Role of ubiquitin-modifying enzymes in regulating the endothelial response to VEGF-A

Cell surface receptors undergo degradation but the biochemical basis for such control remains poorly defined. Endothelial cells express vascular endothelial growth factor receptor 2 (VEGFR2) which binds to circulating VEGF-A, stimulating signal transduction and new blood vessel sprouting i.e. angiogenesis. A central hypothesis is that ubiquitin-modifying enzymes play a key role(s) in the endothelial response to VEGF-A. Work presented in this thesis provides evidence for a novel pathway requiring the E1 ubiquitin-activating enzyme, UBA1, in controlling basal plasma membrane VEGFR2 levels. Evidence suggests that this ubiquitin-linked regulatory pathway controls VEGFR2 levels by modulating constitutive receptor recycling and degradation. Programming basal plasma membrane VEGFR2 levels influences the intensity and duration of the endothelial response to circulating ligands such as VEGF-A. After identification of an UBA1-dependent pathway for modulating VEGFR2 ubiquitination,
the UBA1-interacting E2 ubiquitin-conjugating enzymes were screened. The E2 enzymes, UBE2D1 and UBE2D2, regulate basal VEGFR2 turnover downstream of UBA1. Another feature of regulating VEGFR2 ubiquitination involves a potential role for de-ubiquitinating enzymes (DUBs). There is evidence that a specific DUB enzyme, USP8, regulates VEGFR2 de-ubiquitination, trafficking, signal transduction and proteolysis. Depletion of USP8 caused VEGFR2 accumulation in early endosomes, perturbed VEGFR2 ubiquitination and impaired VEGF-A-stimulated signal transduction. In addition, de-ubiquitination is linked to production of a previously unidentified VEGFR2 cleavage product. Thus, controlling VEGFR2 de ubiquitination has important consequences for the endothelial cell response and vascular physiology. VEGFR2 is the predominant receptor through which VEGF-A regulates pro-angiogenic signal transduction. Conversely, the role of VEGFR1 in endothelial cells is considered
anti-angiogenic. Novel synthetic protein scaffolds called Adhirons were screened for interaction with VEGFR1 or VEGFR2. Adhirons to VEGFR2 inhibited VEGF-Astimulated
signal transduction and endothelial tube formation. In contrast, Adhirons to VEGFR1 increased VEGF-A-stimulated signal transduction, thus promoting increased tubulogenesis. These findings suggest that inhibition of VEGFR1 can stimulate proangiogenic responses. Adhirons represent a new class of synthetic tools with potential
applications in medical diagnostics, disease therapy and biomarker profiling.