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RD Meyer, F Majnoun, N Rahimi; Identification of a Novel SH2-Phosphopeptide Binding site Between VEGFR-2 and Phospholipase-Cg1 . Invest. Ophthalmol. Vis. Sci. 2002;43(13):4022.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: VEGFR-2 plays a crucial role in ocular angiogenesis and many other pathological conditions associated with angiogenesis, but little is known about the molecular mechanisms governing its functions and signal transduction relay in endothelial cells. Here we investigated the molecular mechanism responsible for activation of PLC-γ by VEGFR-2 and impact of its activation in endothelial cell function. Method: PCR-based site directed mutagenesis used to generate point mutations on VEGFR-2. The mutant receptors were expressed in endothelial cells, PAE and variety of biological and biochemical assays were used to define the role of tyrosine sites of VEGFR-2 in activation of PLC-γ and endothelial cell functions. Results: Mutation of tyrosines 799, 820, 952 and 1173 on VEGFR-2 to phenylalanine did not impair its ability to stimulate PLC-γ or MAP kinase activation. Additional site-directed mutagenesis showed that tyrosine 1009, located at kinase domain of VEGFR-2 is responsible for binding to and activation of PLC-γ in endothelial cells. Using variety of molecular and biochemical approaches including use of cells obtained from PLC-γ knock-out mice demonstrated that VEGFR-2 utilizes a unique mechanism to associate with and activate PLC-γ. Further studies revealed that activation of PLC-γ in endothelial cells is required for VEGFR-2 mediated cell differentiation. Conclusion: These results suggest that kinase domain of VEGFR-2, in particular, tyrosine 1009 of VEGFR-2 plays an important role in activation of PLC-γ. Furthermore, our data demonstrates that VEGFR-2 utilizes a novel mechanism to activate PLC-γ and subsequently VEGFR-2 mediated angiogenesis.
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