Dab2 plays a role in the post-endocytic trafficking of VEGFR2

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1 University of Iowa Iowa Research Online Theses and Dissertations Fall 2015 Dab2 plays a role in the post-endocytic trafficking of VEGFR2 Shivangi Makarand Inamdar University of Iowa Copyright 2015 Shivangi Makarand Inamdar This dissertation is available at Iowa Research Online: Recommended Citation Inamdar, Shivangi Makarand. "Dab2 plays a role in the post-endocytic trafficking of VEGFR2." PhD (Doctor of Philosophy) thesis, University of Iowa, Follow this and additional works at: Part of the Cell Biology Commons

2 DAB2 PLAYS A ROLE IN THE POST-ENDOCYTIC TRAFFICKING OF VEGFR2 by Shivangi Makarand Inamdar A thesis submitted in partial fulfillment Of the requirements for the Doctor of Philosophy Degree in Molecular and Cellular Biology in the Graduate College of The University of Iowa December 2015 Thesis Supervisors: Associate Professor Charles Yeaman External Advisor Amit Choudhury

3 Copyright by SHIVANGI MAKARND INAMDAR 2015 All Rights Reserved

4 Graduate College The University of Iowa Iowa City, Iowa CERTIFICATE OF APPROVAL PH.D. THESIS This is to certify that the Ph.D. thesis of Shivangi Makarand Inamdar has been approved by the Examining Committee for the thesis requirement for the Doctor of Philosophy degree in Molecular and Cellular Biology at the December 2015 graduation. Thesis Committee: Charles Yeaman, Thesis Supervisor Amit Choudhury, External Advisor Michael Anderson Frederick Domann John Engelhardt Thomas Rutkowski

5 To my grandfather, parents, late grandmother, Shruti, Shlok, and my dear friends. ii

6 Somewhere, something incredible is waiting to be known Carl Sagan Astronomer,writer iii

7 ACKNOWLEDGEMENTS I would like to thank my mentor Dr. Amit Choudhury to help me begin this project. I would also like to thank Dr. Yeaman for supporting me and mentoring me to complete this project. A special thanks to Dr. Domann and Dr. DeMali for their patience and help they gave towards the progress of my work. I am also grateful to my committee members Dr. Engelhardt, Dr. Rutkowski, and Dr. Anderson for providing me with valuable guidance and encouragement in this project. I would like to thank all my MCB and ACB fellow students who were always around whenever I needed help. I would like to thank my parents, sister, late grandmother, grandfather and all my relatives back in India for being supportive throughout this wonderful journey of mine. A special thanks to my parents who have always encouraged me through my tough times. Finally, I want to express my gratitude to my family in the US, my friends. A special thanks to Raaj, Gopi, Sampada, Pooja, Priyanka, Manali, Mohit, who have supported me and were always there to listen to me cry or laugh about my work. Heartfelt thank you to all of you for this would not have been possible without your support. iv

8 ABSTRACT Angiogenesis is a crucial process under both physiological and pathological conditions. Vascular endothelial growth factor (VEGF) A and its cognate receptor, vascular endothelial growth factor receptor 2 (VEGFR2) are key regulators of angiogenesis. Plasma membrane (PM) levels of VEGFR2 are regulated by de novo synthesis, and by both exocytic and endocytic trafficking. VEGF-binding to VEGFR2 induces phosphorylation of key tyrosine residues located in the cytosolic domain of the receptor, followed by clathrin-mediated endocytosis and signal transduction leading to vascular morphogenesis. Disabled protein 2 (Dab2) is a cytosolic, clathrin-adaptor protein that is known to regulate endocytosis of certain cell surface receptors. Studies of Dab2 function have revealed its role in the development of embryonic vasculature. However, the mechanism of Dab2 function, particularly in conjunction with endosomal VEGFR2, remains poorly understood. Our results show that Dab2 interacts with VEGFR2 and that upon VEGF stimulation the two proteins co-localize within Rab5- positive early endosomes. Knockdown of Dab2 reduces levels of VEGF-induced phosphorylation of VEGFR2 at residue Y1175. This is significant because phosphorylation of VEGFR2-Y1175 is crucial for pro-angiogenic signal transduction. Moreover, knockdown of Dab2 causes an increased trafficking of VEGFR2 to late endosomes (LE). Finally, this altered VEGFR2 trafficking following Dab2 knockdown has major functional consequences for endothelial cells, as they are unable to undergo morphogenesis into tube-like structures in an in vitro assay of angiogenesis. Collectively, our data show that Dab2 plays a crucial role in VEGFR2 trafficking in the endocytic v

9 system and this impacts receptor signaling and endothelial cell morphogenesis during angiogenesis. vi

10 PUBLIC ABSTRACT Blood vessels that are the major carriers of nutrients, blood, and oxygen are formed in two phases. Vasculogenesis, the first phase, occurs during embryonic development whereas; angiogenesis occurs throughout adult life and involves formation of new vessels from existing vessels. Angiogenesis is important for normal processes like wound healing and is also known to cause diseases like cancer. Thus, understanding the molecular basis of this process is important. Endothelial cells (ECs) that form the blood vessel walls are the major participants in the process of angiogenesis. ECs respond to an angiogenic inducer molecule, vascular endothelial growth factor (VEGF), that binds to a receptor VEGF receptor 2 (VEGFR2) that is expressed on the surface of ECs. Binding of the inducer to the receptor initiates downstream signaling that induces ECs to undergo migration and proliferation to form new blood vessels. Activated VEGFR2 does not mediate all its signaling activities at the plasma membrane (PM). In fact, upon activation, VEGFR2 is internalized inside of the cell into structures called endosomes. This transport termed as endocytosis has been demonstrated to play a role in regulating the activity of VEGFR2 downstream signaling. Endocytosis begins with the packaging of the receptor into specialized vesicles that are released into the cell cytoplasm. The proteins that form this packaging vesicle play an important role in the transport of the receptor. Disabled protein 2 (Dab2) is one such protein that functions in the internalization of VEGFR2. In this study, we focused on understanding the details of Dab2 mediated VEGFR2 endocytosis to regulate its function. Here we show that Dab2 interacts with VEGFR2 and colocalizes with it in the early endosomes, the first endocytic station. Absence of Dab2 leads to decrease in the vii

11 VEGFR2 activation. This decrease in VEGFR2 activation was determined to be a result of disrupted trafficking of VEGFR2 to the late endosomes for degradation. This work thus elaborates how Dab2 is required for post-endocytic trafficking of VEFGR2 to impact its function in the development and maintenance of blood vessels. viii

12 TABLE OF CONTENTS LIST OF FIGURES... xi LIST OF ABBREVIATIONS... xii CHAPTER Introduction : Vasculogenesis and angiogenesis : EC behavior in angiogenesis : Regulators of EC behavior in angiogenesis : VEGF and VEGFR in angiogenesis : Receptor endocytosis and its importance in RTK signaling : VEGFR2 endocytosis : Disabled protein 2 structure and function : Research goals CHAPTER Role of Dab2 in VEGFR2 trafficking : Abstract : Introduction : Materials and Methods : Results : Discussion CHAPTER ix

13 General Discussion : Dab2 and VEGFR : Dab2 and other angiogenic receptors REFERENCES APPENDIX x

14 LIST OF FIGURES Figure 1: Vasculogenesis and Angiogenesis... 4 Figure 2: Structure of VEGF and VEGFRs Figure 3: VEGFR2 downstream signaling Figure 4: Clathrin dependent endocytosis (CDE) and caveolin mediated endocytosis (CME) Figure 5: Structure of Dab2 protein Figure 6: Dab2 is localized as discrete punctae in HUVECs Figure 7: Dab2 colocalizes with VEGFR2 in EE upon VEGF stimulation Figure 8: Dab2 co-immunoprecipitates with VEGFR Figure 9: Dab2- specific shrna construct inhibits Dab2 expression in HUVECs Figure 10: Dab2 knockdown decreases VEGFR2 phosphorylation at tyrosine 1175 (Y1175) Figure 11: Dab2 knockdown decreases VEGFR2 phosphorylation at tyrosine 1175 (Y1175) and also alters its localization pattern Figure 12: Dab2 knockdown increases trafficking of VEGFR2 to the LE Figure 13: Quantification showing Dab2 knockdown increases trafficking of VEGFR2 to late endosomes Figure 14: Dab2 knockdown alters endothelial cell morphogenesis Figure 15: Predicted role of Dab2 in VEGFR2 trafficking xi

15 LIST OF ABBREVIATIONS VEGF- Vascular endothelial growth factor VEGFR2- Vascular endothelial growth factor receptor 2 PM- Plasma membrane Dab2- Disabled protein 2 LE- Late endosomes Y- Tyrosine ECs- Endothelial cells BM- Basement membrane Nrps- Neuropilins PlGF- Placental growth factor RTK- Receptor tyrosine kinase PKC- Protein kinase C CDE- Clathrin dependent endocytosis CME- Caveolin-mediated endocytosis APs- Adaptor proteins CCV- Clathrin coated vesicle EE- Early endosomes RE- Recycling endosomes MyoVI- Myosin VI PTP1b- Phosphotyrosine phosphatase1b CLASPs- Clathrin-associated sorting proteins xii

16 Par3- Partitioning defective 3 apkc- Atypical protein kinase C LAMP2- Lysosome-associated membrane glycoprotein 2 HUVECs- Human umbilical vein endothelial cells Nrp1- Neuropilin-1 CCPs- Clathrin coated pits xiii

17 CHAPTER 1 Introduction 1.1: Vasculogenesis and angiogenesis Blood vessels serve as important channels that carry oxygen, nutrients, and immune cells to the different parts of the body and at the same time serve to collect and dispose of waste from the body [1]. Formation of blood vessels occurs in two distinct phases. The formation of blood vessels during embryonic development occurs through the process of vasculogenesis [2]. In the developing embryo, mesoderm differentiates into hemangioblasts which form the first vascular structure known as the primitive blood islands [3]. Hemangioblasts that lie in the center of the island form hematopoietic stem cells whereas those aligned at the periphery of the island differentiate into angioblasts, which serve as the precursors for endothelial cells (ECs) [3-5]. As development of the embryo continues, angioblasts and ECs derived from them migrate and in the process allow fusion of the blood islands into tubular structures knows as the vascular plexus [3, 4]. This primary plexus undergoes further remodeling to form larger vessels that primarily function to supply blood to the growing embryo [3] (Figure 1). Angiogenesis on the other hand is the process of formation of new blood vessels from pre-existing vessels [5] (Figure 1). This is one important mechanism for the formation of new blood vessels in adults [2]. Angiogenesis is a multistep process that requires dynamic and tightly regulated function of ECs [6]. There are two types of angiogenesis: sprouting and non-sprouting angiogenesis [2, 4]. Sprouting angiogenesis involves the 1

18 outgrowth of ECs of preexisting vessels to form branches of new blood vessels [5]. Nonsprouting angiogenesis involves the splitting of vessels into new pillars of functional blood vessels or growth of ECs within a blood vessel, producing a wide lumen that can further split into pillars of new blood vessels [2, 4, 5]. Formation of blood vessels through the angiogenic process plays an important role in several physiological processes such as development, wound healing, and tissue regeneration [3]. Abnormal angiogenesis, however, is also known to participate in several pathological conditions. Insufficient angiogenesis is known to play a major causative role in tissue ischemic diseases, whereas activation of angiogenesis by tumor cells has been known to play a role in cancer metastasis [3, 6]. Since misregulated angiogenic process leading to abnormal vasculature has been observed in several diseases, research focused on developing new strategies to develop drugs that target this process has gained attention. Several drugs that target the angiogenic machinery have been approved by the US Food and Drug Administration (FDA) in treatment of several diseases, including cancer and age-related macular degeneration [1]. The VEGF-neutralizing antibody bevacizumab (Avastin) has been used to treat several cancers [1]. Several others drugs that target the VEGF signaling pathways have also been used to treat cancer patients [1]. Use of such drugs in combination with chemotherapy has shown an improvement in the survival rate of cancer patients [7]. Despite the success of these drugs, limitations of their efficacy arise from patients becoming refractory or resistant to the drug treatment [1, 7]. Therefore, a deeper 2

19 understanding of the molecular basis of angiogenesis could help us discover other novel and effective targets for the treatment of such diseases. 3

20 Figure 1: Vasculogenesis and Angiogenesis As seen above, vasculogenesis occurs during embryonic development and begins with the differentiation of the mesoderm into blood islands and the endothelial precursor cells, hemangioblasts cells. As development progresses hemangioblasts differentiate into ECs and the blood islands undergo remodeling to form the primary plexus that servers to perfuse the growing embryo. Angiogenesis involves the differentiation, migration, and proliferation of ECs of a preexisting vessel to form a new vessel branch. 4

21 1.2: EC behavior in angiogenesis Key regulators of angiogenesis are ECs. In stable blood vessels, ECs are tightly associated with one another, forming a monolayer of polarized cells [6]. Upon angiogenic stimulation, these cells undergo changes that are highly coordinated and tightly regulated to form new blood vessels [6]. An early event following exposure to angiogenic signals involves the loosening of associations between tightly adherent ECs, which subsequently begin to degrade the underlying basement membrane (BM) and become highly motile and invasive [6]. Of the several ECs that are exposed to angiogenic stimuli, only a small population of cells respond by forming wide circular ring structures called podosomes that are rich in F-actin and cortactin [6, 8]. This subset of ECs, called the tip cells, use these podosomes to degrade the underlying basement membrane and migrate in the direction of the angiogenic stimulus [6, 8]. These tip cells can initiate signaling cascades in another subset of EC, termed stalk cells, to prevent them from sprouting and use them to provide guidance signals for sprout growth away from the parent vessel [6, 9]. Unlike tip cells, stalk cells are proliferative and remain attached to the tip cells and trail behind them [6, 9]. Stalk cells keep dividing and organizing as a stalk within the parent vessel allowing the extension of the new blood vessel [6, 9]. As tip cells and stalk cells coordinate the formation of a new vessel, they form a lumen that then merges with the lumen of the parent vessel to form a new sprout [9]. Blood vessel formation is completed by the production of the BM and lining of the new blood vessel by pericytes and smooth muscle cells that help in the stabilization of the newly formed vessels [2]. 5

22 1.3: Regulators of EC behavior in angiogenesis Angiogenesis is a complex, multistep process that requires the dynamic interaction between ECs and their environment [6, 10]. ECs undergo sprouting by coordinately responding to a variety of growth factors, cell adhesion molecules, and associating with mural cells namely pericytes and vascular smooth muscle cells [7, 11]. Angiogenesis is controlled by the balance between several pro-angiogenic and anti-angiogenic factors [2, 12]. Under pathological conditions, as well as in cancer, there is persistent imbalance between these factors that leads to the formation of abnormal vasculature [12]. Understanding the different molecules and their signaling components that balance this process is required and will be briefly discussed in this section. Under normal conditions, quiescent ECs are arranged as a monolayer, in which the ECs remain closely attached to neighboring cells through junctional molecules such as VEcadherin and claudins [1]. Because ECs are also exposed to varying oxygen levels in the blood, they express oxygen sensors and hypoxia sensors namely prolyl hydroxylase domain 2 (PHD2) and hypoxia-inducible factor-2α (HIF-2α) that allow them to function under varying oxygen levels [1]. Moreover, pericytes that stabilize the vasculature prevent EC proliferation under steady state conditions by secreting survival signals which include VEGF and angiopoietin-1 (Ang1) [1]. Several proteins that activate angiogenesis include angiogenin, epidermal growth factor (EGF), estrogen, interleukin 8, prostaglandin E1 and E2, tumor necrosis factor- α (TNF- 6

23 α), and granulocyte colony-stimulating factor, Ang2, and FGFs [1, 13]. However, Vascular endothelial growth factor (VEGF) and its receptors are the most critical stimulators of the angiogenic process [13] and are known to regulate EC migration, proliferation, EC assembly and angiogenic remodeling [14]. These receptors, which are the main topic of this work, will be dealt with in detail in the next section. Upon angiogenic stimulation from factors mentioned above, pericytes that are present on the surface of blood vessels detach from the underlying BM through proteolytic degradation mediated by matrix metalloproteinase (MMPs) and through the action of podosomes formed by the ECs [1, 8]. Following this, vessels dilate and VEGF loosens contacts between adjacent ECs, thereby increasing vascular permeability to plasma proteins [1, 15]. These leaked plasma proteins further serve to provide angiogenic signals to the ECs [1, 15]. Integrins present in this ECM provides means for EC adhesion and migration during this process [1, 15]. ECs can also secrete proteases that provide guidance and support to the sprouting ECs [15]. These proteases also promote release of angiogenic signals embedded in the ECM such as bfgf, VEGF, TGF-β, which further enhance the angiogenic response [1, 15]. Angiogenesis also requires the specification of tip cells that function at the angiogenic front and stalk cells that trail behind them. Specification of tip cells occurs in the presence of VEGFRs, neuropilins (Nrps), and the notch ligands DLL4 and JAGGED1 [1], while stalk cells proliferate to form the stalk of the vessel in the presence of NOTCH, WNTs, placental growth factor (PlGF), and FGFs [1]. As discussed earlier, tip cells 7

24 migrate in the direction of the angiogenic stimulus and this directional migration is brought about by several guidance cues like ephrins and semaphorins [1]. Completion of new blood vessel formation is not complete until pericytes stabilize new sprouts. This is brought about by several factors like the platelet-derived growth factor B (PDGF-B), Ang1, TGF-β, Eph-B2, and NOTCH [1]. Finally secretion of protease inhibitors facilitates deposition of new BM and reestablishment of the cell-cell contacts, completing the process of angiogenesis. Just as proangiogenic factors promote growth of new blood vessels, several antiangiogenic factors are required to ensure that blood vessels develop with the right density [16]. It has also been suggested that anti-angiogenic factors are necessary to maintain vasculature quiescence at steady state, and also to inhibit transitory angiogenic events under physiological conditions like wound healing [17]. Several protein fragments deriving from the ECM or the BM have been documented to function as anti-angiogenic factors [18]. Some of the matrix-derived inhibitors include arresten, canstatin, collagen fragments, endostatin, fibronectin fragments, tumstatin, thrombospondin1 and 2. Several growth factors and cytokines can also serve to inhibit angiogenesis such as interferons, interleukins, and platelet factor 4. [7, 13, 18]. Recently, vasohibin, derived from the endothelium was found to function as an endogenous inhibitor serving as a feedback regulator of angiogenesis [7]. 8

25 1.4: VEGF and VEGFR in angiogenesis Of all the different positive regulators discussed in the previous section, VEGFs and their corresponding receptors, VEGFRs, are the best understood inducers of angiogenesis. VEGFs comprise a family of secreted, dimeric glycoproteins [19]. In mammals there are several members belonging to this family of growth factors. This includes VEGF-A, VEGF-B, VEGF-C, VEGF-D, endocrine gland VEGF (EG-VEGF), VEGF-E, VEGF-F, VEGF-b, and placental growth factor (PlGF) (Figure 2). These different members serve to regulate several developmental activities. VEGF-A, also known as VEGF, is the principal regulator of vasculogenesis and angiogenesis [10, 19, 20]. In vivo studies have shown that homozygous null VEGF mice die embryonically due to severe impairment in blood vessel formation [21], suggesting the importance of VEGF in blood vessel formation. VEGF-B is known to play a role in cardiac development [22], while VEGF-C and D have been studied to regulate lymphatic vessel development [20, 23]. EG-VEGF, as the name suggests plays a major role in the ECs that line the endocrine glands [20]. VEGF-b, an isoform derived from VEGF gene, has been shown to have anti-angiogenic properties where as PlGF has been mainly studied in association with pathological disorders [20]. VEGF-A also has 4 splice variant forms of which VEGF-165 is known to have to most potent mitogenic activity [20]. VEGF family members mediate their activity by binding in an overlapping manner to three main receptors: VEGF receptors 1, 2, and 3 (VEGFR-1, 2, and 3) (Figure 2). These receptors belong to the receptor tyrosine kinase (RTK) superfamily [19] and share an 9

26 overall similar structure. Each of the three receptors has seven immunoglobulin- (IgG)- like domains forming the extracellular domain of the receptor except for VEGFR-3, which has a disulphide bond instead of the fifth IgG domain [19]. Following the extracellular domain is a single transmembrane domain, a tyrosine kinase domain that is split by an kinase insert domain, and finally the C-terminal tail [19] (Figure 2). Functionally, VEGFR1 has been shown to positively regulate monocyte and macrophage migration and negatively regulate angiogenesis through VEGFR2 signaling [19]. VEGFR2 is known to be the key regulator of EC function, in both physiological and pathological angiogenesis. Importance of VEGFR2 signaling in blood vessel formation was demonstrated in in vivo studies where mice deficient in VEGFR2 gene died embryonically due to lack of blood island formation and vasculogenesis [24]. VEGFR3 on the other hand is important for development of ECs of the lymphatic system [19]. VEGF, which is the principal stimulator of angiogenesis, can bind to either VEGFR-1 or VEGFR-2 [20]. However, VEGF can stimulate angiogenesis most prominently through signaling downstream of VEGFR2 [1]. Binding of VEGF to VEGFR2 causes receptor dimerization that leads to strong autophosphorylation of VEGFR2 at key tyrosine (Y) sites (Figure 3). These phosphorylated residues in turn provide binding sites for downstream signaling molecules that regulate VEGFR2 function [20]. Of the several phospho-tyrosine residues, Y1059 and Y1054 are required for the maximal kinase activity of VEGFR2 [19, 20, 25]. Y1175 is the most important since this residue provides a binding site for several downstream signaling molecules that regulate EC proliferation, migration, and survival that are necessary for blood vessel formation (Figure 3). 10

27 Interestingly, in vivo studies have shown that mice expressing a mutation of tyrosine (Y) to phenylalanine (Phe) at 1175 residue, died due to vascular defects [26]. Moreover these vascular defects observed were similar to those observed in VEGFR2 null mice, suggesting that signaling through just Y1175 is central to vascular development. Mechanistically, Y1175 serves as a docking site for phospholipase C-γ (PLC-γ), which activates mitogen activated protein kinase (MAPK) and is responsible for regulating EC proliferation [20, 27] (Figure 3). PLC-γ can also lead to activation of protein kinase C (PKC), which also plays a role in mediating angiogenic responses [20]. Phosphatidylinositol 3 kinase (PI3K) can also be activated through Y1175 residue and this, in turn, activates the Akt/PKB pathway to regulate EC survival and vascular permeability through endothelial nitric oxide synthase (enos) [19, 20, 28-30] (Figure 3). In addition to the numerous signaling functions of Y1175, phosphorylation at Y951 opens a binding site for T-cell specific adaptor (TsAD), which can interact with Src to stimulate endothelial migration and vascular permeability [20, 31] (Figure 3). Thus, binding of VEGF to VEGFR2 and subsequent downstream signaling cascades thereby initiated represents a central component regulating vascular development and angiogenesis. 11

28 Figure 2: Structure of VEGF and VEGFRs VEGF and VEGFRs are the principle regulators of blood vessel formation. There are six members belonging to the family of VEGFs, VEGFA, B, C, D, E, and PlGF. These can bind in an overlapping manner to either VEGFR1, 2, or 3. All the VEGFRs have a similar structure. The extracellular domain is made up of seven IgG-like domains except for VEGFR2 where the 5 th domain is replaced by a disulphide bond. A transmembrane domain follows the extracellular domain, and two kinase domains (kinase domain I and 12

29 II) that are interrupted by a kinase insert domain. Finally, kinase insert domain II is followed by C-terminal domain. 13

30 Figure 3: VEGFR2 downstream signaling VEGFR2 signaling plays an important role in vasculogenesis and angiogenesis. VEGFA (or VEGF) binding leads to VEGFR2 dimerization that stimulates autophosphorylation of several tyrosine (Y) residues. Shown above are the key residues that are important in VEGFR2 signaling in blood vessel formation. As highlighted in the figure in red, phosphorylation of Y1175 is the most important since it allows binding of several downstream signaling molecules that function to regulate EC migration, survival, proliferation, and vascular permeability. 14

31 1.5: Receptor endocytosis and its importance in RTK signaling After discussing the importance of VEGFR2 signaling in angiogenesis, I will now discuss how membrane bound receptors, including VEGFR2, are regulated by the process of endocytosis. Receptors present on the cell surface respond to variety of environmental signals. Once activated, these receptors mediate physiological output by activating several other downstream cascades [32]. It is now clear that receptor signaling is not restricted to the PM, but in many cases accompanies receptor internalization and entry into the endocytic pathway [32, 33]. Endocytosis involves the sorting and trafficking of receptors to various endosomal compartments [33]. Although it was initially thought to function only as a means of signal attenuation, it is now accepted that endocytosis can serve as a platform for active downstream signaling and also to regulate levels of receptor and ligand expression at the cell surface [32, 33]. Several physiological abnormalities are caused by defects in receptor endocytosis and trafficking, including improper processing of amyloid precursor protein (APP) [32, 34], anti-inflammatory response [32], several developmental abnormalities and even cancer [35]. There are multiple modes of endocytosis, and these are dictated by the cargo that is being internalized [36]. Two major pathways of internalizing material from the environment are phagocytosis and pinocytosis [36]. Phagocytosis involves the internalization of large particles mainly by using the actin cytoskeleton [36]. It occurs in specialized cells like 15

32 macrophages, monocytes, and neutrophils [37]. In contrast, pinocytosis refers to the uptake of fluids and nutrients from the extracellular medium [36]. Pinocytosis can further be grouped into four mechanisms- clathrin dependent endocytosis (CDE), caveolinmediated endocytosis (CME), micropinocytosis, and dynamin-and clathrin-independent endocytosis [36]. CDE is essentially seen in every cell kind but CME has been shown to play a role in endocytosis of activated receptors only in a few cell types [36]. Because CDE and CME play a role in transmembrane receptor endocytosis, they will be described in detail below. CDE is a common mode of endocytosis that occurs constitutively in all mammalian cells. It has been shown that RTKs undergo rapid endocytosis via CDE upon activation [33]. Moreover, it has been shown that CDE plays a role in tissue and organ development throughout the life of the organism and has the ability to modulate signal transduction by regulating both receptor levels and stability [37]. In CDE, activated transmembrane receptors and their ligands are concentrated into specialized regions in the PM called coated pits [37] (Figure 4). These pits are formed by assembly of the cytosolic coat protein clathrin [37]. Clathrin is a protein made up of three heavy chains, and each heavy chain is tightly associated with a light chain [37]. Clathrin cannot by itself bind to the membrane or the receptor but does so with the help of adaptor proteins (APs) and accessory proteins. AP2 is the prototypical clathrin adaptor protein complex [38, 39]. It is a tetrameric complex made up of small (σ2), medium (µ2) and large (α and β2) subunits [39]. In addition to binding clathrin, these different subunits have other functions, such as recognizing sorting and internalization signals on cargo (cargo selection), membrane 16

33 binding, and binding accessory proteins [39]. The combination of AP2 and accessory proteins is responsible for selecting cargo during endocytosis [40]. After the initial binding of clathrin, AP2, and the cargo-specific accessory protein to the receptor at PM, clathrin polymerization continues to form a clathrin coated vesicle (CCV) structure [40-42]. Clathrin polymerization eventually leads to the deformation of the attached membrane and formation of the vesicle neck [41]. Finally, the large GTPase dynamin hydrolyzes GTP to promote membrane scission near the vesicle neck, thereby releasing the CCV with the receptor contained in it inside of the cell [37, 41, 42]. During CME, receptor internalization occurs through structures called caveolae (Figure 4). These are flask-shaped invaginations of the PM, and are most prominent in several cell types such as smooth-muscle cells, fibroblasts, ECs, and adipocytes [37, 43]. The membrane proteins responsible for caveolae formation are called caveolins [43]. Mammalian cells express three caveolin proteins, caveolin 1-3 (CAV1-3). Of these, CAV1 and CAV3 are important in the formation of caveolae [43]. Caveolins are dimeric proteins that bind to cholesterol and do so by inserting themselves as a loop into the inner leaflet of the PM [37]. Within the PM, caveolins then self-associate to form a caveolin coat at the PM [37]. Endocytosis of the activated receptor complex through the caveolae involves invagination of the caveolae from the PM using dynamin GTPase activity [44]. Receptor-containing caveolae bud from the plasma membrane and are subsequently released into the cytoplasm. 17

34 Once inside the cell, CCVs are first uncoated and then fuse with early endosomes (EE) [45]. Cargo contained in budded caveolae can also fuse with either caveosomes or EE [43] (Figure 4). EE are membranous structures that have a tubular morphology and are located in the cell periphery [46]. These organelles are rich in phosphatidylinositol-3- phosphate (PtdIns3P) and proteins like early endosome antigen-1 (EEA-1), and Rab5 that can bind the incoming cargo and regulate its trafficking and activity within the cell [45, 46]. From EEs, cargo can be redirected to the Rab-11 positive recycling endosomes (RE) to be trafficked back to the cell surface or may be routed to multivesicular endosomes or late endosomes (LE) for lysosomal degradation [45] (Figure 4). 18

35 Figure 4: Clathrin dependent endocytosis (CDE) and caveolin mediated endocytosis (CME) Receptors following activation undergo rapid internalization via either CDE or CME. In CME, activated receptors are present in coated pits, which are formed by the cytosolic protein clathrin. Clathrin forms a coat around the activated receptor in association with adaptor protein, AP2, and accessory proteins. Such clathrin-coated vesicles (CCVs) are pinched from the PM to the cell s interior. In CME, the activated receptor is contained in flask shaped invaginations seen at the PM. These are formed by the protein caveolin. Receptors lodged in these caveolin-coated vesicles are then internalized within the cells. 19

36 Once inside the cell, the cargo in first delivered to the EE from where it gets sorted to the RE for recycling back to the PM or to the late endosomes for degradation. 20

37 1.6: VEGFR2 endocytosis As discussed in the previous section, several membrane receptors undergo rapid endocytosis upon activation. Endosomes with their small volumes provide a conducive environment for protein-protein interactions, assembly of signaling complexes and a vehicle for rapid transportation to various intracellular destinations [47]. Endocytosis of VEGFR2 has also been shown to be important in regulating VEGFR2 signaling and blood vessel formation [48] [49]. In this section we discuss different aspects of VEGFR2 endocytosis. Unlike most RTKs that undergo rapid internalization after ligand stimulation, subsets of receptors (i.e. T-cell receptor, epidermal growth factor receptor (EGFR), and VEGFR2) have been shown to undergo constitutive recycling even in the absence of VEGF stimulation [47, 50]. Thus, at steady state in ECs, inactive VEGFR2 is distributed between the PM and an endocytic storage compartment, mainly EEs and a Rab4 positive RE [50]. It is from these compartments that VEGFR2 is transported continuously to the PM [50]. Ligand mediated stimulation and subsequent endocytosis of VEGFR2 is important for maximizing the amplitude of kinase activation of this receptor [50]. Ligand binding to VEGFR2 is followed by its dimerization and autophosphorylation of key tyrosine (Y) residues. VEGFR2 activated in this way is then internalized by either the CDE [51] or by CME [52]. Under normal conditions, the clathrin-mediated, dynamindependent pathway is the prevalent mode of internalization of VEGFR2 [47, 51, 53]. VEGFR2 has several binding partners at the PM that aid in its internalization and 21

38 signaling. These include a wide variety of proteins, including VEGFR1/3, Nrp-1 [54], Eph B2 ligand [55], adhesion molecules (i.e. VE-cadherin [50] and integrins) and components of the ECM (i.e. fibronectin and collagen [47]) that function my enhancing ligand binding to the receptor. Similarly, on the cytoplasmic side, VEGFR2 may be associated with a variety of protein tyrosine phosphatases (s) including VE-PTP, PTP1b, DEP1-/CD148, and TCPTP [47], all of which regulate VEGFR2 endocytosis and signaling [47]. Once activated, VEGFR2 is internalized via CDE and is initially trafficked to EEs or the sorting compartment. Trafficking of VEGFR2 to the EE has been shown to depend on Rab5a, a Ras-related GTPase that regulates vesicle fusion with the EE and is thus abundantly found residing on that compartment [56]. In addition, recent studies have shown that synectin, a scaffold protein, binds the retrograde motor protein myosin VI (MyoVI) and this complex is necessary to carry activated VEGFR2 from the plasma membrane to the EE [48]. In the absence of this assisted transport by synectin-myovi, phosphorylated VEGFR2 fails to traffic to the EE and in the process becomes dephosphorylated at Y1175 through the action of the PTP1b phosphatases [48]. Also, recent work has shown that Nrp1 is necessary to connect phosphorylated VEGFR2 to the synectin-myovi machinery to carry the cargo to the EE to activate downstream ERK signaling [49, 54]. From the EE, VEGFR2 may be recycled to the PM in a Rab4- and Rab11-dependent manner [57]. VEGFR2 may also be recycled in association with Nrp1 from Rab11 associated vesicles [58, 59]. Studies by Gampel et al have also shown that 22

39 upon VEGF stimulation, VEGFR2 and Src are uniquely recycled to the PM in a pathway that requires Src activation [50]. In addition to these recycling pathways, VEGFR2 may be targeted for degradation, and this pathway is important to limit the sensitization of ECs to VEGF stimulation [60]. Studies have shown that upon activation VEGFR2 is ubiquitinated and targeted to LE and subsequently to lysosomes for degradation [61]. Research has also shown that this degradative pathway is Rab7-dependent [59], and that sorting in the LE is important for VEGFR2 signaling and EC function [56]. VEGFR2 may also be misrouted to the Rab7 positive LE when it is endocytosed in the absence of Nrp1 [59]. Thus, VEGFR2 has multiple fates along the endocytic route that influence VEGFR2 function and activity to finally affect blood vessel formation. 23

40 1.7: Disabled protein 2 structure and function CME is one of the most common modes of receptor internalization, and activated VEGFR2 follows this route into cells [50]. CME requires the coordination of several molecular events that lead to the formation of a CCV containing the activated receptor [62]. Adaptor proteins are essential to this process. These adaptor proteins select receptors to be internalized based on internalization signals present on the cytoplasmic tail [39]. Adaptor proteins simultaneously bind to clathrin and the membrane, via phosphatidylinositol (4, 5)-bisphosphate (Ptdlns (4, 5) P2) [38, 39]. In addition to these binding sites, the prototypical adaptor protein AP2 also carries sites that bind several accessory proteins that regulate formation and budding of CCVs [39]. Certain cargoes use alternative mechanisms, such as monomeric adaptor proteins called clathrinassociated sorting proteins (CLASPs) [38, 39]. Disabled protein 2 (Dab2) is one such CLASP that is known to play a role in CME in several cell types [38, 39]. Dab2 is a multivalent scaffolding protein, with separate binding sites for the cargo internalization signal FxNPxY, PtdIns (4,5) P2, AP2 and clathrin [38] (Figure 5). It also carries five asparagine-proline-phenylalanine (NPF) motif (Figure 5), two aspartic acidproline-phenylalanine (DPF), a PTB domain at its N terminus, and a binding site for Myo VI that is known to participate in endocytosis [38] (Figure 5). With such functionally important binding sites, Dab2 has been shown to play a role in the endocytosis of receptors in a cargo selective manner and has been shown to do so even in the absence of the AP2 complex [63]. It has been demonstrated to function in endocytosis of LDLR 24

41 [64], CFTR [65], β-integrin [39], megalin [38], and TGFβ receptor [66]. Dab2 is known to bind to CIN85, Myo VI, and Grb2, further supporting its role in endocytosis of a wide variety of cargo [39, 67-69]. Additionally, Dab2 expression is required for visceral endoderm formation, cell differentiation and also functions as a tumor suppressor [64, 67]. However, interesting to this work is the study done by Cheong et al., where they showed that Dab2 played a role blood vessel formation in the Xenopus early embryogenesis [70]. Recent work by Nakayama et al., demonstrated that Dab2, in a complex with polarity protein Par3 and ehprin B (EphB), functions to regulate VEGFR endocytosis and that the activity of this protein complex is regulated by atypical protein kinase C (apkc) [71]. However, the exact step at which Dab2 influences endocytic trafficking of VEGFR2 to affect blood vessel formation remains unknown. This work was thus performed to understand the exact step at which Dab2 influences VEGFR2 endocytosis and trafficking to affect its signaling and function. 25

42 Figure 5: Structure of Dab2 protein Dab2 is a clathrin associated sorting protein (CLASP) that is known to play a role in CDE. Dab2 has a multidomain structure. As seen here, Dab2 carried FXNPXY, PTB, and PRD domains with which it can selectively bind to cargo. It also has clathrin binding domains with which it interacts with clathrin and helps in the formation of the clathrin coat. Dab2 is also known to interact with the motor protein myosin VI (MyoVI), a retrograde motor protein. 26

43 1.8: Research goals Angiogenesis is an important biological process in adults, in which new blood vessels are formed from the pre-existing vessels. While angiogenesis is required for normal physiological processes such as wound healing, it is also known to be important during the pathogenesis of several diseases. As such, inadequate angiogenesis that leads to inadequate vessel maintenance and growth is known to cause ischemia in diseases like myocardial infarction, stroke, and neurodegenerative and obesity-related disorders [5]. On the other hand, excessive growth of blood vessels or inefficient remodeling of blood vessels through angiogenesis can lead to cancer, inflammatory diseases and eye disorders [5]. Studies have also shown that the cancer cells can take over the host s angiogenic machinery and utilize it to supply nutrients and oxygen to the growing cancer cells [5] allowing the cells to metastasize. ECs that line the blood vessel are the critical players of this process. As described in section 1.1, ECs undergo a highly complex yet tightly regulated differentiation program leading to formation of new blood vessels [10]. ECs mediate this process by responding to several pro-angiogenic stimuli, the activity of which is counterbalanced by antiangiogenic factors that leads to the formation of normal vasculature [12]. Of the different angiogenic stimulators, VEGF binding VEGFR2 to stimulate downstream signaling has been shown to be the most important signaling event during angiogenesis [1, 20, 24, 26]. As reviewed in Section 1.5, binding of VEGF to VEGFR2 leads to its dimerization and phosphorylation at tyrosine residues, specifically Y1175, that is known to be critical for 27

44 angiogenesis [26]. Moreover, binding of VEGF to VEGFR2 triggers its rapid internalization via the clathrin-mediated pathway and trafficking through the endosomes. This endocytic trafficking of VEGFR2 has been demonstrated to play a role in downstream VEGFR2 signaling to regulate angiogenesis [48-50]. Dab2, a CLASP, that is known to function as a cargo-selective accessory protein in CME, has been shown to play a role in blood vessel formation [70]. Specifically, formation of intersomitic veins (ISVs) which arise from the process of angiogenesis was inhibited in Xenopus that lacked Dab2 expression [70]. Moreover, recent studies have shown that Dab2 in a complex with polarity protein PAR3 and ehprin B (EphB) functions to regulate VEGFR endocytosis [71]. However, the exact step at which Dab2 influences VEGFR2 trafficking within the cell still remains unclear. Thus, in this work I concentrated on testing how Dab2 regulates the movement of internalized VEGFR2 through the different endocytic destinations. In this work I first studied if Dab2 played a role in the internalization and trafficking to the EE and saw that upon VEGF stimulation VEGFR2 and Dab2 colocalize with each other in the EE. Moreover I show that Dab2 coimmunoprecipitates with VEGFR2 further explaining the colocalization of VEGFR2 and Dab2 in the EE. To understand the role of Dab2 in VEGFR2 signaling, I utilized lentiviruses to knockdown endogenous Dab2 function and tested its effect on VEGFR2 phosphorylation at Y1175 residue upon VEGF stimulation. This study revealed that in the absence of Dab2 there is a reduction in VEGFR2 phosphorylation at Y1175 in Dab2 knockdown cells (shdab2) cells as compared to the control cells (wt) over the entire time course of VEGF stimulation. This is significant because phosphorylation of VEGFR2-Y1175 is crucial for pro-angiogenic 28

45 signal transduction. Further analysis of the role of Dab2 on VEGFR2 trafficking showed that in the absence of Dab2 there is an increased trafficking of VEGFR2 to the LE suggesting that Dab2 may play a role in the post-endocytic trafficking of VEGFR2. Finally, this defect in post-endocytic trafficking impairs VEGFR2 signaling function as seen as the inability of endothelial cells (ECs) to undergo morphogenesis in an in vitro assay. Thus, our results show that Dab2 is required for the proper sorting of VEGFR2 within the endocytic pathway. Endocytosis was initially thought to be a process that was functionally separate from receptor signaling, and was considered to serve only to inhibit signaling [32, 33]. However it is now known that endocytosis can regulate receptor signaling by regulating signal down regulation, maintenance, and even allow crosstalk [33]. Thus, the improved understanding of VEGFR2 endocytosis provided by this study will help us understand how trafficking can impact VEGFR2 signaling and function in blood vessel formation. This improved understanding can also provide new therapeutic targets to treat angiogenesis-related diseases such as cancer and ischemia. 29

46 CHAPTER 2 Role of Dab2 in VEGFR2 trafficking 2.1: Abstract Angiogenesis is a crucial process under both physiological and pathological conditions. Vascular endothelial growth factor (VEGF) A and its cognate receptor, vascular endothelial growth factor receptor 2 (VEGFR2) are key regulators of angiogenesis. Plasma membrane (PM) levels of VEGFR2 are regulated by de novo synthesis, and by both exocytic and endocytic trafficking. VEGF-binding to VEGFR2 induces phosphorylation of key tyrosine residues located in the cytosolic domain of the receptor, followed by clathrin-mediated endocytosis and signal transduction leading to vascular morphogenesis. Disabled protein 2 (Dab2) is a cytosolic, clathrin-adaptor protein that is known to regulate endocytosis of certain cell surface receptors. Studies of Dab2 function have revealed its role in the development of embryonic vasculature. However, the mechanism of Dab2 function, particularly in conjunction with endosomal VEGFR2, remains poorly understood. Our results show that Dab2 interacts with VEGFR2 and that upon VEGF stimulation the two proteins co-localize within Rab5-positive early endosomes. Knockdown of Dab2 reduces levels of VEGF-induced phosphorylation of VEGFR2 at residue Y1175. This is significant because phosphorylation of VEGFR2- Y1175 is crucial for pro-angiogenic signal transduction. Moreover, knockdown of Dab2 causes an increased trafficking of VEGFR2 to late endosomes. Finally, this altered VEGFR2 trafficking following Dab2 knockdown suggests having major functional consequences for endothelial cells, as they are unable to undergo morphogenesis into 30

47 tube-like structures in an in vitro assay of angiogenesis. Collectively, our data show that Dab2 plays a crucial role in VEGFR2 post-endocytic trafficking and that this trafficking may be important for VEGFR2 signaling and function during angiogenesis. 31

48 2.2: Introduction Blood vessels that carry oxygen and nutrients throughout the body are crucial for organ development and repair [72]. Blood vessel formation occurs in two distinct phases. The first phase occurs during embryonic development by a process known as vasculogenesis, during which endothelial precursor cells undergo differentiation to form a primitive network [2]. In the next phase of development, known as angiogenesis, this preexisting primitive network undergoes expansion and remodeling to form mature blood vessels [2]. Angiogenesis is a process that continues throughout adult life and provides blood supply for physiological processes such as wound healing and tissue regeneration, and supports aberrant growth of tissues in diseases such as cancer [3]. Angiogenesis is an intricate process that requires dynamic and regulated behavior of endothelial cells (ECs) [6]. Quiescent ECs respond to angiogenic signals and undergo proliferation, migration, and morphogenesis to give rise to new blood vessels [6]. Quiescent ECs respond to angiogenic signals and undergo proliferation, migration, and morphogenesis to give rise to new blood vessels [6]. ECs respond to diverse angiogenic cues, but vascular endothelial growth factors (VEGFs) are the principal regulators of EC function during angiogenesis [19]. There are five different splice variants of VEGF ligand, VEGFA, B, C, D and placenta growth factor (PLGF)[19]. These ligands bind in an overlapping fashion to their cognate receptors known as VEGF receptor-1, -2 and -3 (VEGFR1-3)[19]. Of these different ligand-receptor combinations, VEGFA binding to VEGFR2 has been shown to have the most angiogenic potential regulating EC behavior 32

49 during blood vessel development [6]. VEGFR2 belongs to the family of RTKs and shares several regulatory mechanisms with them [19]. VEGFA binding to VEGFR2 is accompanied by receptor dimerization and activation leading to autophosphorylation of several tyrosine residues [6]. These phosphorylated residues serve as binding sites for several downstream signaling proteins that in turn regulate angiogenesis [19]. Of the different phosphorylated tyrosine residues, signaling downstream of tyrosine 1175 (Y1175) has been shown to be most important during blood vessel development. Knockin mice carrying a mutation in this residue die due to vascular defects [26]. Ligand-mediated activation and downstream signaling through these tyrosine residues is also accompanied by internalization and endocytic trafficking of activated receptors [46]. Endocytic trafficking of signaling receptors involves multiple organelles, including PM and several cytoplasmic compartments [36]. First, activated receptors are internalized through either a clathrin-mediated pathway (CME) or a clathrin-independent pathway (CIE). Regardless of the mode of entry, endocytosed cargo merges in early endosomes (EEs), from where receptors can be recycled back to the PM either directly or via recycling endosomes (REs) [32] or be delivered to late endosomes (LE) / lysosomes for degradation. Endocytic trafficking of receptors plays an important regulatory role in signal transduction by controlling signal attenuation, amplitude and/or duration of downstream signaling. Ultimately, this controls the amplitude of the biological response [49]. Prior studies on endocytic trafficking of VEGFR2 have revealed that this receptor undergoes constitutive recycling even in the absence of stimulating ligand [47, 58]. Upon stimulation, VEGFR2 is internalized via CME and is first trafficked to EE, a step critical 33

50 for activation of downstream signaling proteins [58]. From the EE compartment, VEGFR2 is sorted either to LE for signal attenuation or to RE for return to the PM [58]. Because it is known that endocytosis of VEGFR2 is important for its downstream signaling and angiogenesis [49, 73], it is crucial to understand how different proteins that function along the endocytic route may influence its activity. Studies by Lanahan et al., have shown that a protein complex containing synectin and myosin VI functions in trafficking of activated VEGFR2 and protects phosphorylated Y1175 from dephosphorylation, thereby stimulating blood vessel formation [48]. Also, recent work by Nakayama et al., shows that angiogenesis requires temporally and spatially regulated VEGFR endocytosis. They showed that Dab2, a clathrin-associated sorting protein (CLASP), is in a complex with the polarity protein Par3, ehprin-b2 and atypical protein kinase C (apkc), which functions to regulate VEGFR endocytosis [71]. While it is known that Dab2 plays a role in cargo-selective endocytosis [63], and blood vessel formation [70, 71], the exact step(s) at which Dab2 influences endocytic trafficking and activity of VEGFR2 remains unknown. In this study we show that Dab2 interacts with VEGFR2 and that the two proteins colocalize in EEs. Suppression of Dab2 expression alters VEGFR2 trafficking by misrouting it to LEs, where it colocalizes with the LAMP2. These results indicate that in the absence of Dab2, VEGFR2 is misrouted to the degradative pathway. Finally, this misrouting reduces VEGFR2 signaling and disrupts the ability of endothelial cells (ECs) to undergo morphogenesis in an in vitro angiogenesis assay. Thus, our results show that 34

51 Dab2 is required for post-endocytic sorting of VEGFR2 following its internalization into EEs. 35

52 2.3: Materials and Methods Reagents: Rabbit monoclonal antibodies against human VEGFR2 (55B11) and phospho- VEGFR2 (Y1175, Y951, Y1205) were purchased from Cell Signaling Technology (Danvers, MA). Purified mouse anti-disabled-2/p96 (#610464), and mouse-anti early endosomal antigen (EEA-1) antibody were purchased from BD Transduction Laboratories. Anti-rabbit Dab2 (H-110) and anti-goat Dab2 (C-20) were purchased from Santa Cruz Biotechnology, Inc. (Dallas, Texas). Monoclonal antibody against lysosomeassociated membrane protein-2 (Lamp-2) was obtained from Developmental Studies Hybridoma Bank at the University of Iowa. Antibodies to α tubulin and α-actin were purchased from Sigma-Aldrich, while that for GAPDH was purchased from EMD Millipore (Massachusetts, USA). Plasmid expressing RFP-tagged Rab5 protein was obtained from Addgene (Cambridge, MA). The ligand VEGF-A 165 (VEGF) was purchased from R&D Systems (Minneapolis, MN). Dab2 was inhibited using short hairpin RNAs (shrnas) delivered to cells using lentiviruses (proprietary sequences from GenTarget Inc., San Diego, CA). X-treme 6 HD transfection reagent, protease inhibitor, and phosphatase inhibitor cocktail tablets were obtained from Roche Diagnostics (Indianapolis, IN). Alexa Fluor-conjugated Abs were obtained from Invitrogen, Molecular Probes. Growth factor-reduced Matrigel for in vitro tube formation assays was purchased from BD Biosciences. Protein G Sepharose 4 Fast flow beads were obtained from GE Healthcare. 36

53 Cell Culture and lentivirus infection: Primary human umbilical vein endothelial cells (HUVECs) (Lonza, Walkersville, MD) were cultured in MCDB131 media supplemented with 7.5% fetal bovine serum (Life Technologies; Grand Island, NY), 25ng/ml endothelial cell growth supplement (Corning; Bedford, MA), 10ng/ml epidermal growth factor, 2 mm L-glutamine (Life Technologies; Grand Island, NY), 1 µg/ml hydrocortisone, and 250 ng/ml fungizone. MCDB131 media was used because it has been shown to be the most effective medium for promoting HUVEC proliferation [74]. Cultured cells were maintained at 37 C in a humidified incubator in the presence of 5% CO 2. Cells were seeded and maintained on Type I collagen-coated (rat-tail collagen) surfaces and used for 3-7 passages. For most of the experimental procedures, HUVECs were serum starved for 3 hours in MCB131 media without any supplements (MCDB131 - /- ) and then stimulated with VEGF-A at a concentration of 50 ng/ml. For lentiviral transduction, HUVECs were grown to 70% confluency, washed and incubated in MCDB131 -/- containing polybrene (8 µg/ml) for 2 hours. Lentiviral vectors ( TU/ml) containing either a Dab2-specific hairpin (shdab2) or a scrambled hairpin control (Scr sh) were added to cultures and incubated for 24 hours. Media was then replaced with growth media and 48 hours later transduced cells were used for experiments. Immunoblotting and Immunoprecipitation: Confluent HUVECs, control and shdab2 cells, were serum starved and treated with VEGF-A. Cells were then washed, mechanically dissociated from plates in PBS, and then centrifuged at 10,000-x g for 10 37

54 minutes to sediment them. Cells were lysed in RIPA buffer (50 mm Tris ph 7.4, 150 mm NaCl, 0.5% sodium deoxycholate, 0.1% SDS, 1% NP-40) supplemented with protease inhibitors and phosphatase inhibitors for 25 minutes on ice. Lysates were centrifuged at 13,000x g for 20 minutes, supernatants were collected, and protein concentrations determined using a DC Protein Assay Kit (Bio-Rad). Equal protein amounts (30 µg) were resolved by SDS-PAGE, and subsequently transferred to a polyvinylidene difluoride membrane. Membranes were blocked with 5% (wt/vol) nonfat milk in TBS containing Tween-20 (TBST) and incubated overnight at 4 0 C with antibodies. After a second round of washing with TBST, membranes were incubated with an HRPconjugated secondary antibody for 1 hour at room temperature. Following a final round of washes, proteins were visualized with enhanced chemiluminescence reagents using a ChemiDocIt instrument (UVP). Intensities of protein bands were quantified using ImageJ 1.42q software. For immunoprecipitation, control cells were serum starved, treated with VEGF and at each time point, cells were lysed and protein was extracted as described above. These lysates were first precleared with protein G sepharose beads and then incubated overnight at 4 0 C with protein G sepharose beads bound to VEGFR2 antibody (1µg antibody/reaction). After overnight incubation, immune complexes were washed in RIPA buffer and then analyzed by SDS-PAGE and immunoblotting. Immunofluorescence and quantification: Cells were grown on coverslips, fixed with 4% paraformaldehyde for 25 minutes at room temperature (RT) and quenched in 100 mm glycine in PBS for 10 minutes at RT. Cells were permeabilized in 0.1% Triton X-100 in PBS for 5 minutes, washed with PBS, and blocked in PBS containing 5% glycine and 5% 38

55 normal goat or donkey serum for 60 minutes at RT. Cells were then incubated with indicated primary antibodies in blocking buffer overnight at 4 0 C, washed with PBS, and then incubated with Alexa Fluor 488-, Alexa Flour 594- or Alexa Fluor 647-conjugated secondary antibodies for 1 hour at RT at a 1:200 dilution. Cells were again washed in PBS and coverslips were mounted onto a slide with Vectashield mounting medium containing DAPI. Fluorescence images were acquired using a Zeiss LSM 700 inverted microscope (Carl Zeiss) with a Plan-Apochromat 63 /1.40 oil objective in the x-, y-, and z- planes using an identical exposure time. Obtained images were processed with ImageJ and MetaMorph image processing software (Molecular Devices Corp., Downingtown, PA). Quantification of colocalization was performed as previously described [75]. Briefly, images of individual cells were first converted into composite images using ImageJ. Merged images representing fluorescence excitation at two or three different wavelengths were then separated into individual channels using MetaMorph software and then the threshold was adjusted such that backgrounds were identically reduced. This threshold was maintained constant for a given experiment. Images were then analyzed for colocalization using the quantitative colocalization function in MetaMorph. The total pixel intensity of the threshold protein 1 that colocalized with protein 2 was displayed as a percentage. This method was applied to 8 cells per condition from 3 separate experiments. 39

56 Tube formation assay: Assays were performed as previously described by Manickam et al., 2011 [76]. Briefly, 5 µl of growth factor reduced Matrigel was applied to the bottom of 8-chambered slides and permitted to solidify at 37 0 C. Serum-starved HUVECs, both control and shdab2, were harvested and resuspended in MCDB131 -/- media and cells were added to the top chamber. Cells were incubated at 37 0 C for 45 minutes, at which time, the media was replaced with MCDB131 -/- media containing VEGF-A (50 ng/ml). After 12 hours of incubation with the VEGF-A, media was aspirated and cells in each chamber were fixed with paraformaldehyde and imaged using Nikon Microphot-FX microscope, with a 5X objective lens. Tube formation was quantified by counting the number of branch points in x fields of view. The experiment was repeated three times independently. Statistical Analysis Statistical significance of data was determined by performing either two-way ANOVA or student s t-test analysis using GraphPad Prism software (GraphPad Software, version 6.0e; San Diego, CA), as indicated in figure legends. For each experiment, a p value of <0.05 was considered significant. 40

57 2.4: Results Dab2 binds VEGFR2 in EEs: Dab2, an endocytic adaptor protein, is important for embryonic angiogenesis [70]. Dab2 binds ephrin-b2 and Par3 and these interactions contribute to VEGFR2 endocytosis and signaling [71]. The precise mechanism by which Dab2 facilitates endocytic trafficking of VEGFR2 is unclear, however. To begin to address this question, I sought to determine the subcellular compartment(s) in which Dab2 and VEGFR2 co-localize. As previously observed, Dab2 appeared as discrete puncta throughout HUVECs [77] (Figure 6). Similarly, VEGFR2 was distributed in puncta throughout cells. There were areas where the two proteins co-localized and these tended to be along cell peripheries as seen in Figure 6, where phalloidin was used to label the cell surface. I next wanted to determine whether this pattern of co-localization changed upon VEGF stimulation and where within the endocytic pathway Dab2 might associate with activated VEGFR2. HUVECs over-expressing an early endosomal marker, RFP-tagged Rab5, were serum starved, stimulated with VEGF, and examined by immunofluorescence. Dab2 co-localized with VEGFR2 in early EEs in the absence of VEGF stimulation, and this colocalization increased during the course of a 60 minutes exposure to the ligand (Figure 7). To determine if Dab2 is physically associated with VEGFR2, I examined coimmunoprecipitation of the two proteins. Dab2 co-precipitated with VEGFR2, even in the absence of ligand (Figure 8). Following VEGF stimulation, Dab2 remained associated with VEGFR2 during the time at which receptors were activated and trafficked through EEs. We note that the level of Dab2 that was associated with VEGFR2 was 41

58 somewhat reduced suggesting that the interaction of Dab2 with activated VEGFR2 might be dynamic. 42

59 Merge Inset VEGFR2 Dab2 Phalloidin Figure 6: Dab2 is localized as discrete punctae in HUVECs HUVECs were cultured on coverslips, fixed, permeabilized, and labeled with antibodies against VEGFR2 and Dab2. Phalloidin dye was used to label cortical actin structures. Representative images are shown. 43

60 Merge Inset VEGFR2 Dab2 Rab5 VEGF stimulation (mins) * * % Rab5 vesicles colocalizing with Dab % VEGFR2 colocalizing % VEGFR2 colocalizing with Dab * VEGF STIMULATION (minutes) VEGF STIMULATION (minutes) VEGF STIMULATION (minutes) Figure 7: Dab2 colocalizes with VEGFR2 in EE upon VEGF stimulation Confluent RFP-Rab5 expressing HUVECs were serum starved for 3 hours and stimulated with VEGF-A. Top panel] At indicated times, cells were fixed, permeabilized and immunostained for VEGFR2 and Dab2. Lower panel] Quantification of the percentage of Rab5 positive compartment colocalizing with VEGFR2 and Dab2, and percentage of VEGFR2 colocalizing with Dab2 was achieved using MetaMorph. Values represent mean ± SD, n=5 cells per time point. Statistical significance for each analysis was done by one way anova test where p<0.05 was considered significant. 44

61 Figure 8: Dab2 co-immunoprecipitates with VEGFR2 Confluent HUVECs were serum starved for 3 hours and stimulated with VEGF-A for indicated times. Cells were lysed and immunoprecipitated with VEGFR2 antibody. Immunoprecipitates were resolved by SDS-PAGE and immunoblotted with antibodies against VEGFR2, phospho-vegfr2 (Y1175), Dab2 or actin. Input protein lysates were immunoblotted as controls. 45

62 Dab2 knockdown decreases levels and phosphorylation of VEGFR2 at Y1175: To better understand the function of Dab2 in VEGFR2 trafficking and signaling, we performed loss-of-function studies. For this, I used lentiviruses expressing an shrna to transiently inhibit Dab2 expression in HUVECs and evaluated Dab2 expression by immunoblotting. Using this approach, we reproducibly inhibited Dab2 expression by ~80%, compared to cells that expressed control Scr sh (Figure 9). I next examined whether Dab2 was required for VEGFR2 signaling and trafficking. In vivo studies have shown that of the different VEGFRs, VEGFR2 and signaling downstream of this receptor is most important for blood vessel formation [24, 26]. Activation of VEGFR2 promotes receptor dimerization and autophosphorylation at key tyrosine residues [19]. Phosphorylation specifically at the Y1175 residue provides binding sites for several downstream signaling proteins that regulate endothelial cell function during angiogenesis [19, 26]. Further, Lanahan et al., showed that phosphorylation of VEGFR2 at Y1175 coupled with its internalization and trafficking away from the PM is crucial for arterial morphogenesis [48]. To understand the role of Dab2 in VEGFR2 signaling, either control or shdab2 HUVECs were stimulated with VEGF and the amount of phosphorylated receptor in lysates was examined using an antibody that recognizes VEGFR2 phosphorylated only at Y1175. As shown in Figure 10, Dab2 knockdown led to decreased VEGFR2 levels and reduced levels of Y1175 phosphorylation upon VEGF stimulation when compared to control cells. To determine whether the observed reduction in Y1175 phosphorylation was due to an overall decrease in receptor levels in shdab2 cells, we calculated the ratio of phospho-y1175 VEGFR2 to 46

63 total VEGFR2 levels. This analysis revealed that upon VEGF stimulation, there is a noticeable reduction in normalized levels of phospho-y1175 VEGFR2 during the entire course of VEGF stimulation in shdab2 cells as compared to control cells. Reduced levels of VEGFR2 phosphorylation are also observed by immunofluorescence (Figure 11). Little activated receptor is observed in cells under basal conditions. Within 5 minutes of VEGF stimulation, a robust PM-associated pool of phospho-y1175 VEGFR2 is observed in control cells, but little if any is seen in shdab2 cells. This may be due to the lower overall receptor expression in these cells, but also the lower normalized level of activated receptors. Phospho-Y1175 VEGFR2 is observed in cytoplasmic vesicles in both control and shdab2 cells, but some differences are noted. In control cells, levels of PM-associated activated receptor decrease as levels of endosomeassociated activated receptor increase between 5 and 30 minutes of stimulation. However, in shdab2 cells, endosome-associated activated receptors are observed at both 5 and 30 minutes of stimulation, but there appears to be a qualitative, rather than a quantitative change in endosome-associated pools. At 5 minutes, activated receptors appear to be in numerous small vesicles, while at 30 minutes they appear to be in larger, more heterogeneously shaped structures. Collectively, these data indicate that Dab2 controls both VEGFR2 trafficking and activation specifically at the Tyr1175 residue 47

64 Figure 9: Dab2- specific shrna construct inhibits Dab2 expression in HUVECs Left panel] HUVECs were transduced with lentiviral vectors encoding either a control scrambled shrna (Scr sh) or a Dab2-specific shrna (shdab2) for 72 hours. Cells were lysed and lysates were immunoblotted with antibodies against Dab2 to show the level of inhibition. GAPDH was used as a loading control. Right panel] Densities of western blot bands were quantified using ImageJ and normalized to those of a loading control (GAPDH). 48

65 Relative Intensity of pvegfr2/vegfr VEGF STIMULATION (minutes) wt sh p value < Figure 10: Dab2 knockdown decreases VEGFR2 phosphorylation at tyrosine 1175 (Y1175) Control and shdab2 cells were serum starved and stimulated with VEGF for indicated times. Right panel] Cells were lysed and lysates were immunoblotted with antibodies against total VEGFR2, phospho-vegfr (Y1175) or GAPDH. Left panel] Quantification of immunoblots was performed by measuring band intensities using ImageJ. Phospho- VEGFR2 (Y1175) levels were normalized to total VEGFR2 levels. Statistical analysis measured by two-way ANOVA from four independent experiments shows that upon VEGF stimulation there is a reduction in VEGFR2 phosphorylation at Y1175 in shdab2 cells as compared to the control cells over the entire time course of VEGF stimulation. 49

66 VEGF (mins) Control VEGF (mins) pvegfr2 (Y1175) shdab2 Figure 11: Dab2 knockdown decreases VEGFR2 phosphorylation at tyrosine 1175 (Y1175) and also alters its localization pattern Control and shdab2 cells were serum starved and stimulated with VEGF for indicated times. Cells were fixed, permeabilized, and processed for immunofluorescence to study the effect of Dab2 knockdown on localization of phospho-vegfr2 (Y1175) upon VEGF stimulation. 50

67 Knockdown of Dab2 causes misrouting of VEGFR2 to late endosomes: Previous studies showed that Dab2 is required for VEGFR2 endocytosis [71]. However, it is unknown whether and where Dab2 is required for post-endocytic trafficking of VEGFR2. To gain insight into trafficking events that require Dab2, I monitored VEGFR2 movement through the endocytic pathway in cells expressing either normal or reduced levels of Dab2. To study the underlying reasons for reduced phosphorylation levels of VEGFR2 in shdab2 cells, I tested whether Dab2 knockdown caused misrouting of VEGFR2 to the LEs for degradation. To analyze this possibility, serum starved control and shdab2 cells were stimulated with VEGF for different lengths of time and the degree to which VEGFR2 colocalized with lysosomal-associated membrane protein-2 (LAMP2) was examined. When compared to control cells, shdab2 cells exhibited an increased colocalization of VEGFR2 with LAMP2 positive LEs 10 minutes after VEGF stimulation, suggesting that Dab2 knockdown leads to misrouting of VEGFR2 to Les (Figure 12 and 13). 51

68 VEGF VEGFR2 LAMP2 DAPI VEGFR2 LAMP2 DAPI Control shdab2 Figure 12: Dab2 knockdown increases trafficking of VEGFR2 to the LE Control and shdab2 cells were serum starved and then stimulated with VEGF for indicated times. Cells were fixed, permeabilized, and then processed for immunofluorescence using antibodies against VEGFR2 and LAMP2 (marker of LEs) and DAPI. Representative images showing colocalization of VEGFR2 with LAMP2. 52

69 * % of VEGFR2 colocalizing in LAMP2 positivecompartment Control shdab VEGF Stimulation (min). 4. Dab2 knockdown increases trafficking of VEGFR2 to the LEs: Control and Figure 13: Quantification showing Dab2 knockdown increases trafficking of VEGFR2 to late endosomes Quantification of Figure 12, which shows the percentage of VEGFR2 colocalizing within LAMP2 positive cellular compartments that was achieved using MetaMorph software. Values represent mean ± SD, n= 8 cells per time point for three independent experiments and p<0.05 as analyzed two way ANOVA. 53

70 Dab2 knockdown impairs the ability of endothelial cells to undergo morphogenesis: We next wanted to test if the alteration in VEGFR2 trafficking that was caused by loss of Dab2 impairs endothelial cell function. Trafficking of VEGFR2 along the endocytic pathway is critical for its role in angiogenesis [49]. We therefore tested if Dab2 knockdown impaired angiogenesis. We monitored in vitro tube formation, an assay that reports endothelial cell function by monitoring the ability of cells to undergo morphogenesis into tube-like structures. For this, control and shdab2 cells were seeded at the same density on growth factor reduced Matrigel. Cells were then stimulated with VEGF and analyzed for their ability to form tube-like structures. As seen in Figure 14, Dab2 knockdown significantly reduced the ability of endothelial cells to undergo morphogenesis into tube like structures. Collectively, these results indicate that in the absence of Dab2, VEGFR2 is trafficked to LEs with faster kinetics than it is in control cells, and that this may affect VEGFR2 downstream function in endothelial cells. 54

71 Avg number of branch points Control Control+ VEGF p= shdab2 +VEGF Figure 14: Dab2 knockdown alters endothelial cell morphogenesis Serum starved control and shdab2 cells were seeded on Matrigel and stimulated with VEGF. After 18 hours of stimulation the ability of these cells to form tube-like structures was analyzed. Top panel] Representative images of control and shdab2 cells grown on the Matrigel. Lower panel] Quantification of tube-like structures was achieved by counting branch points (shown as red spots in A). Values represent mean ± SD, n=3 55

72 different focal planes for each of the 3 independent experiments and p<0.05 as determined by student s t test. 56

73 2.5: Discussion VEGFR2 and its downstream signaling pathways play a central role in the process of blood vessel development. Like other receptor tyrosine kinases, VEGFR2 undergoes autophosphorylation at key tyrosine residues upon ligand stimulation. Signaling following phosphorylation of VEGFR2-Y1175, in particular, has been shown to be especially important for blood vessel formation [26]. Moreover it has been shown that VEGFR2 activation is accompanied by receptor endocytosis and that this is important for downstream activation of the ERK1/2 pathway, which is also known to play an important role in angiogenesis [49]. Endocytosis entails the movement of cargo within endosomes in a highly regulated fashion to control signaling duration, magnitude and outcome [51]. Dab2 is a clathrin-coat adaptor molecule that facilitates endocytosis in a cargo-specific manner [66] and has been shown to play an important role in blood vessel formation [70]. Recent work by Nayakama et al., has shown that Dab2, in a complex with Par3 and ephrin-b2, interacts with VEGFR to regulate its endocytosis [71]. However, the exact step(s) at which Dab2 regulates VEGFR2 trafficking and activity are not entirely clear. Our work shows that Dab2 plays a crucial role in post-endocytic trafficking of VEGFR2, and that in its absence VEGFR2 is misrouted to LEs. Ultimately, loss of Dab2 affects VEGFR2 signaling activity and impairs its function in promoting angiogenesis. In our initial efforts to understand whether Dab2 is involved in VEGFR2 trafficking, we tracked movement of VEGFR2 along the endocytic route upon VEGF stimulation. We observed that Dab2 colocalized with VEGFR2 in the EE (Figure 7) and co- 57

74 immunoprecipitated with phosphorylated VEGFR2 upon ligand stimulation (Figure 8). Association of Dab2 with phosphorylated VEGFR2 was sustained as receptors moved through EEs, although interaction levels were somewhat reduced following 10 mins of VEGF stimulation (Figures. 7 and 8). Previous work has shown a similarly interaction pattern between Dab2 and EGFR. Dab2 was shown to dissociate from EGFR upon EGF stimulation to allow binding of other downstream signaling proteins [50]. Thus, a transient interaction of Dab2 with phosphorylated VEGFR2 might be necessary to allow binding of regulatory proteins to aid in VEGFR2 signaling. Whether this association is lost before or after arriving at EE, and whether Dab2 re-binds VEGFR2 to facilitate postendocytic trafficking following signal transduction, are questions that need to be addressed in order to further understand the role of Dab2 in regulating VEGFR2 signaling and trafficking. As mentioned earlier, signaling of VEGFR2 downstream of Y1175 phosphorylation is important for blood vessel formation [26]. Our present work shows that knockdown of Dab2 is associated with reduced levels of phospho-y1175 VEGFR2 (Figure 10). This suggests that the interaction of Dab2 with VEGFR2 is important for receptor activation and subsequent signaling. Previous work by Lanahan et al. have shown that VEGFR2 requires synectin and the motor protein myosin VI to ferry activated receptors away from the PM in order to prevent their dephosphorylation by phosphatase PTP1b and preserve their activity [48]. Furthermore, it has been shown that VEGFR2 cannot bind the synectin-myosin VI complex directly, but does so through its coreceptor neuropilin-1 (Nrp1) [54]. Nrp1 provides a binding site for VEGFR2-Nrp1 complex to synectin protein 58

75 via its PDZ domain and this facilitates its movement to EEs [54]. Our results show that Dab2 colocalizes with VEGFR2 in Rab5 positive endosomes and that knockdown of Dab2 reduces VEGFR2 phosphorylation at Y1175 residue. It is possible that the reduced phosphorylation levels of VEGFR2 at Y1175 observed upon Dab2 knockdown may be due the phosphatase activity of PTP1B, suggesting that Dab2 may function along with Nrp1-synectin-myosinVI complex in the trafficking of VEGFR2 to the EE while protecting it from the attack of the PTP1b phosphatase. Reduction of Dab2 expression also dramatically reduced total levels of VEGFR2 in HUVECs. Although the reasons for this are not yet understood, a likely explanation is that Dab2 is required for returning a constitutively recycling pool of VEGFR2 to the PM. If Dab2 functions exclusively in regulating VEGFR2 endocytosis, then reducing Dab2 levels should cause an elevated level of PM-associated receptors to accumulate in cells. This is not observed. We suggest that VEGFR2 continues to undergo constitutive endocytosis in non-stimulated shdab2 cells (likely at a reduced rate compared to control cells), but that instead of returning to the PM it is drawn off for degradation in LEs and lysosomes. This would account for both the lower observed VEGFR2 expression levels in non-stimulated shdab2 cells and the much weaker labeling of PM-associated activated receptors at early times following stimulation. If this is the case, it supports our conclusion that Dab2 is required for post-endocytic sorting of VEGFR2. 59

76 In conclusion, Dab2, a clathrin adaptor protein, plays an important role in post-endocytic VEGFR2 trafficking. In the absence of Dab2, VEGFR2 is misrouted to the LEs and this impacts VEGFR2 downstream signaling, and ultimately endothelial cell morphogenesis. 60

77 CHAPTER 3 General Discussion 3.1: Dab2 and VEGFR2 Angiogenesis is the formation of new blood vessels from preexisting vasculature. Angiogenesis is known to play an important role in several physiological processes in adults and is also known to play a role in the pathogenosis of several diseases like cancer. In angiogenesis, ECs that line the blood vessels undergo a highly regulated morphogenetic proceudre to form new blood vessels. ECs mediate this morphogenetic process in response to the angiogenesis stimulating ligand, VEGF that binds to the VEGFR2 receptor. Because it is known that the host s angiogenic machinery is compromised in diseases like cancer, several drugs that target the VEGF and VEGFR are avaliable [1, 7]. However the efficacy of these drugs continues to remain a problem. Thus, understanding the biology of VEGFR2 in angiogenesis at the molecular level will help us find alternate therapeutic targets for diseases like cancer with better efficiancy. Apart from signals through downstream signaling molecules, VEGFR2 is also regulated by the process of endocytosis [49] In this work we show that Dab2, which is a clathrin adaptor protein plays an important role in VEGFR2 post-endocytic trafficking. We show that in the absence of Dab2, there is an increased trafficking of VEGFR2 to the LE upon VEGF stimulation. Thus we suggest that Dab2 may play a critical role in determining the postendocytic fate of VEGFR2 that may ultimately affect the process of angiogenesis. 61

78 Our initial data shows that Dab2 has a diffused distribution throughout the cell and is occasionally observed colocalizing with VEGFR2 at the PM (Figure 6). Following VEGF stimulation, Dab2 colocalizes with VEGFR2 in the EE (Rab5-positive compartment) (Figure 7), the sorting station in the cell from where cargo may be redirected to the LE for degradation or recycled back to the PM. Quantification of colocalization between VEGFR2 and Dab2 shows that even in the absence of VEGF stimulation, both VEGFR2 and Dab2 colocalize with the Rab5 positive EE compartment (Figure 7). Upon 10 minutes of VEGF stimulation, there is an increase in the percentage of VEGFR2 colocalizing with the EE and a simultaenous decrease in Dab2 colocalizing with VEGFR2 in the EE (Figure 7). This reduction in Dab2 association with VEGFR2 at 10 minutes of VEGF stimulation is also obsesrved biochemically, because the amounts of these proteins that co-immunoprecipitate decreases at this time point (Figure 8). Based on these observations, we hypothesize that the association of Dab2 with VEGFR2 at the PM under non-stimulated conditions might provide a mechanism by which the cell prepares itself for immediate response to VEGF stimulation. Upon stimulation, the associated Dab2 might thus help in VEGFR2 clustering and initiation of the formation of clathrin coated pits (CCPs). Following CCP formation and VEGFR2 accumulation in the pits, the release of Dab2 from the complex might be a prerequisite for the downstream proteins to bind to the activated receptor in order to mediate its signaling. A similar transient interaction of Dab2 with EGFR upon EGF stimulation has been previously studied to be a prequisite for for EGFR downstream signaling. 62

79 In this study we performed Dab2 knockdown studies using lentiviruses to understand the role of Dab2 in VEGFR2 signaling,. As shown Figure 10, Dab2 knockdown led to a reduction in the levels of phosphorylated VEGFR2 at Y1175 residue over the entire time course of VEGF stimulation when compared to control cells. Previous work has shown that trafficking of activated VEGFR2 from the PM to the cell s interior by Myo VI motor protein is important to restore VEGFR2 Y1175 phosphorylation from the attack of PTP1b phophstase [48]. Based on this observation, it is possible that Dab2 with its Myo VI binding motif mediates this trafficking and that in its absense phosphovegfr2 (Y1175) is attcked by PTP1b phosphatases leading to the reduction in its phosphorylation at Y1175 residue. Using PTP1b specific inhibitors or PTP1b sirna and studying its effect on phopsho-y1175 VEGFR2 levels in control and shdab2 cells, will help us understand the whether Dab2 mediated traffikcing of VEGFR2 to EE is required to protect phospho- Y1175 VEGFR2 from the action of PTP1b phosphatases. Interestingly, Dab2 knockdown also led to the reduction in the total VEGFR2 levels (Figure 10). One plausible explanation for this is that Dab2 might be involved in regulating VEGFR2 receptor turnover. Since we obsesrved that Dab2 is associated with VEGFR2 even in the absence of the stimulating ligand (Figures 6, 7, 8, 10), we suspect that Dab2 might play a role in mediating the constitutive recycling of VEGFR2 from the EE to PM in the absence of VEGF to ultimately regulate the receptor expression at the PM. In the absence of Dab2 we hypothesize that this recycling of VEGFR2 is impaired leading to its degradation. One approach to answer this hypothesis would be to inhibit protein synthesis using cycloheximide (CHX) in both control and shdab2 cells to monitor receptor stability when Dab2 is present or absent in cells. While this approach was attempted, a conclusive result 63

80 was not obtained since we did not achieve robust Dab2 knockdown (Figure A.1). Using an efficient technique such CRISPR-Cas system to obtain robust and stable Dab2 knockout or isolating endothelial cells from Dab2 knockout mice can help overcome this issue. Alternately Cheong et al., have shown that Dab2 can influence VEGF expression through activin-like signaling pathways [70]. It is possible that Dab2 regulates VEGFR2 expression levels in a similar way [70]. Thus it will be important to study how Dab2, an endocytic adaptor protein, might affect the mrna levels of VEGFR2. We next studied whether the reduction of phosphorylated VEGFR2 was due to trafficking defects caused by Dab2 knockdown. Because we have shown that Dab2 colocalizes with VEGFR2 in the EE upon VEGF stimulation (Figure 7), we wanted to study whether trafficking of VEFR2 was affected in Dab2 knockdown cells. Thus, we performed an experiment to label the surface VEGFR2 with an antibody and then chased it for different lenghts of time after VEGF stimulation to the study the rate at which the cargo (tagged- VEGFR2) was trafficked to the EE in control and shdab2 cells (Figure A.2). Unfortunately, because the antibody used to label VEGFR2 during trafficking is poorly characterized, results obtained with this assay were not sufficiently reproducible to achieve conclusive results. One alternative approach to answer this question is would be perform cell surface biotinylation assay. In this assay, biotinylation of cell surface would biotinylate cell surface proteins including VEGFR2 which in the presence of VEGF ligand can be allowed to undergo internalization. Precipitating this internalized pool of biotinylated VEGFR2 (protected from the quenching action) in both control and shdab2 cells will help us understand the rate at which surface VEGFR2 is internalized into the 64

81 cell. While this approach was attempted (Figure A.3), we could not make a conclusive statement on the rate of VEGFR2 internalization since we failed to obtain efficient Dab2 knockdown. Thus, along with finding ways to efficiently knockdown Dab2 as mentioned above, another approach would be to clone VEGFR2 to carry a fluorescent tag that does not interfere with VEGF binding and that can be expressed in HUVECs to study the kinects of its transport to the EE. Despite the inabiltity to achieve conclusive results regarding the effect of Dab2 knockdown on the rate of VGFR2 internalization, some indication for the role of Dab2 in VEGFR2 internalization can be deduced from data presented in Figure 11 where the both control and shdab2 cells were processed by immunofluorescene to study the localization of phosph- Y1175 VEGFR2 upon VEGF stimulation. As seen in this figure, compared to the control cells, in shdab2 cells there is no sustained signal of phospho-y1175 VEGFR2 signal at the cell periphery but rather phospho VEGFR2 (Y1175) appears as punctae within the cell s interior and that with 60 minutes of VEGF stimulation, these puncate get larger in size. This suggests in the absence of Dab2, activated VEGFR2 is internalized at a faster rate as compared to the control cells and the increased size of punctae with time suggests that cells are impaired in their ability to sort the incoming cargo. This observation is however in contrast to work of Nakayama et al., in which they showed that Dab2 knockdown led to an inhibition of VEGFR2 internalization [34]. Several methodologial differences between our studies and those of Nakayama et al., might explain this discerepancy. First, our study uses lentiviral vectors to knockodown endogenous Dab2 protein. Lentivirus-mediated knockdown gave us <100% knockdown 65

82 (~80%), allowing 20% of Dab2 to mediate normal function. Previous work has also shown that depending on its cellular level, Dab2 can selectively choose the mode of cargo internalization and can switch from either the caveolin or the clahtrin mediated pathway [67]. Therefore it is possible that under low levels of Dab2, VEGFR2 gets internalized via the CME. Finding out the involvement of Dab2 in clustering VEGFR2 in caveolin rich membranes to mediate CME, will help us gain further knowledge about the role of Dab2 in the process of endocytosis. Another explanation for the unhindered internalization is the ability of the cell to compensate for the loss of Dab2 by using other adaptor proteins like autosomal recessive hypercholestrolemia (ARH), to mediate VEGFR2 internalization. Therefore, studies of cells in which expression of both ARH and Dab2 are both silenced should help us understand the role of Dab2 in VEGFR2 internalization [78]. To test the idea whether Dab2 knockdown affects post-endocytic trafficking, allowing VEGFR2 misrouting it to the LE, we performed immunofluorescence assays. As seen in Figures 12 and 13, we observed that in shdab2 cells there was an increase in the trafficking of VEGFR2 to the LE at 10 minutes of VEGF stimulation as compared to the control cells. This suggests that Dab2 is important for VEGFR2 post-endocytic trafficking. While this observation is primarily based on the immunofluorescence data, it will be ideal to show the same effect of Dab2 knockdown by performing some biochemical analysis to study the accumulation of VEGFR2 in control and shdab2 cells in the presence of lysosomal enzyme inhibitors. 66

83 Finally, we have shown that trafficking defects caused by Dab2 knockdown impair the ability of EC to undergo morphogenesis, a crucial step in angiogenesis (Fig 14). It will be interesting to identify which downstream signaling protein function is affected upon Dab2 knockdown using a simple western blot technique. Moreover, it will be of great importance to test whether these in vitro effects of Dab2 knockdown can be recapitulated under in vivo conditions using a Dab2 conditional knockout mice [79]. More interestingly, designing peptide sequences that can block Dab2 interaction with VEGFR2 in patient derived xenograft models will help us understand the role od Dab2 as a therapeutic target in cancer. Based on my current findings, we propose the following model for the role of Dab2 in VEGFR2 trafficking and signaling. As seen in Fig 10, under steady state condtions, VEGFR2 is distributed between the EE compartment and at the PM. Dab2 has a diffuse distribution through out the cell with occasional association with VEGFR2 and the EE. Under non-stimulated conditions, Dab2 associates with VEGFR to mediate its constituitve recycling from the EE to PM thereby regulating the levels of VEGFR2 at the cell surface. Upon ligand stimulation, binding of Dab2 with VEGR2 brings about the clustering of activated receptors to enhance its activity and at the same time initiate the formation of CCPs. All these steps denote events that occur in the earliest moments of VEGF stimulation. Once the VEGF-phospho-VEGFR2-Dab2 commplex is collected within CCPs, internalized vesicles are trafficked to the EE and away from the PM, and Dab2 via its Myo VI-binding domain is responsible for mediating this transport. This trafficking is also a key step in maintaining the phosphorylation of VEGFR2 (Y1175) and 67

84 protecting it from the attack of cellular phosphatases such as PTP1b. Upon arrival of phospho-vgefr2 (Y1175) at the EE, Dab2 dissociates from the complex and this is likely to be a prerequisite for the recruitment and activation of other downstream signaling proteins that bind to activated receptors at EE. Once activation and signaling of those proteins has occurred, Dab2 reassociates with VEGFR2 to now regulate its trafficking to the recycling endosome in order to return VEGFR2 to PM to mediate further rounds of signaling. 68

85 Figure 15: Predicted role of Dab2 in VEGFR2 trafficking Based on our current findings, we suggest that the following model for the role of Dab2 in VEGFR2 traffiking in the absence (left panel) and presence (right panel) of VEGF. Left panel] In the absence of the stimulating ligand VEGF, Dab2 functions in the constitutive recycling of VEGFR2 from the EE to PM thereby maintaining the surface level expression of VEGFR2. In the absence of Dab2, this recyling of VEGFR2 from EE is impaired and VEGFR2 is targeted to LE for degradation. Right panel] Upon VEGF stimulation, Dab2 at the PM mediates VEGFR2 clustering and the initiation of the CCP formation. Dab2 present in these CCP s functions in the trafficking of VEGFR2 to the EE 69

86 via its interaction with the MyoVI motor protein and during this transport, protects phospho- VEGFR2 (Y1175) from the action of PTP1b phosphatase. Shortly after VEGFR2 is delivered to the EE, Dab2 falls off allowing the phospho-vegfr2 (1175Y) to now interact with downstream signaling molecules. Once activation of downstream signaling molecules is achieved, Dab2 once again re-associates with VEGFR2 to now traffick it to the RE for its transport back to the PM. In the absence of Dab2, internalization and trafficking of phospho-vegfr2 (Y1175) to the EE is not affected due to the compensatory action of the other adaptor proteins like AP2. However during the trafficking of phosp-vegfr2 (Y1175) to the EE, the phopho VEGFR2 (Y1175) is targeted by PTP1b phosphatases leading to the reduction of phospho VEGFR2 (Y1175) levels. Once in the EE, in the absence of Dab2, the cells are unable to recyle VEGFR2 back to the PM leading to its degradation. 70

87 3.2: Dab2 and other angiogenic receptors VEGF-VEGFR2 receptor complexes are the crucial regulators of angiogenesis. However, there are several other receptors that function in this process and it will be interest to study if Dab2 can regulate their trafficking. VEGF, which is considered to be the most imporant inducer of angiogenesis, has in fact a greater binding affinity to VEGFR1, another member of the family of VEGFRs [80]. Binding of VEGF to VEGFR1 is followed by autophosphorylation at several tyrosine residues [80]. However, signaling downstream of these residues has been studied to produce only weak responses of EC proliferation, migration, and tube formation [80]. In contrast to these in vitro results, vegfr1 knock-out mice are embryonically lethal and die due to the disorganization of the vasculature caused by overgorwth of ECs [80]. This suggests that VEGFR1 might play a substantial role in maintaining a normal vasculature. Since VEGFR1 and VEGFR2 have a similar structure, it will be interesting to see if Dab2 also functions in the endocytosis of VEGFR1 receptor. Under certain circumstances upon ligand stimulation, studies have also shown that VEGFR1 can heterodimerize with VEGFR2 to transactivate VEGFR2 to postitively regulate angiogenesis [81, 82]. Understanding whether Dab2 recognizes this complex for internalization will further help us understand the post-endocytic trafficking and thus its function in angiogenesis. Because VEGF has greater binding affinity for VEGFR1 than VEGFR2, it is thought that VEGFR1 competes with VEGFR2 to bind to the ligand and thus downregulate VEGFR2 downstream function [82]. Understanding if Dab2 is capable of similarly competing for 71

88 cargo selection between VEGFR1 and VEGFR2 will help us better understand how Dab2 can selectively choose its cargo for internalization during angiogenesis. Another receptor-ligand complex that is known to play a role in angiogenesis is TGFβ and its receptors [83]. Mice defecient in TGF-β1 gene die in utero due to defects in vascular system development. Of the several diverse functions mediated by TGF-β1 in angiogenesis, the function of TGF-β1 to oppose VEGF prosurvival signaling and induce EC apotosis is important [83]. This TGF-β1 induced EC apotpsis has been studied to play an important role in vascular sprout pruning and remodeling oh the vessel [83]. Recent studies have shown that VEGFR2 signaling is important to stimulate TGF-β1 signaling to induce EC apoptosis [83]. Moreover, it has been shown that Dab2 is required for the trafficking of TGF-β receptors from the EE to the RE [66]. Our results have shown Dab2 knockdown reduces signaling of VEGFR2 affecting the phohporylation of Y1175 residue. It is possible that this reduced VEGFR2 signaling at Y1175 residue caused by Dab2 knockdown may affect TGF-β1 signaling to affect EC bheaviour. Performing simple protein lysate analysis by immunoblotting to study the different activation patterns of the two receptors or live cell imaging of cells overexpressing differentially tagged VEGFR2 and TGF-β1 will help test the idea of Dab2 cargo switch to regulate EC apoptosis. Conversely it will of interest to see if Dab2 can mediate TGF-β1 trafficking independent of VEGFR2 to affect angiogenesis. Similar to receptor molecules like VEGFR1 and TGF-β receptors that can influence VEGFR2 signaling in angiogeneis, integrins (β1, αvβ3) have been studied to cooperate with VEGFR2 signaling [84, 85]. Work done by Teckchandani et al have shown that Dab2 regulates intergrin endocytosis to affect cell 72

89 migration, as important step in angiogenesis [85]. It will be imortant to know how within the EC, different populations of intracellular Dab2 molecules are dedicated to interact with these different VEGFR2 signal mediators and coordinate its signaling. As such, live cell imaging to track the function of Dab2 at different stages of endocytosis will be really helpful. Apart from the transmembrane receptors mentioned above, the activity of VEGFR2 is also regulated by coreceptors. Nrp1 is one such coreceptor that is known to aid in VEGFR2 signaling [54]. Work done by Lanahan et al has shown that Nrp1 via its C- terminal PDZ domain binds to synectin-myovi complex [54]. This binding of this Nrp1- synectin-myovi complex has been studied to important for VEGFR2 trafficking which is turn is important to protect VEGFR2 phosphorylation (Y1175) from the attack of PT1b phosphatases [48, 54]. Since Dab2 has a MyoVI binding domain, it will be of interest to see if Dab2 also functions as a part of Nrp1-synectin-MyoVI to traffick VEGFR2 away from the PM and to the EE. Nrp1 in association with plexin D receptor is also known to bind semaphorin 3A (Sema3A). Sema3A is known to function in the late stages of angiogenesis where it functions in the pruning and remodelling of the blood vessel sprouts [86]. Sema3A in association with Nrp1-plexin D receptor is known to induce inhibitory signals to negatively regulate EC adhesion to the integrins [86]. Since it is known that Nrp1 can bind synectin-myovi complex [54] and that Dab2 carries a MyoVI binding domain, it will be of great importance to study if Dab2 functions in the endocytosis and traffikcing 73

90 of this complex. These studies will thus help us understand how Dab2 can function to balance between (Nrp1-VEGFR2) proangiogenic receptor and antiangiogenic receptor (Nrp1-plexin D receptor) endocytosis and signaling. In conclusion, my work here presents that Dab2, a CLASP, functions to regulate trafficking of VEGFR2. Knockdown of Dab2 decreases in VEGFR2 phosphorylation at the Y1175 residue, a residue known to regulate VEGFR2 signaling in angiogenesis. Ultimately, in the absence of Dab2, there is increased traffikcing of VEGFR2 to the LE, which may be affecting its function in EC morphogenesis. Thus, this suggests that trafficking by Dab2 is required for VEGFR2 signaling and function. Thus, this study provides further understanding of VEGFR2 trafficking, and identifies Dab2 as a possible therapeutic target for several angiogenesis-related diseases. 74

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96 APPENDIX Figure A.1: Effect of Dab2 knockdown on VEGFR2 protein half-life Both Control and shdab2 cells were treated with cycloheximide (10µg/ml) (protein synthesis inhibitor) for the different lengths of time as indicated in the figure. At each time point, cells were lysed and VEGFR2, Dab2, and GAPDH levels were quantified. As 80

97 seen in the figure, there is no significant difference in VEGFR2 half-life was observed between control and shdab2 cells. Moreover, based on our hypothesis that Dab2 is required for VEGFR2 recycling at steady state, we did not detect significantly lower levels of VEGFR2 in shdab2 cells (0 min). However, it is to be noted that robust levels of Dab2 knockdown were not achieved. Thus is this result needs further validation. 81

98 VEGF VEGFR2 EEA1 wt shdab2 % EEA1colocalization HUVEC wt HUVEC sh Dab mins 30 mins VEGF treatment 60 mins Figure A.2: Dab2 knockdown causes a delay in the trafficking of VEGFR2 to the early endosomes Wt (control) and shdab2 were serum starved and then labeled for surface VEGFR2 with an E-tagged -VEGFR2 antibody (ScFv) at 4 0 C for 30 minutes. Cells were then washed and provided with VEGFA for additional 30 minutes at 4 0 C. Cells in the presence of the antibody and VEGF, were chased at 37 0 C for different time points to allow the tagged 82

99 VEGFR2 to undergo endocytosis. At the end of each time point, cells were fixed, permeabilized and processed for immunofluorescence as described in materials and methods. A] Representative images showing colocalization of VEGFR2 to the early endosomes antigen-1 (EEA1), a marker for the early endosomes B] Quantification of the percent of tagged-vegfr2 colocalizing with the early endosomes (EEA1) was performed using MetaMorph software. As seen in this figure, we see that while there was no difference in the trafficking of VEGFR2 to the EE in control and shdab2 cells, there was a rapid release of VEGFR2 from the EE in shdab2 cells 30 mins post-vegf stimulation suggesting that suppression of Dab2 may affect sorting of VEGFR2 from the EE. However, since the surface-labeling antibody failed to show consistent labeling pattern, we could not arrive at a conclusion. 83

100 Figure A.3: Effect of Dab2 knockdown on VEGFR2 internalization Both control and shdab2 cells were labeled with Biotin at 4 0 C that labels all the surface proteins including VEGFR2. After binding, the cells were treated VEGF and then internalization of biotinylated VEGFR2 was allowed for different time points at 37 0 C. At the end of each time point, the cells were treated with a reducing agent to remove all VEGFR2 that failed to undergo internalization. Following this treatment, the cells were lysed and the lysate that contains the internalized and hence protected fraction of VEGFR2 (from the action of the reducing agent) was immunoprecipitated with 84