Vascular endothelial growth factor receptor (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. Mutation of tyrosine 1212 on VEGFR-2 to phenylalanine severely impaired the ligand-dependent autophosphorylation of VEGFR-2 and its ability to associate with and activate Src. This mutation also reduced the VEGFR-2 ability to phosphorylate phospholipase C␥1 and mitogen-activated protein kinase (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, phospholipase C␥1, 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 liganddependent autophosphorylation. Collectively, these results suggest that phosphorylation of tyrosine 1212 of VEGFR-2 plays a crucial role in the activation of VEGFR-2 and subsequently VEGFR-2-mediated angiogenesis.

VEGFR-1 is devoid of ligand-dependent tyrosine autophosphorylation and its activation is not associated with proliferation of endothelial cells. The molecular mechanism responsible for this characteristic of VEGFR-1 is not known. In this study, we show that VEGFR-1 is devoid of ligand-dependent downregulation and failed to stimulate intracellular calcium release, cell migration and angiogenesis in vitro. To understand the molecular mechanisms responsible for the poor tyrosine autophosphorylation of VEGFR-1, we have either deleted the carboxyl terminus of VEGFR-1 or exchanged it with the carboxyl terminus of VEGFR-2. The deletion of carboxyl terminus of VEGFR-1 did not reverse its defective ligand-dependent autophosphorylation. The carboxyl terminus-swapped VEGFR-1, however, displayed ligand-dependent autophosphorylation, downregulation and also conveyed strong mitogenic responses. Thus, the carboxyl tail of VEGFR-1 restrains the ligand-dependent kinase activation and downregulation of VEGFR-1 and its ability to convey the angiogenic responses in endothelial cells.