Structural Mechanism of Activation and Modulation of Tie2-mediated Angiogenesis

PROJECT SUMMARY The cardiovascular system is an essential organ key for development. This highly efficient network of vasculature structures constantly undergoes expansion and trimming to maintain vessel homeostasis. Angiogenesis – the budding of new vasculature structures from preexisting vessel systems, is one of the

primary processes contributing to network expansion. Multiple cellular receptors function together to ensure successful proliferation and motility of endothelial cells for angiogenesis. One of these receptors is Tie2, a receptor tyrosine kinase (RTK) expressed exclusively on the endothelial cell surface. Tie2's primary role, upon

binding to angiopoietin-1 (Ang-1), is to initiate and maintain tight cell-cell junctions across the endothelial monolayer. Like other RTKs, Tie2's proposed activation pathway involves the dimerization of the receptors to propagate a signal across the cell membrane to phosphorylate downstream targets. While the role of Tie2 in

angiogenesis is clear, little is known about the structural changes that Tie2 undergoes that allow for dimerization. The Ang-1 binding site is distant from the proposed dimerization site. Therefore, information must travel across multiple domains, and there is currently no mechanism for this transduction. In addition, our

understanding of cell signaling pathways has been described as most straightforward possible, often rendering them as exclusively linear with a single ligand and receptor. However, within the cellular context, proteins and ligands are constantly engaging with other signaling pathways in addition to their own. Integrins, a membrane

receptor that has a role in cellular adhesion and motility, have been observed to engage with Tie2 on the cellular level. But the molecular details remain elusive. Therefore, a structural investigation into Tie2's activation and modulation with angiopoietin and integrin would contribute to a more complete picture of Tie2

biology. To this end, I will combine cutting-edge structural biology techniques with traditional cellular biology to dissect these structural changes in Tie2. My goals are to elucidate the molecular mechanism of Tie2 dimerization and the receptor cross-talk that occurs with integrin α5β1. I have designed experiments with

two specific aims to understand how Tie2 is structurally impacted by angiopoietin-1 and integrin α5β1. Aim 1 will examine and describe the impact of Ang-1 binding on the full-length Tie2 receptor using cryogenic electron microscopy (cryoEM) and cell-based assays. The results from this aim will push the limits of our understanding

of Tie2's structural architecture and ligand-induced dimerization of RTKs. Aim 2 will determine the structural role of integrin α5β1 in modulating Tie2's signaling axis. I will use a combination of cryoEM and Förster resonance energy transfer to identify the key residues of this receptor-crosstalk. Results from this aim will shed

light on the functional importance of receptor cross-talk and how structures of the receptors can influence communication across signaling pathways. By studying Tie2, we can better understand its structure and potentially develop new therapies targeting this receptor.