Date of Degree
PhD (Doctor of Philosophy)
Molecular and Cell Biology
First Committee Member
Second Committee Member
Third Committee Member
Fourth Committee Member
Fifth Committee Member
Sixth Committee Member
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 system and this impacts receptor signaling and endothelial cell morphogenesis during angiogenesis.
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 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.
Dab2, Early endosome, Endocytosis, Late endosome, tube formation assay, VEGFR2
xiii, 85 pages
Includes bibliographical references (pages 75-79).
Copyright © 2015 Shivangi Makarand Inamdar
Inamdar, Shivangi Makarand. "Dab2 plays a role in the post-endocytic trafficking of VEGFR2." PhD (Doctor of Philosophy) thesis, University of Iowa, 2015.