and processed under identical conditions to evaluate the extent of
background labeling. In 293F cells expressing Tie-2, Ang-1 was seen as clusters of silver
particles bound predominately to the microvilli extending from the cell body (Fig. 22A) with few silver particles seen on the surfaces of cells not incubated with Ang-1 (Fig. 22
B).
87 A
B
Figure 22: Validation of the Ang-1 labeling strategy. 293F cells overexpressing Tie-2 were grown on glass coverslips that were pre-coated with poly-l-lysine. Cells were incubated on ice for 90 min in media with or without Ang-1 (800ng/ml). The cells were washed, fixed, and treated with mouse monoclonal anti- Ang1 antibodies (1μg/ml) followed by donkey anti-mouse secondary antibodies conjugated to 0.8nm gold particles. The sizes of the gold particles were enhanced with silver. Cells were imaged using the Hitachi S-5200 scanning electron microscope. Backscatter images of (A) Ang-1 on the cell surface, and (B) background control.
On the HUVEC plasma membrane, Ang-1 was observed as discrete but variably sized clusters of particles (Fig. 23 A, B). The clusters of Ang-1 appeared to be spread
88 over the HUVEC plasma membrane with clusters seen near endothelial microvilli (Fig.
23B). The micrographs of Tie-2 and Ang-1 suggest that the oligomerization state of Tie-
2 may allow more efficient binding and activation by Ang-1. Background control micrographs for Ang-1 are shown in Fig. 23C. In the absence of Ang1, some silver particles were observed on the HUVEC cell surface indicating that the anti-Ang1 antibody may have slight cross-reactivity with other proteins on the cell surface.
Conventional scanning electron micrographs showing the structure of the 293F and
HUVEC plasma membranes for the corresponding fields of view are shown in Appendix
F and Appendix G.
89 A
B
Figure 23: Ang-1 is oligomerized on the endothelial cell surface. HUVECs were incubated on ice with Ang-1 for 90 min. The cells were washed to remove unbound ligand, fixed, exposed to mouse monoclonal anti-Ang1 antibodies followed by donkey anti-mouse secondary antibodies conjugated to 0.8nm gold particles. The sizes of the gold particles were enhanced with silver. Cells were viewed using the Hitachi S- 5200 scanning electron microscope. (A, B) Backscatter images showing clusters of Ang-1 on the endothelial cell surface.
90 C
Figure 23: (C) Background control for the Ang-1 labeling strategy in HUVECs. Cells were incubated on ice for 90min in the absence of Ang-1. The cells were washed, fixed, and exposed to mouse monoclonal anti-Ang1 antibodies followed by donkey anti-mouse secondary antibodies conjugated to 0.8nm gold particles. The sizes of the gold particles were enhanced with silver. Cells were viewed using the Hitachi S- 5200 scanning electron microscope.
3.4.4 Tie-2 Internalization
As shown in Chapter 2, using a cell surface biotinylation assay, Tie-2 is internalized
upon activation by Ang-1. In this assay, the amount of Tie-2 remaining at the plasma
membrane is evaluated upon the addition of Ang-1 (Fig. 24A). After incubating
HUVECs with Ang-1 for 20min at 37°C; the amount of Tie-2 remaining at the cell surface is considerably reduced compared to unstimulated cells suggesting that Tie-2 has
internalized into the cell (Fig. 24A).
91 To demonstrate that Tie-2 is internalized into intracellular compartments in response to Ang-1 using an alternate method, proteins on the cell surface were cleaved by trypsin.
In this assay, cells were stimulated with Ang-1 for various times to induce receptor internalization after which extracellular proteins were cleaved with trypsin. After stimulation with Ang-1, a portion of Tie-2 progressively becomes resistant to trypsin over time indicating that the receptor has been sequestered into intracellular compartments
(Fig. 24B). A control Western blot showing the position of Tie-2 using non-trypsinized cell lysates is shown in Appendix H. A
Tie-2
Ang-1 - +
B 150- Tie-2 Tie-2 100- NS
50- β-actin ß-actin
Ang-1 (min) 0 10 20 Ang1 (min) 0 20 Figure 24: (A) Tie-2 disappears from the plasma membrane upon Ang-1 stimulation. HUVECs were stimulated with or without Ang-1 for 20 min at 37°C followed by cell surface biotinylation. Cell surface proteins were isolated by streptavidin agarose and resolved by SDS-PAGE followed by Western blotting using mouse monoclonal anti-Tie2 antibodies. (B) Left and Right pannels Tie-2 accumulates within endothelial cells upon activation by Ang-1. HUVECs were stimulated with Ang-1 for various times at 37°C. The cells were washed and incubated on ice for 90 min in PBS containing 1 mg/ml trypsin. The cells were collected in HUVEC growth media and centrifuged. The cell pellet was lysed and cell lysates resolved by SDS-PAGE and Western blotted using mouse monoclonal anti-Tie2 and anti-β-actin antibodies. Numbers indicate molecular mass in kDa shown on the left. NS refers to non- specific bands.
92 Internalization of Tie-2 in the presence of Ang-1 can also be observed by scanning
electron microscopy. In the presence of Ang-1, the number silver particles representing
Tie-2 seen on the endothelial cell surface decrease over time (Fig. 25).
90 80 70 60 50 40 Surface 30 20 10 0 Number of Spheres on the Cell 12340 15 30 120 Time (min)
Figure 25: The number of silver particles on the HUVEC plasma membrane decrease in the presence of Ang-1. HUVECs were stimulated with Ang-1 for the indicated times. The cells were fixed and Tie-2 labeled as previously described using the mouse monoclonal antibody 33. The cells were imaged using the Hitachi S-5200 scanning electron microscope and silver particles were counted over 20µm2.
Inhibiting cellular endocytosis by potassium depletion or by acidifying the cytosol
blocked the internalization of Tie-2 induced by Ang-1 (Fig. 26).
93
Tie-2
Ang-1 - + - + - +
Control K+ NH4Cl Depleted
Figure 26: Inhibiting cellular endocytosis blocks the internalization of Tie-2 in response to Ang-1. Cellular endocytosis was inhibited by depleting cellular potassium (K+ Depleted) or by acidifying the cytosol (NH4Cl). HUVECs were stimulated with Ang-1 for 20 min at 37°C followed by biotinylation of the cell surface. Surface proteins were isolated by streptavidin agarose and subjected to SDS-PAGE followed by Western blotting using mouse monoclonal anti-Tie2 antibodies.
3.4.5 Tie-2 Localizes to Clathrin-Coated Pits in Response to Ang-1
Many cell surface receptors internalize via clathrin-coated pits and vesicles (Sorkin
and Waters 1993). Receptors targeted for internalization localize to regions within the
plasma membrane that invaginate (or form pits) and eventually pinch off into the cell
(reviewed by Mousavi et al. 2004). The protein clathrin, composed of heavy and light
chains, self-assembles into a lattice that surrounds the budding vesicle and remains
associated with the newly formed vesicle as it enters the cell (Young 2007). The clathrin
coat is then shed and the vesicles are delivered to early endosomes (Young 2007).
To determine whether Tie-2 is internalized through clathrin-coated pits, we
performed double immunolabeling experiments targeting Tie-2 and the clathrin heavy
chain (CHC) to determine whether Tie-2 co-localized with the CHC upon activation by
Ang-1. To label Tie-2 and the CHC, a similar labeling strategy was employed for both
94 antigens as already described for Tie-2 and Ang-1. To distinguish Tie-2 from the CHC,
the silver enhancement reaction was performed twice for the Tie-2 label, and only once for the CHC label. This generated large and small populations of silver particles as described by Yi et al. (2001). For the Tie-2 label, the particle sizes ranged from 16 to 39 nm (mean diameter = 26 nm, determined from measuring 42 particles) shown in Fig.
27A. Double silver enhancement of the Tie-2 label likely resulted in the fusion of multiple particles generating larger single particles. For the CHC label, the particles were considerably smaller ranging in size from 3 to 12 nm (mean diameter = 8 nm, determined from measuring 39 particles) and are shown in Fig. 27B.
Upon stimulation with Ang-1, Tie-2 co-localized with the CHC (Fig. 27C to 27J).
In Fig. 27C to 27J, larger particles representing Tie-2 were surrounded by numerous smaller particles representing the CHC. The smaller particles formed a cage, or lattice, around the larger particles. Tie-2 was seen co-localized with the CHC at both the apical
and basolateral plasma membranes (Fig. 27C, D, E, F), and also within the cell (Fig. 27G,
H, I, J). The number of receptors within clathrin- coated pits and vesicles was quantified
over 4mm of length across a confluent cell monolayer by examining the number of
aggregates of large and small particles. In un-stimulated cells, 6 co-localizations of large
and small particles were observed after examining portions of 45 cells. After stimulation
with Ang-1 for 2 min at 37°C the number of receptors associated with the CHC increased
to 30 after examining portions of 70 cells, indicating that Tie-2 engages the clathrin-
coated pit machinery upon activation by Ang-1.
95 A
B
Figure 27: HUVECs were grown as a monolayer to confluence and stimulated with Ang-1 for 2 min at 37°C. The cells were fixed, permeabilized, treated with goat polyclonal anti-Tie2 antibodies and anti- clathrin heavy chain (CHC) antibodies followed by secondary antibodies conjugated to 0.8nm gold particles. The sizes of the gold particles were enhanced with silver. For the CHC label (B), the gold particles were silver enhanced once and for the Tie-2 label (A) the particles were silver enhanced twice. The two labels were distinguished by size. The cells were embedded, sectioned (90nm thick), and stained with uranyl acetate. (A) Tie-2 label alone showing large silver particles. (B) CHC label alone showing small silver particles.
96 C
Figure 27: Tie-2 localizes to clathrin-coated pits in response to Ang-1. HUVECs were grown as a monolayer to confluence and stimulated with Ang-1 for 2 min at 37°C. (C) Tie-2 and CHC co-localized at the apical plasma membrane. Image was obtained using the Hitachi S-5200 transmission electron microscope.
97 D
Figure 27: Tie-2 localizes to clathrin-coated pits in response to Ang-1. HUVECs were grown as a monolayer to confluence and stimulated with Ang-1 for 2 min at 37°C. (D) Tie-2 and CHC co-localized at the apical plasma membrane. Image was obtained using the Hitachi S-5200 scanning electron microscope.
98 E
Figure 27: Tie-2 localizes to clathrin- coated pits in response to Ang-1. HUVECs were grown as a monolayer to confluence and stimulated with Ang-1 for 2 min at 37°C. (E) Tie-2 and CHC co-localized at the basolateral plasma membrane. Image obtained using the Hitachi H-7000 transmission electron microscope.
99
F
Figure 27: Tie-2 localizes to clathrin-coated pits in response to Ang-1. HUVECs were grown as a monolayer to confluence and stimulated with Ang-1 for 2 min at 37°C. Sections were viewed using the Hitachi H-7000 transmission electron microscope. (F) Higher magnification of the image shown in (E).
100 G
Figure 27: Tie-2 localizes to clathrin-coated pits in response to Ang-1. HUVECs were grown as a monolayer to confluence and stimulated with Ang-1 for 2 min at 37°C. (G) Tie-2 and CHC double label co-localized inside endothelial cells. Image was obtained using the Hitachi S-5200 electron microscope.
101 H
Figure 27: Tie-2 localizes to clathrin-coated pits in response to Ang-1. HUVECs were grown as a monolayer to confluence and stimulated with Ang-1 for 2 min at 37°C. (H) Tie-2 and CHC double label co-localized inside endothelial cells. Image was obtained using the Hitachi H-7000 transmission electron microscope.
102 I
J
Figure 27: Tie-2 localizes to clathrin-coated pits in response to Ang-1. HUVECs were grown as a monolayer to confluence and stimulated with Ang-1 for 2 min at 37°C. (I) Tie-2 and CHC double label co-localized inside endothelial cells. (J) Magnified image of the boxed area shown in (I). Images were obtained using the Hitachi S-5200 electron microscope.
103 Interestingly, knocking down cellular expression of the CHC by 70-90% using siRNA did not block the internalization or degradation of Tie-2 in response to Ang-1 (Fig. 28).
A CHC
β-actin
B Tie-2
Ang-1 - + - + - + - +
Mock siRNA siRNA Control 1 2 siRNA
C Tie-2
Ang-1 - + - +
Mock siRNA 2
Figure 28: Knocking down the expression of the clathrin heavy chain (CHC) does not inhibit Tie-2 internalization or degradation in response to Ang-1. HUVECs (30-50% confluent) were transfected using Lipofectamine RNAiMAX with 50nm siRNA. After 48h post-transfection, cells were stimulated with or without Ang-1 for 20 min at 37°C. (A) Cells were lysed and proteins resolved by SDS-PAGE followed by Western blotting using anti-CHC (TD.1) and anti-β-actin antibodies. (B) Following stimulation with or without Ang-1, the HUVEC cell surface was biotinylated. Surface proteins were isolated using streptavidin agarose and resolved by SDS-PAGE followed by Western blotting using mouse monoclonal anti-Tie2 antibodies. Mock Cells transfected in the absence of siRNA. siRNA 1 and siRNA 2 Cells transfected using two different CHC siRNAs. Control siRNA Cells transfected with siRNA not targeting any known RNA sequence. (C) HUVECs were stimulated with or without Ang-1 (800ng/ml) for 4 h at 37°C. The cells were lysed and proteins resolved by SDS-PAGE followed by Western blotting using mouse monoclonal anti-Tie2 antibodies.
104 Knocking down the cellular expression of the GTPase Dynamin II by 80-90% also had no effect on the internalization of Tie-2 in response to Ang-1 (Fig. 29).
DYN2
β-actin
Tie-2
Ang1 - + - +
Mock DYN2 siRNA
Figure 29: Knocking down the expression of dynamin II (DYN2) does not inhibit Tie-2 internalization in response to Ang-1. HUVECs (30-50% confluent) were transfected using Lipofectamine RNAiMAX with 50nM siRNA. After 48h post-transfection, cells were stimulated with or without Ang-1 for 20 min at 37°C. Top Panel Cells were lysed and proteins resolved by SDS-PAGE followed by Western blotting using anti-dynamin II antibodies and Middle Panel anti- β-actin antibodies. Lower Panel Following stimulation with or without Ang-1, the HUVEC cell surface was biotinylated. Surface proteins were isolated using streptavidin agarose and resolved by SDS-PAGE followed by Western blotting using mouse monoclonal anti-Tie2 antibodies. Mock Cells transfected in the absence of siRNA. DYN2 siRNA Cells transfected using siRNA (Duplex 2 from Huang et al. 2004).
105 3.4.6 Tie-2 Does Not Reside Within Endothelial Caveolae
It was previously shown using confocal microscopy that Tie2 localized within
endothelial caveolae (Yoon et al., 2003). Using electron microscopy, caveolae can be readily observed on both the apical and basolateral plasma membranes of HUVECs (Fig.
30A and B). Although some Tie-2 labeling was observed around the necks of endothelial caveolae (Fig. 30C), this represented a minor fraction (probably <5%). The vast majority of Tie-2 labeling was observed on the smooth portion of the plasma membrane (both
apical and basolateral).
106 A
Figure 30: (A) Flask shaped caveolae are seen (indicated by arrows) on the apical surface of HUVECs. Tie-2 labeling not seen within apical caveolae. Image taken using the Hitachi H-7000 transmission electron microscope.
107 B
Figure 30: (B) Caveolae are seen on the basolateral side of HUVECs (indicated by the arrow). Tie-2 labeling was not seen within basolateral caveolae. Image taken using the Hitachi H-7000 transmission electron microscope.
108 C
Figure 30: (C) Tie-2 (large particle indicated by arrow) is seen close to the neck of a single caveolae (indicated by arrow) seen on the apical surface of HUVECs. Tie-2 close to the necks of caveolae can be observed but the vast majority of Tie-2 labeling was seen on the smooth portion of the HUVEC plasma membrane. Smaller particles representing the clathrin heavy chain can be seen near the larger particle representing Tie-2.
3.5 Discussion
In the electron micrographs showing Tie-2 on the cell surface, the number of receptors represented by one silver particle is difficult to determine. Under optimal binding conditions where all antigenic sites are available for antibody binding, a single
109 antibody molecule could bind to two receptors. It is possible that not all receptors were available for antibody binding due to factors such as steric hindrance or chemical fixation. Therefore, one silver particle could represent an area on the cell surface containing one, two, or more than two receptors. Another layer of complexity is added by the secondary antibody. This issue was partially resolved by cross-linking surface proteins with the chemical cross-linker BS3 which indicated that only a small fraction of
Tie-2 was monomeric and that the minimal oligomeric state of Tie-2 on the surface of
endothelial cells is a dimer. Therefore, in the electron micrographs of Tie-2 on the
endothelial cell surface, a single silver particle, which was the most frequent observation
in the micrographs of Tie-2, most likely represents at least two receptors. The possibility
remains that in addition to Tie2-Tie2 dimers, the sharp band with a molecular mass > 250
kDa observed in Tie-2 Western blots after cell surface protein cross-linking, also
included Tie2-Tie1 dimers, as Tie-2 has been reported to heterodimerize with Tie-1
(Marron et al. 2000; Saharinen et al. 2005). Tie-2 has also been shown to heterodimerize
with the vascular endothelial protein tyrosine phosphatase (VE-PTP) (Fachinger et al.
1999) and with integrin α5β1 (Cascone et al. 2005). The heterodimerization of Tie-2
with either VE-PTP or α5β1 would not likely be observed in Tie-2 Western blots after
cell surface protein cross-linking since the combined molecular mass of either Tie2-VE-
PTP or Tie2-α5β1 would exceed 400 kDa which would not be resolved by SDS-PAGE
under the conditions of the experiment.
According to the classical view of receptor tyrosine kinase activation; receptors
exist as monomers at the plasma membrane. Ligand binding induces receptor
dimerization or oligomerization; a step necessary for trans phosphorylation of the
110 receptor intracellular domains (Hubbard and Till 2000). Increasing evidence indicates
that in the absence of ligand, some tyrosine kinase receptors are assembled into pre- formed clusters. Pre-formed receptor dimers and higher order oligomers have been reported for the EGFR (Moriki et al. 2001; Saffarian et al. 2007) and TrkA (Mischel et al.
2002) while the platelet derived growth factor receptor – β (PDGFR-β) exists mainly as a pre-aggregated tetramer (Wiseman and Petersen 1999). Our results indicate that on the surface of endothelial cells, the tyrosine kinase receptor Tie-2 is also arranged in pre- formed clusters containing various numbers of receptors (dimers and higher order oligomers). The arrangement of receptors in pre-formed clusters likely facilitates ligand binding and receptor activation since many ligands themselves are either bivalent (one ligand molecule binds to two receptors) (Schlessinger 2000), homodimeric (Schlessinger
2000), or multimeric (Toth et al. 2001). All angiopoietins (Ang-1, Ang-2, and Ang-3/4) form variably sized multimers in solution with Ang-1 forming larger clusters than Ang-2
and Ang-3/4 (Kim et al. 2005; Lee et al. 2004; Procopio et al. 1999). On the endothelial
cell surface, the arrangement of Tie-2 in pre-formed clusters would position the receptor
for efficient binding and activation by Ang-1. At the present time, it is not known what
significance the sizes of the various Tie-2 clusters have for downstream signal
transduction.
Numerous reports have shown that endothelial cells, when grown in culture,
synthesize and secrete an extracellular matrix and establish apical (luminal) and
basolateral (abluminal) polarity (Beer Stolz et al. 1992; Muller and Gimbrone 1986).
Clusters of Tie-2 were observed on all plasma membrane surfaces of HUVECs; on the
apical surface, on the tips of microvilli, and on the basolateral plasma membrane. The
111 distribution of Tie-2 on the plasma membrane of endothelial cells would indicate where
Tie-2 signaling is initiated following angiopoietin binding. The observation that Tie-2 is expressed on the basolateral plasma membrane is consistent with the widely accepted view that endothelial cells interact with smooth muscle cells (SMC)/pericytes and that
SMC/pericytes synthesize and secrete Ang-1 which binds to Tie-2 in a paracrine manner
(Armulik et al. 2005; Sundberg et al. 2002). The binding of Ang-1 to Tie-2 strengthens or maintains contacts between endothelial cells and between endothelial cells and the extracellular matrix (reviewed by Morisada et al. 2005; Suri et al. 1996). The presence of
Tie-2 on the apical surface of endothelial cells and on the tips of microvilli suggests that
Tie-2 signaling could be initiated on the luminal surface of blood vessels. Ang-1 and
Ang-2 can be detected in the blood (Caine et al. 2003). Ang-2 is synthesized by endothelial cells and stored in Weibel-Palade bodies together with the von Willebrand factor (vWF) (Fiedler et al. 2004). Stored Ang-2 is rapidly released upon endothelial activation following treatment with PMA (Fiedler et al. 2004). Released Ang-2 is believed to bind to Tie-2 in an autocrine manner. Activation of Tie-2 by Ang-2 may sensitize endothelial cells to inflammatory stimuli (reviewed by Fiedler and Augustin
2006). Interestingly, in response to PMA, van Buul-Wortelboer et al. (1989) have shown that vWF secretion from Wiebel-Palade bodies occurs on the luminal surface of endothelial cells. It is possible that in response to certain stimuli, Ang-2 could be secreted preferentially to the luminal surface and bind to Tie-2 specifically within that plasma membrane domain. Differential signaling between basolateral and apical plasma membranes for a given receptor-ligand pair has been previously reported. For example, the EGFR, in response to EGF, activates SHC and ERK1/2 on both apical and basolateral
112 plasma membranes while activation of FAK and β-catenin occurred preferentially on
basolateral and apical plasma membranes respectively (Kuwada et al. 1998).
Compartment specific signaling for Tie-2 in response to Ang-1 has recently been shown
(Fukuhara et al. 2008; Saharinen et al. 2008). The specific role of Tie-2 on the tips of endothelial microvilli is presently not known. Endothelial microvilli have been shown to express IL-8 important for endothelial-leukocyte interactions (Middleton et al. 2002).
Since microvilli are extensions of the plasma membrane that project outward from the cell surface, Middleton et al. (2002) have suggested that expression of IL-8 on the tips of microvilli may form the initial points of contact for leukocyte interaction with the endothelium. Ang-1 has been shown to inhibit IL-8 production by endothelial cells
(Pizurki et al. 2003)
In response to Ang-1, Tie-2 is internalized into the cell and targeted for degradation.
Tie-2 internalization in response to Ang-1 can be inhibited by depleting cellular
potassium or by acidifying the cytosol. These two treatments were initially shown to
inhibit clathrin-mediated endocytosis but subsequently were shown to inhibit other
endocytic pathways within cells (Carpentier et al. 1989; Cosson et al. 1989; Larkin et al.
1983; Sandvig et al. 1987). Here we show that at least one mechanism mediating the
internalization of Tie-2 in endothelial cells is through the clathrin-coated pit pathway.
Receptors at the cell surface are recruited into clathrin-coated pits by binding to clathrin adaptor proteins via their intracellular domains triggered possibly by ligand induced
receptor phosphorylation or ubiquitination (Mousavi et al. 2004 Kirchhausen 1999).
Mutation of tyrosine 1100 or 1106 on Tie-2 (mouse); tyrosine residues implicated in
downstream signaling of the receptor, did not impair the internalization of Tie-2 in
113 response to Ang-1 when expressed in NIH 3T3 cells (data not shown) indicating that
phosphorylation of these residues is not required for ligand induced receptor
internalization. Knocking down the expression of the CHC did not have any effect on the
internalization or degradation of Tie-2 in response to Ang-1 compared to Mock
transfected cells or cells transfected with control siRNA. One explanation for these
observations is that essential membrane turnover can proceed normally with the
remaining 10-30% of the CHC. In cells where the CHC was knocked down we did not
observe any changes in the growth or morphology of the cells compared to Mock
transfected or control siRNA transfected cells (data not shown). Hinrichsen et al. (2003)
have shown that in cells where the CHC was depleted by 90%, the cytosolic CHC pool
was significantly depleted while the amount of CHC associated with the membrane
increased. Perhaps the membrane associated CHC may be able to mediate some of the
cellular processes that are dependent on clathrin. Huang et al. (2004) have shown that
depleting cellular CHC by >95% was required to inhibit the internalization of the EGFR
in response to EGF. Another possibility is that in the face of drastically reduced levels of the CHC, the cell may relocate the remaining clathrin to particular processes or Tie-2 may internalize through clathrin independent mechanisms. Internalization through caveolae has been described as a clathrin independent mechanism of receptor endocytosis
(Cohen et al. 2004). Although we did observe some Tie-2 labeling around the necks of endothelial caveolae, the vast majority of Tie-2 was observed on the smooth portion of the plasma membrane. Therefore, internalization of Tie-2 through caveolae would not likely account for the loss of receptor from the plasma membrane in response to Ang-1.
Recently, clathrin- and caveolar- independent mechanisms of receptor internalization
114 have begun to emerge and are beginning to be examined (reviewed by Sandvig et al.
2008). The possibility exists that in addition to the clathrin-coated pits, Tie-2 could also
be internalized through an alternate clathrin independent pathway(s) ensuring that Tie-2
activity remains regulated by the endothelial cell.
Knocking down the cellular expression of the GTPase dynamin II also had no effect
on the internalization of Tie-2. Similar to the CHC, the possibility exists that normal
membrane turnover can proceed with the remaining 10-20%.
The findings reported by Saharinen et al., (2008) and Fukuhara et al., (2008)
published in the same issue showed that stimulation of confluent endothelial cells with
Ang-1 induced the translocation of Tie-2 to the edges of the cell where Ang-1 bound and bridged Tie-2 receptors on adjacent cells. The authors concluded that the multimeric nature of Ang-1 could allow it to bind to Tie-2 receptors on opposing cells. In sparse cells, the addition of Ang-1 induced either the internalization of Tie-2 or localization to cell-substratum contacts. In migrating cells, Ang-1 induced the translocation of Tie-2 to the rear or the trailing edge of cells.
The conclusions of these two papers differ significantly from our research which
indicated that in the presence of Ang-1 Tie-2 is internalized into cells and targeted for
degradation. Two other published papers also showed that upon stimulation of
endothelial cells with Ang-1, Tie-2 is internalized and degraded (Hashimoto et al., 2004;
Marron et al., 2007). At the present time it is not clear why the research on Tie-2 is
yielding vastly different results and conclusions. During the course of our research we
regularly observe the disappearance of Tie-2 from the cell surface upon the addition of
Ang-1. This process was found to be dependent on temperature, similar to the cell’s
115 endocytic processes, and independent of cell confluence. Appendix I shows confluent
human umbilical vein endothelial cells that were stimulated with Ang-1 for 20 min at
37°C. The amount of Tie-2 remaining at the cell surface was assessed, revealing that
after Ang-1 stimulation for 20 min the majority of Tie-2 has internalized.
We have chosen to use electron microscopy to examine the distribution of Tie-2 in
endothelial cells in the absence or presence of Ang-1 due to the higher sensitivity and
resolution of the electron microscope and also because we have not been able to produce
reliable and reproducible images of Tie-2 in endothelial cells using immunofluorescence
and confocal microscopy. We found that it was difficult to discern background from specific Tie-2 labeling likely due to low endogenous levels of the receptor. Tie-2 could readily be imaged using immunofluorescence when the receptor was overexpressed or when the signal from the endogenous receptor was amplified using the tyramide signal amplification kit (Molecular Probes). One concern with amplifying the signal using tyramide was the potential for the amplified product to diffuse away from the target preventing the accurate assessment of Tie-2 localization in endothelial cells.
When Tie-2 was imaged by electron microscopy in endothelial cells, Tie-2 was
distributed on all surfaces of the cell including the cell edges (Appendix J). Upon
stimulation with Ang-1 we did not observe an accumulation of Tie-2 at the cell edges
even when the cells were stimulated for 1 hour with Ang-1. Since Tie-2 is present at the cell edges it may be possible that Ang-1 could bind to Tie-2 receptors on adjacent cells.
At the present time we cannot explain why our observations differ from those observed by Fukuhara et al. (2008) and Saharinen et al. (2008).
116 CHAPTER 4
Conclusion
To summarize the main findings from Chapters 2 and 3, on the endothelial cell surface the receptor Tie-2 is arranged in discrete groupings containing various numbers of receptors on all surfaces of the cell. Upon binding Ang-1, the main activating ligand for Tie-2, the rate of Tie-2 internalization is increased resulting in the rapid removal of receptors from the cell surface (both apical and basolateral). Clusters of Tie-2, when internalized, are targeted for degradation while the ligand Ang-1 is released from the cell surface into the surrounding medium. One pathway mediating the internalization of Tie-
2 in HUVECs is through clathrin-coated pits. The work in this thesis has mainly focused on receptors that were activated by the ligand while the internalization of non-activated or un-occupied receptors was minimally addressed.
Timely degradation of activated RTKs appears to be a common mechanism employed by cells to regulate the signaling of receptors that is critical for proper organism function. Many members of the RTK superfamily are highly mitogenic and when mutated or overexpressed have shown to contribute to the progression of various types of cancer (Moscatello et al., 1995; Yarden and Skiwkowski, 2001) exemplifying the need to regulate RTK signaling. Several RTKs are now targeted in anti-cancer therapies; some examples include the EGFR, ErbB2/Her2, VEGFR2, IGF-1R, c-kit,
FGFR, and PDGFR (Gschwind et al., 2004). Herceptin, a drug approved for metastatic breast cancer targeting the RTK ErbB2/Her2, binds to the receptor and induces receptor internalization thereby stimulating the downregulation program that leads to enhanced degradation of the receptor (Gschwind et al., 2004). In addition, it is now thought that
117 the ability of mitogenic RTKs such as the ErbB RTK subfamily (EGFR (ErbB1), ErbB2,
ErbB3, ErbB4) to evade ligand-induced downregulation may have relevance in cancer
(Roepstorff et al., 2008). Impairments in the downregulation machinery for the EGFR
were also suggested to contribute to cell transformation (Roepstorff et al., 2008). As
mentioned in Chapter 2, evidence that Tie-2 signaling is under regulation within
organisms comes from patients manifesting vascular abnormalities termed venous
malformations (Vikkula et al., 1996; Calvert J T et al., 1999). These patients carry an
activating mutation within Tie-2 and display large, dilated, and tortuous vessels (Vikkula et al., 1996). From these examples it is clear that receptor endocytosis and downregulation are fundamental mechanisms employed by the cell to tightly regulate the response of the cell to external stimuli.
Accumulating evidence suggests that receptor internalization upon ligand binding
can also facilitate downstream signal transduction where some receptor signals could be
generated within intracellular compartments while the receptor is en route to the
lysosome (Sorkin and von Zastrow, 2002). This is entirely possible since ligand induced receptor internalization occurs faster than receptor degradation resulting in the
intracellular accumulation of activated receptors (Wiley, 2003). In this thesis, the
relationship between Tie-2 internalization and downstream signal transduction was not directly evaluated although two experiments were performed to obtain some insight into
Tie-2 signaling and trafficking. First, after activation by Ang-1, Tie-2 is progressively
dephosphorylated but this process takes up to 1 h indicating that Tie-2 internalized in
response to Ang-1 remains active within intracellular compartments (data not shown).
Therefore, the possibility exists that some Tie-2 signals could be generated or facilitated
118 within the endosomal compartments. Second, the compartmentalization of Tie-2
signaling was evaluated by examining the activation of Akt/PKB in response to Ang-1.
In HUVECs stimulated with Ang-1, Akt/PKB was primarily activated at the plasma
membrane judged by phosphorylation of S473 of Akt/PKB, with little activation
observed in the cytosol or internal membranes determined by subcellular fractionation
(data not shown). This likely reflects the mechanism of Akt/PKB activation within cells,
where Akt/PKB binds the lipid products of PI 3-kinase generated at the plasma
membrane.
The adapter proteins Dok-R, Shc, ABIN-2, and the p85 subunit of PI 3-kinase have
all been shown to bind to Tie-2 (Jones and Dumont, 1998, Kontos et al., 1998; Hughes et
al., 2003; Audero et al., 2004). Dok-R and the p85 subunit of PI 3-kinase bind to Tie-2 at the cell surface (Kontos et al., 1998; Master et al., 2001) indicating that Tie-2 signaling is
initiated at the plasma membrane. Whether Dok-R or the p85 subunit of PI 3-kinase
remain associated with Tie-2 within intracellular compartments, or whether ABIN-2 or
Shc bind to Tie-2 intracellularly remains to be determined. For example, the EGFR has
been shown to bind the adapter protein Shc at the plasma membrane and also within
endosomes (Burke et al., 2001).
The increased rate of Tie-2 internalization in response to Ang-1 appears to be
dependent on the kinase activity of Tie-2 (Fukuhara et al., 2008). As mentioned,
however, mutating tyrosines 1100 or 1106 to phenylalanine did not block the
internalization of Tie-2 in response to Ang-1 when expressed in NIH 3T3 cells (data not
shown). When NIH 3T3 cells were transfected with kinase inactive (kinase dead)
constucts of Tie-2 (Tie2-KD) where alanine at position 853 in the kinase insert was
119 substituted for lysine (Dumont et al., 1994), Tie2-KD appeared to be expressed by NIH
3T3 cells but not properly transported/inserted into the plasma membrane precluding the evaluation of Tie2-KD (data not shown) although Fukuhara et al., (2008) showed that kinase-inactive Tie-2 constructs failed to internalize in response to Ang-1 when expressed in CHO cells. Recently, it has been demonstrated that Tie-2 becomes ubiquitinated in response to Ang-1 and that ubiquitination may trigger receptor internalization (Christina Wehrle, unpublished data)
The clathrin-coated pit pathway appears to be one pathway mediating the internalization of Tie-2 in response to Ang-1. Many RTKs have shown to internalize through clathrin-coated pits (reviewed by Mukherjee et al., 2006) and molecular details regarding how receptors are recruited into clathrin-coated pits have now emerged.
Receptors internalized through clathrin-coated pits are delivered to early endosomes
(Sorkin and Waters, 1993). From early endosomes, receptors can be sorted to a recycling compartment and recycled back to the plasma membrane, or trafficked to late endomes/lysosomes for degradation (Stahl and Schwartz, 1986). Attempts to localize
Tie-2 within early endosomes were unsuccessful for several reasons. First, to preserve the silver Tie-2 label when imaging cells using electron microscopy, post-fixation with osmium tetroxide and post-staining with lead compounds was omitted since these treatments destroyed/obscured the Tie-2 label. Post-fixation with osmium tetroxide preserves membrane ultrastructure and post-staining with lead compounds allows the visualization of membranes. As a result of omitting these two treatments, localization of
Tie-2 within a clearly defined organelle was not possible. Second, co-localization experiments using antibodies recognizing Tie-2 and the endosomal markers Rab5 and
120 EEA1 where the labels were silver enhanced either once or twice (as described for Tie-2
and the CHC in Chapter 3) were ambiguous and difficult to interpret since Rab5 and
EEA1 appeared as single particles and did not form a lattice-like structure as that
observed for the CHC which was easily recognized. An alternate strategy when
performing co-localization studies at the electron microscopy level would be to use two
distinct sizes of gold particles to distinguish between antigens. Unfortunately, using
secondary antibodies conjugated to larger gold particles than 0.8 nm (Chapter 3), for
example, 6 and 10 nm gold particles, significantly reduced the amount of Tie-2 labeling
observed. In addition, using larger gold particles may not be ideal for labeling
intracellular antigens in permeabilized cells (Baschong and Stierhof, 1998). Attempts
were made to perform co-localization experiments using the silver label for one antigen
and a horse radish peroxidase (HRP) label for the second antigen with DAB as a substrate
(HRP-DAB). These attempts were also unsuccessful since the HRP-DAB reaction
required using reduced osmium tetroxide to impart contrast to the DAB product and that
destroyed the silver label marking the first antigen. In addition, the DAB product may
diffuse away from the site of production preventing precise co-localization of the two
antigens (Baschong and Stierhof, 1998).
Although clathrin- coated pits represent one pathway mediating the internalization
of Tie-2, other endocytic pathways exist within cells and mechanisms regulating these pathways are beginning to emerge (Sandvig et al., 2008). The suggestion that other endocytic pathways exist or that there may be functional redundancy of proteins within a
given endocytic pathway came from studies where the expression of the CHC or
components of the clathrin pathway were knocked down using siRNA. When the
121 internalization of the EGFR in response to EGF was examined, knocking down the
expression of the CHC by 90-95% had no effect on the internalization of EGFR
(Hinrichsen et al. 2003). Subsequently, Huang et al. (2004) showed that virtually all of the CHC content had to be eliminated in order to fully block EGFR internalization.
Knocking down the expression of the clathrin adaptor protein AP-2 also had no effect on
EGFR internalization in response to EGF indicating that other adaptor proteins may
function in this process (Hinrichsen et al. 2003; Motley et al. 2003). When the
expression of dynamin II was knocked down by 80-90% using siRNA in endothelial
cells, the internalization of Tie-2 in response to Ang-1 was unaffected. Dynamin II is a
ubiquitously expressed GTPase responsible for vesicle scission from the plasma
membrane (Sontag et al., 1994). These results suggest that either protein turnover can
proceed normally with the remaining 10-20% dynamin II or that alternate proteins may
perform a similar function. Further experiments are needed to address these possibilities.
It remains to be seen what other endocytic pathways exist for Tie-2 within endothelial
cells.
It is unlikely that Tie-2 is recycled back to the plasma membrane after
internalization by Ang-1 since the metabolic half-life of Tie-2 in the presence of Ang-1
indicates that after activation by Ang-1, Tie-2 is targeted for degradation. Degradation of
Tie-2 more than likely occurs within the lysosome as described for other cell surface
receptors (Sorkin and Waters, 1993). Since non-activated or un-occupied receptors were
not examined in this study it remains possible that in the basal state Tie-2 undergoes
cycles of internalization and recycling as has been shown for the EGFR (Burke and
Wiley, 1999).
122 The finding that activated Tie-2 is targeted for degradation indicates that Tie-2 signaling is regulated by the endothelial cell. Angiogenesis, the formation of new blood vessels from existing vessels, is dependent on the action of numerous receptors and angiogenic factors acting together in a coordinated fashion (Jain 2003; Adams and
Alitalo, 2007). In this context, in the developing organism, the activity of Tie-2 would need to be controlled to ensure co-operation with other receptors and molecules. In addition, the blood vasculature is believed to be maintained by a balance between pro- angiogenic and anti-angiogenic factors (Folkman 1992; Ribatti et al., 2002). If the presence of pro-angiogenic factors exceeds the anti-angiogenic factors then the process of angiogenesis is favored. Conversely, if anti-angiogenic factors dominate then angiogenesis would be inhibited. Therefore, regulating the activity of receptors and other molecules maintains the balance or equilibrium within the vasculature.
The observation that Ang-1 and Ang-2 do not internalize into endothelial cells together with Tie-2 was surprising since many ligands have been shown to co-internalize together with the receptor. Internalization of the ligand together with the receptor and degradation of the ligand-receptor complex within lysosomes would clear the ligand from the extracellular space. The fact that Ang-1 and Ang-2 do not internalize into endothelial cells and instead accumulate in the surrounding medium prompts the question of how the angiopoietins are cleared from the extracellular space. Findley et al. (2007) have shown that shedding of the Tie-2 ectodomain into the surrounding medium as soluble Tie-2
(sTie-2) bound to Ang-1 and Ang-2 in the medium and this prevented Ang-1 and Ang-2 from binding to Tie-2 on the surface of endothelial cells. As mentioned in Chapter 2, the extracellular domain of Tie-2 is constitutively shed by endothelial cells and can be
123 detected in the blood (Reusch et al., 2001). Future research will determine the biological
significance of released Ang-1 and Ang-2 and how the activity of these ligands is regulated within the vasculature.
The first tyrosine kinase receptor to be identified was the EGFR and details
regarding the activation, regulation, and trafficking of this receptor are now known
(Gschwind et al., 2004). The regulation and trafficking of other RTKs have shown to be similar to that of the EGFR. Although many receptors display very similar internalization and trafficking mechanisms, differences do exist. Given the various
functions and tissue distributions of the members of the RTK family, it is likely that at
the molecular level, there will be differences in how the cell regulates the activity of the
various RTKs as has been suggested for the G-protein coupled receptor superfamily
(Wolfe and Trejo, 2007). The findings presented in this thesis provide a general view of
how Tie-2 is arranged, distributed, and regulated by the endothelial cell. The novel
contributions of this thesis to the Tie2/Angiopoietin field of study are: (1) Ang-2 behaves
as a partial agonist for Tie-2, (2) in the presence of Ang-1, Tie-2 localizes to clathrin-
coated pits, (3) Ang-1 and Ang-2 do not appear to internalize into endothelial cells
together with Tie-2, and (4) Tie-2 appears to be arranged in variably sized clusters on the
endothelial cell surface. The specific molecular mechanisms involved and the
significance of these observations for Tie-2 signaling in endothelial cells should be the
focus of future research.
124 Appendix A
182-
116-
182-
116-
Non Ang-1 Ang-2 Ang-1 Ang-2
anti-polyHis
Appendix A: Cross-linking Ang-2 does not enhance Tie-2 activation. EA.hy926 endothelial cells were stimulated with either Ang-1 or Ang-2 (800ng/ml of each ligand) for 15 min at 37°C. Prior to some cell stimulations, Ang-1 and Ang-2 were pre-incubated with an anti-polyhistidine (anti-polyHis) antibody (10μg/ml) that cross-links and multimerizes the ligands. The cells were lysed and proteins resolved by SDS-PAGE followed by Western blotting using anti- phosphotyrosine antibodies (top panel) or anti-Tie2 antibody 33.1 (lower panel). Non refers to un-stimulated cells. Molecular mass standards are shown on the left.
125 Appendix B
A
B
Appendix B: Conventional scanning electron micrograhs showing the structure of the 293F cell plasma membrane. The cells were fixed as described for HUVECs in Section 3.3.3. Both 293F cells and 293F cells expressing Tie-2 were treated with mouse monoclonal anti-Tie2 antibodies (10μg/ml) followed by goat anti-mouse secondary antibodies conjugated to 0.8nm gold particles.The sizes of the gold particles were enhanced with silver. Cells were viewed using the Hitachi S-5200 scanning electron microscope showing the surface of (A) 293F cells, and (B) 293F cells expressing Tie-2.
126 Appendix C
A
B
Appendix C: Conventional scanning electron micrographs showig the structure of the 293F plasma membrane. 293F cells and 293F cells stably expressing Tie-2 were grown on glass coverslips that were pre-coated with poly-l-lysine. The cells were fixed as described for HUVECs in Section 3.3.3. Both 293F cells and 293F cells expressing Tie-2 were treated with goat polyclonal anti-Tie2 antibodies (2μg/ml) followed by rabbit anti-goat secondary antibodies conjugated to 0.8nm gold particles. The sizes of the gold particles were enhanced with silver. Cells were viewed using the Hitachi S-5200 scanning electron microscope showing the surface of (A) 293F cells, and (B) 293F cells expressing Tie-2.
127 Appendix D
A
B
Appendix D: Conventional scanning electron micrographs showing the structure of the HUVEC plasma membrane. Cells were fixed as described in 3.3.3 and treated with primary anti-Tie2 antibodies followed by secondary antibodies conjugated to 0.8nm gold particles. Cells were imaged using the Hitachi S-5200 scanning electron microscope showing Tie-2 labeling using (A) goat polyclonal anti-Tie2 antibodies followed by rabbit anti-goat secondary antibodies, and (B) mouse monoclonal anti-Tie2 antibodies followed by goat anti-mouse secondary antibodies.
128 Appendix E
A
B
Appendix E: Conventional scanning electron micrographs showing the structure of the HUVEC plasma membrane. Background control images for Tie-2 labeling. HUVECs were fixed as described in 3.3.3 and treated with secondary antibodies conjugated to 0.8nm gold particles followed by silver enhancement. Cells were imaged using the Hitachi S-5200 scanning electron microscope showing background labeling using (A) rabbit anti-goat secondary antibodies, and (B) goat anti-mouse secondary antibodies.
129 Appendix F
A
B
Appendix F: Conventional scaninng electron mirographs showing the structure of the 293F cell plasma membrane. 293F cells expressing Tie-2 were grown on glass coverslips that were pre- coated with poly-l-lysine. Cells were incubated on ice for 90 min in media with or without Ang-1 (800ng/ml). The cells were washed, fixed, and treated with mouse monoclonal anti-Ang1 antibodies (1μg/ml) followed by donkey anti-mouse secondary antibodies conjugated to 0.8nm gold particles. The sizes of the gold particles were enhanced with silver. Cells were imaged using the Hitachi S-5200 scanning electron microscope showing (A) Ang-1 on the cell surface, and (B) background control.
130 Appendix G
Appendix G: Conventional scanning electron micrograph showing the structure of the HUVEC plasma membrane. Background control for Ang-1 labeling in HUVECs. Cells were incubated on ice for 90min in the absence of Ang-1. The cells were washed, fixed, and exposed to mouse monoclonal anti-Ang1 antibodies (1µg/ml) followed by donkey anti-mouse secondary antibodies conjugated to 0.8nm gold particles. The sizes of the gold particles were enhanced with silver. Cells were viewed using the Hitachi S-5200 scanning electron microscope.
131 Appendix H
250- 150- Tie-2 100- 75- NS
50-
Non 0 10 20 Tryp. Tryp. Ang-1 (min)
Appendix H: HUVECs were stimulated with Ang-1 (800ng/ml) for the indicated times at 37°C. After stimulation with Ang-1, cell surface proteins were cleaved by exposing the cells to trypsin as described in section 2.3.7. The cells were lysed and cell lysates resolved by SDS-PAGE followed by immunoblotting using anti-Tie2 antibody 33. Non- trypsinized cell lysates were included to confirm the position of Tie-2 on the Western blot. Numbers indicate molecular mass in kDa. NS refers to non-specific bands.
132 Appendix I
A
B
Tie-2
Ang-1 - +
Appendix I: Tie-2 internalizes in response to Ang-1 in confluent endothelial cell cultures. (A) Human umbilical vein endothelial cells were grown to confluence (4.2 X 105 cells/cm2) fixed with formaldehyde (4%) and photographed using the Leica TCS-SP2-X confocal microscope in transmission mode (maginification 20x). Scale bar = 100μm. (B) In parallel samples, cells were stimulated for 20 min at 37°C in the presence (+) or absence (-) of Ang-1 (800ng/ml). The cell surface was biotinylated, cell surface proteins were isolated with streptavidin conjugated to agarose, resolved by SDS-PAGE and Western blotted using anti-Tie2 antibody 33. In confluent cells, the addition of Ang-1 induced the internalization of Tie-2. Micrograph in (A ) was taken by Ilya Gourevich from the Centre for Nanostructure Imaging at the Department of Chemistry, University of Toronto.
133 Appendix J
A B
Appendix J: (A and B) Tie-2 localizes to the edges of the cell. Silver particles representing Tie-2 in human umbilical vein endothelial cells are shown localized at the cell edges indicated by the arrows in un-stimulated cells. Sections were made through the cell monolayer perpendicular to the plane of the cells and imaged using the Hitachi H-7000 transmission electron microscope. Scale bar represents 100nm.
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