Abstract
VEGFR-2 is the primary regulator of angiogenesis, the development of new blood vessels from pre-existing ones. VEGFR-2 has been hypothesized to be monomeric in the absence of bound ligand, and to undergo dimerization and activation only upon ligand binding. Using quantitative FRET and biochemical analysis, we show that VEGFR-2 forms dimers also in the absence of ligand when expressed at physiological levels, and that these dimers are phosphorylated. Ligand binding leads to a change in the TM domain conformation, resulting in increased kinase domain phosphorylation. Inter-receptor contacts within the extracellular and TM domains are critical for the establishment of the unliganded dimer structure, and for the transition to the ligand-bound active conformation. We further show that the pathogenic C482R VEGFR-2 mutant, linked to infantile hemangioma, promotes ligand-independent signaling by mimicking the structure of the ligand-bound wild-type VEGFR-2 dimer.
Highlights
Angiogenesis, the development of new blood vessels from pre-existing ones, plays a critical role in embryogenesis, organ development, and wound healing (Qutub et al, 2009; Nessa et al, 2009; Adams et al, 2007)
We investigated whether full-length VEGFR-2 is capable of forming dimers in the absence of ligand using an established quantitative FRET method (Chen et al, 2010)
The interactions between VEGFR-2 molecules, labeled with the fluorescent proteins YFP or mCherry, were characterized in plasma membrane-derived vesicles generated from transfected Chinese Hamster Ovary (CHO) cells in response to osmotic stress (Del Piccolo et al, 2012; Sarabipour et al, 2015)
Summary
Angiogenesis, the development of new blood vessels from pre-existing ones, plays a critical role in embryogenesis, organ development, and wound healing (Qutub et al, 2009; Nessa et al, 2009; Adams et al, 2007). Angiogenesis is associated with diabetic and age-related macular degeneration and is required for the growth of solid tumors, which recruit blood vessels to ensure adequate supply with oxygen and nutrients (Luo et al, 2008; Tsuzuki et al, 2001). Angiogenesis is regulated by the ligands and receptors of the vascular endothelial growth factor (VEGF) signaling network (Ferrara et al, 2003; Matsumoto et al, 2001; Koch et al, 2011; Olsson et al, 2006; Shibuya et al, 2006). Of the three VEGF receptors, VEGFR-2 is the primary regulator of endothelial cell proliferation and migration (Takahashi et al, 2005; Gerhardt et al, 2003). Aggressive cancerous phenotypes correlate with enhanced VEGFR-2 signaling (Chatterjee et al, 2013)
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