Research on angiogenesis has been focused on developing anti-angiogenic therapies to target endothelial cell-specific signalling pathways, as a mean to limit tumour outgrowth and metastasis. One of the main targets is the endothelial cell-specific Tie2 receptor and its ligands, the angiopoietins, which controls the later stages of angiogenesis. Although the angiopoietin/Tie2 signalling pathways have been well characterized, the molecular mechanism by which the ligands regulate Tie2 activity remains unclear. To address this question, we determined whether the activation mechanism of Tie2 is induced by dimerisation alone, or whether subsequent relative rotation of the kinase domain is required. Here we employed a coiled-coiled based protein engineering approach to identify the relative orientations of the kinase domains that are optimal for Tie2 activation. By replacing the extracellular domain of Tie2 with the dimeric parallel coiled-coil motif Put3cc, we generated ligand-independent homodimers of the kinase domains Put3cc-Tie2 I-VII that have distinct orientations. We show that dimerisation is sufficient to induce Tie2 activation and downstream activation of Akt, and that varying the interface of the kinase domain in Tie2 dimers can increase its catalytic efficiency. In addition we examined for the presence of potential dimerisation within the transmembrane and intracellular domain of Tie2. We show that the KD and potentially the TM contain dimerisation motifs that stabilise Tie2 in the inactive and active conformations. In addition, we show that deletion of the potential coiled-coil motif in the JM does not disrupt dimerisation but decreases the catalytic efficiency of Tie2. Finally, we propose that the activation mechanism of Tie2 may be similar to the previously described asymmetric dimer formation of EGFR and FGFR receptors.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:740240 |
| Date | January 2013 |
| Creators | Nyamay'Antu, Alengo |
| Contributors | Ballestrem, Christoph ; High, Stephen |
| Publisher | University of Manchester |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | https://www.research.manchester.ac.uk/portal/en/theses/elucidating-the-mechanism-of-angiopoeitinmediated-tie2-signalling(4269f3c3-fc71-455d-ae5b-4bc47dda78ca).html |
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