Different signal transduction properties of KDR and Flt1, two receptors for vascular endothelial growth factor

Vascular endothelial growth factor (VEGF) is a homodimeric peptide growth factor which binds to two structurally related tyrosine kinase receptors denoted Flt1 and VEGFR2/KDR/Flk-1. In order to compare the signal transduction via these two receptors, the human Flt1 and VEGFR2/KDR/Flk-1 proteins were stably expressed in porcine aortic endothelial cells. Binding analyses using 125I-VEGF revealed Kd values of 16 pM for Flt1 and 760 pM for VEGFR2/KDR/Flk-1. Cultured human umbilical vein endothelial (HUVE) cells were found to express two distinct populations of binding sites with affinities similar to those for Flt1 and VEGFR2/KDR/Flk-1, respectively. The VEGFR2/KDR/Flk-1 expressing cells showed striking changes in cell morphology, actin reorganization and membrane ruffling, chemotaxis and mitogenicity upon VEGF stimulation, whereas Flt1 expressing cells lacked such responses. VEGFR2/KDR/Flk-1 was found to undergo ligand-induced autophosphorylation in intact cells, and both Flt1 and VEGFR2/KDR/Flk-1 were phosphorylated in vitro in response to VEGF, however, VEGFR2/KDR/Flk-1 much more efficiently than Flt1. Neither the receptor-associated activity of phosphatidylinositol 3'-kinase nor tyrosine phosphorylation of Phospholipase C-gamma were affected by stimulation of Flt1 or VEGFR2/KDR/Flk-1 expressing cells, and phosphorylation of GTPase activating protein was only slightly increased. Members of the Src family such as Fyn and Yes showed an increased level of phosphorylation upon VEGF stimulation of cells expressing Flt1 but not in cells expressing VEGFR2/KDR/Flk-1. The maximal responses in VEGFR2/KDR/Flk-1 expressing porcine aortic endothelial cells were obtained at higher VEGF concentrations as compared to HUVE cells, i.e. in the presence of Flt1. This difference could possibly be explained by the formation of heterodimeric complexes between VEGFR2/KDR/Flk-1 and Flt1, or other molecules, in HUVE cells.