Biochemical, cellular and structural studies on vascular receptors and ligands

Vascular endothelial growth factor receptors (VEGFRs) regulate fundamental cellular responses such as cell proliferation, survival, and migration, and thus play key roles in angiogenesis and vascular physiology in higher eukaryotes such as vertebrates. The intracellular signaling pathways associated with VEGFRs have been well studied and characterised. However, there is a lack of understanding of their mechanism of activation. In this study, mammalian cell lines for tetracycline-inducible expression of VEGFR1, VEGFR2 and VEGFR3 were developed. A strategy was devised for expression, solubilisation and purification of VEGFR2. The first negative stain electron microscopy (neg stain-EM) observation of full-length VEGFR2 produced various negative stain class averages of VEGFR2 dimer bound to VEGF-A ligand. By biochemical and structural means, we also unraveled the process of differential activation of VEGFR2, its modulation on the cell membrane in multiple states against the previously reported ‘one structure’ model. Also implicated in cardiovascular disease, lectin-like low-density lipoprotein receptor-1 (LOX-1) is a member of the scavenger receptor family that binds to oxidized low-density lipoprotein (OxLDL). It is also proposed to activate downstream signal transduction that causes pro-atherogenic processes such as endothelial dysfunction, foam cell formation and apoptosis. LOX-1 does not recognize the native form of OxLDL, and the mechanism of ligand recognition by LOX-1 is unknown. Moreover, the structural changes brought about by LDL oxidation are also unknown. This study revealed that LOX-1 forms higher order multimers with OxLDL. Using cryo-electron microscopy (cryo-EM), the first 3D de novo model of the OxLDL particle was proposed. These studies also investigated the structural differences between native LDL (nLDL) and OxLDL, and identified that oxidation of LDL causes tighter packing of lipids and unravels hithero inaccessible binding domains of ApoB-100 (protein component) of the lipoprotein, which is recognized by LOX-1 receptor. This starts to provide some insight into a crucial event in the progression of atherosclerosis.