KDR activation is crucial for VEGF165-mediated Ca2+ mobilization in human umbilical vein endothelial cells.

We have prepared a polyclonal mouse antibody directed against the first three immunoglobulin-like domains of the kinase insert domain-containing receptor (KDR) tyrosine kinase. It possesses the ability to inhibit binding of the 165-amino acid splice variant of vascular endothelial cell growth factor (VEGF 165 ) to recombinant KDR in vitro as well as to reduce VEGF 165 binding to human umbilical vein endothelial cells (HUVEC). These results confirm that the first three immunoglobulin-like domains of KDR are involved in VEGF 165 interactions. The anti-KDR antibody is able to completely block VEGF 165 -mediated intracellular Ca 2+ mobilization in HUVEC. Therefore, it appears that binding of VEGF 165 to the fms-like tyrosine kinase (Flt-1) in these cells does not translate into a Ca 2+ response. This is further exemplified by the lack of response to placental growth factor (PlGF), an Flt-1-specific ligand. Additionally, PlGF is unable to potentiate the effects of submaximal concentrations of VEGF 165 . Surprisingly, the VEGF-PlGF heterodimer was also very inefficient at eliciting a Ca 2+ signaling event in HUVEC. We conclude that KDR activation is crucial for mobilization of intracellular Ca 2+ in HUVEC in response to VEGF 165 .