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

We have prepared apolyclonal mouse antibody directed against the first threeimmunoglobulin-like domains of the kinase insert domain-containingreceptor (KDR) tyrosine kinase. It possesses the ability to inhibitbinding of the 165-amino acid splice variant of vascular endothelialcell growth factor (VEGF₁₆₅) torecombinant KDR in vitro as well as to reduceVEGF₁₆₅ binding to human umbilical vein endothelial cells (HUVEC). These results confirm that the firstthree immunoglobulin-like domains of KDR are involved in VEGF₁₆₅ interactions. The anti-KDRantibody is able to completely blockVEGF₁₆₅-mediated intracellularCa²⁺ mobilization in HUVEC.Therefore, it appears that binding of VEGF₁₆₅ to the fms-like tyrosinekinase (Flt-1) in these cells does not translate into aCa²⁺ response. This is furtherexemplified by the lack of response to placental growth factor (PlGF),an Flt-1-specific ligand. Additionally, PlGF is unable to potentiatethe effects of submaximal concentrations ofVEGF₁₆₅. Surprisingly, theVEGF-PlGF heterodimer was also very inefficient at eliciting aCa²⁺ signaling event in HUVEC. Weconclude that KDR activation is crucial for mobilization ofintracellular Ca²⁺ in HUVEC inresponse to VEGF₁₆₅.