KDR activation is crucial for VEGF(165)-mediated Ca2+ mobilization in human umbilical vein endothelial cells

We have prepared a polyclonal mouse antibody directed against the first thr ee immunoglobulin-like domains of the kinase insert domain-containing recep tor (KDR) tyrosine kinase. It possesses the ability to inhibit binding of t he 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) bin ding to human umbilical vein endothelial cells (HUVEC). These results confi rm that the first three immunoglobulin-like domains of KDR are involved in VEGF(165) interactions. The anti-KDR antibody is able to completely block V EGF(165)-mediated intracellular Ca2+ mobilization in HUVEC. Therefore, it a ppears that binding of VEGF(165) to the fms-like tyrosine kinase (Flt-1) in these cells does not translate into a Ca2+ response. This is further exemp lified 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 heterodi mer was also very inefficient at eliciting a Ca2+ signaling event in HUVEC. We conclude that KDR activation is crucial for mobilization of intracellul ar Ca2+ in HUVEC in response to VEGF(165).