Vascular endothelial growth factor (VEGF)-stimulated nitric oxide (NO) release from endothelial cells is mediated through the activation of VEGF receptor-2 (VEGFR-2). Herein, we have attempted to determine which autophosphorylated tyrosine residue on the VEGFR-2 is essential for VEGF-mediated endothelial nitric-oxide synthase (eNOS) activation and NO production from endothelial cells. Tyrosine residues 801, 1175, and 1214 of the VEGFR-2 were mutated to phenylalanine, and the mutated receptors were analyzed for their ability to stimulate NO production. We show, both in COS-7 cells cotransfected with the VEGFR-2 mutants and eNOS and in bovine aortic endothelial cells, that the Y801F-VEGFR-2 mutant is unable to stimulate NO synthesis and eNOS activation in contrast to the wild type, Y1175F-VEGFR-2, and Y1214F-VEGFR-2. However, the Y801F mutant retains the capacity to activate phospholipase C-␥ in contrast to the Y1175F-VEGFR-2. Interestingly, the Y801F-VEGFR-2, in contrast to the wild type receptor, does not fully activate phosphatidylinositol 3-kinase or recruit the p85 subunit upon receptor activation. This results in a complete incapacity of the Y801F-VEGFR-2 to stimulate Akt activation and eNOS phosphorylation on serine 1179 in endothelial cells. In addition, constitutive activation of Akt or a phosphomimetic mutant of eNOS (S1179D) fully rescues the inability of the Y801F-VEGFR-2 to induce NO release. Finally, we generated an antibody that specifically recognizes the phosphorylated form of tyrosine 801 of the VEGFR-2 and demonstrate that this residue is actively phosphorylated in response to VEGF stimulation of endothelial cells. We thus conclude that autophosphorylation of tyrosine residue 801 of the VEGFR-2 is essential for VEGFstimulated NO production from endothelial cells, and this is primarily accomplished via the activation of phosphatidylinositol 3-kinase and Akt signaling to eNOS.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.