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N Rahimi, F Majnoun, V Dayanir, RD Meyer; The Presence of a Single Tyrosine Residue at the Carboxyl Domain of Vascular Endothelial Growth Factor Receptor-2/FLK-1 Regulates Its Activation and Association With Signaling Molecules . Invest. Ophthalmol. Vis. Sci. 2002;43(13):2750.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: VEGFR-2 plays a critical role in vasculogenesis during embryonic development and pathological angiogenesis, but little is known about the molecular mechanisms governing its functions. Here we investigated the role of tyrosine 1212 on mouse VEGFR-2 autophosphorylation and its signal transduction relay in endothelial cells. Methods: PCR-based site directed mutagenesis used to generate point mutation 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 1212 of VEGFR-2 in endothelial cell functions. Results: Mutation of tyrosine 1212 on VEGFR-2 to phenylalanine severely impaired the ligand-dependent autophosphorylation of VEGFR-2, its ability to associate with and activate Src. This mutation also reduced the VEGFR-2’s ability to phosphorylate PLC-γ1, and MAPK. Unlike, mutation of tyrosine 1212 to phenylalanine, replacement of tyrosine 1212 with glutamic acid, preserved the ligand-dependent activation of VEGFR-2 and activation of VEGFR-2 associated signaling proteins including, Src, PLC-γ1 and MAPK. Further analysis showed that Src activation is not required for activation of VEGFR-2, since cells co-expressing wild type receptor with kinase dead Src or wild type Src displayed no apparent effect in the ligand-dependent autophosphorylation of VEGFR-2. Similarly, expression of wild type VEGFR-2 in fibroblast (SYF) cells obtained from the triple knockout Src family kinases showed normal ligand-dependent autophosphorylation. Conclusion: These results suggest that phosphorylation of tyrosine 1212 of VEGFR-2 plays a crucial role in activation of VEGFR-2 and subsequently VEGFR-2 mediated angiogenesis.
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