Molecular regulation of VEGFR-2 expression and activation in endothelial cells

Angiogenesis, a hallmark step in tumor growth and metastasis, is primarily driven by the action of vascular endothelial growth factor (VEGF) on its receptor, VEGFR-2. Central to its regulation, the abundance of VEGFR-2 on the surface of endothelial cells determines VEGF's ability to stimulate vasculogenesis and pathological angiogenesis. By using a yeast two-hybrid system we have identified PDCL3 (Phosducin like 3) as a novel VEGFR-2 interacting protein that serves as a chaperone protein. PDCL3 expression is highly up-regulated in pathological neovascularization in a mouse model of oxygen-induced proliferative retinopathy and its expression correlates with VEGFR-2 expression. Knockdown of PDCL3 in endothelial cells significantly reduced hypoxia-induced expression of VEGFR-2. Further studies showed that PDCL3 binds to both nascent and mature VEGFR-2 through recognition of its juxtamembrane (JM) domain. Preventing binding of PDCL3 to the JM domain, and manipulating expression of PDCL3 by over-expression and siRNA in endothelial cells, showed that PDCL3 controls the abundance of VEGFR-2 through inhibition of its ubiquitination and degradation. Various in vivo and in vitro angiogenesis assays showed that PDCL3 activity is required for VEGF-mediated endothelial cell proliferation, capillary tube formation, and angiogenesis in developing zebrafish embryos. Further investigation of the molecular mechanisms of VEGFR-2 protein homeostasis revealed that its PEST domain controls degradation and ubiquitination of mature VEGFR-2. The data demonstrate that beta-Trcp1 ubiquitin E3 ligase is recruited to the PEST domain and mediates ubiquitination and degradation of VEGFR-2. The PEST domain via phospho-Y1173 also contributes to activation of p38 mitogen-activated protein kinase (p38 MAPK), which results in the stabilization of VEGFR2. The work demonstrates that VEGFR-2 protein homeostasis in endothelial cells is controlled at multiple levels involving co-chaperone functions of PDCL3, betaTrcp1 ubiquitin E3 ligase and p38 MAPK pathway.