The second immunoglobulin-like domain of the VEGF tyrosine kinase receptor Flt-1 determines ligand binding and may initiate a signal transduction cascade.

Abstract

Vascular endothelial growth factor (VEGF) is an angiogenic inducer that mediates its effects through two high affinity receptor tyrosine kinases, Flt‐1 and KDR. Flt‐1 is required for endothelial cell morphogenesis whereas KDR is involved primarily in mitogenesis. Flt‐1 has an alternative ligand, placenta growth factor (PlGF). Both Flt‐1 and KDR have seven immunoglobulin (Ig)‐like domains in the extracellular domain. The significance and function of these domains for ligand binding and receptor activation are unknown. Here we show that deletion of the second domain of Flt‐1 completely abolishes the binding of VEGF. Introduction of the second domain of KDR into an Flt‐1 mutant lacking the homologous domain restored VEGF binding. However, the ligand specificity was characteristic of the KDR receptor. We then created chimeric receptors where the first three or just the second Ig‐like domains of Flt‐1 replaced the corresponding domains in Flt‐4, a receptor that does not bind VEGF, and analyzed their ability to bind VEGF. Both swaps conferred upon Flt‐4 the ability to bind VEGF with an affinity nearly identical to that of wild‐type Flt‐1. Furthermore, transfected cells expressing these chimeric Flt‐4 receptors exhibited increased DNA synthesis in response to VEGF or PlGF. These results demonstrate that a single Ig‐like domain is the major determinant for VEGF‐PlGF interaction and that binding to this domain may initiate a signal transduction cascade.

Novel anti-VEGF chimeric molecules delivered by AAV vectors for inhibition of retinal neovascularization

“…In this study, we have developed several novel hybrid molecules that inhibit VEGF in vitro and are capable of inhibiting ocular neovascularization in vivo. It has been reported that domain 2 of VEGFR Flt-1 requires the presence of flanking sequences from Novel anti-VEGF chimeric molecules P Pechan et al dimerization of the soluble receptor, we constructed Flt-1 domain 2 linked directly to IgG1-Fc fragment (molecule D2-Fc), but such a strategy did not enhance its binding to VEGF, in agreement with Davis-Smyth et al 13 We then have linked the second domain of Flt-1 to Fc through a 9Gly linker, and thus generated a novel molecule, sFLT01, with potent VEGF binding. Our results show, for the first time, that Flt-1 domain 2 does not require presence of other VEGFR domains for high-affinity VEGF binding.…”

“…13 The molecule Flt(1-3)-IgG was reported to have the same VEGF-binding affinity as Flt(1-7)-IgG, however Flt(2)-IgG that contains only second domain was not capable of inhibiting VEGF. 13,17 Another molecule called VEGF-Trap, generated by the second domain of Flt-1 fused to the third domain of KDR and human IgG1-Fc region, has been shown to be a very potent VEGF binder. 18 Adeno-associated virus (AAV) vectors offer an attractive tool for intraocular gene delivery because of their nonpathogenic nature, low toxicity, minimal immunogenicity and long-term persistence.…”

“…11,12 The VEGF-binding function of Flt-1 has been mapped to the second domain. [13][14][15][16] There have been previous studies with two truncated soluble receptor hybrids, Flt(1-3)-IgG and Flt(1-7)-IgG, consisting of either the first three domains or all seven domains fused to human IgG1-Fc region. 13 The molecule Flt(1-3)-IgG was reported to have the same VEGF-binding affinity as Flt(1-7)-IgG, however Flt(2)-IgG that contains only second domain was not capable of inhibiting VEGF.…”

“…[13][14][15][16] There have been previous studies with two truncated soluble receptor hybrids, Flt(1-3)-IgG and Flt(1-7)-IgG, consisting of either the first three domains or all seven domains fused to human IgG1-Fc region. 13 The molecule Flt(1-3)-IgG was reported to have the same VEGF-binding affinity as Flt(1-7)-IgG, however Flt(2)-IgG that contains only second domain was not capable of inhibiting VEGF. 13,17 Another molecule called VEGF-Trap, generated by the second domain of Flt-1 fused to the third domain of KDR and human IgG1-Fc region, has been shown to be a very potent VEGF binder.…”

Emodin inhibits vascular endothelial growth factor‐A‐induced angiogenesis by blocking receptor‐2 (KDR/Flk‐1) phosphorylation

“…KDR/Flk-1 is required mainly for mitogenic and chemotactic responses, whereas Flt-1 contributes to endothelial cell morphogenesis. 12,13 Recently, the intracellular signaling pathways mediating these effects downstream of KDR/Flk-1 activation have been identified. KDR/Flk-1 induces proliferation through activation of the extracellular signalregulated kinase (ERK) 1/2 pathway leading to gene transcription 14 and endothelial cell survival through phosphatidylinositol 3-kinase activation, resulting in increased lipid phosphatidylinositol (3,4,5)P 3 and activation of several important intracellular molecules, such as Akt and the small GTP-binding protein, Rac.…”