Bruce I. Terman

Identification of the KDR tyrosine kinase as a receptor for vascular endothelial cell growth factor

Vascular endothelial cell growth factor (VEGF), also known as vascular permeability factor, is an endothelial cell mitogen which stimulates angiogenesis. Here we report that a previously identified receptor tyrosine kinase gene, KDR, encodes a receptor for VEGF. Expression of KDR in CMT-3 (cells which do not contain receptors for VEGF) allows for saturable 125I-VEGF binding with high affinity (KD = 75 pM). Affinity cross-linking of 125I-VEGF to KDR-transfected CMT-3 cells results in specific labeling of two proteins of M(r) = 195 and 235 kDa. The KDR receptor tyrosine kinase shares structural similarities with a recently reported receptor for VEGF, flt, in a manner reminiscent of the similarities between the alpha and beta forms of the PDGF receptors.

VEGF Receptor Subtypes KDR and FLT1 Show Different Sensitivities to Heparin and Placenta Growth Factor

Vascular endothelial growth factor (VEGF) is an angiogenic growth factor which binds to two structurally related tyrosine kinase receptors denoted KDR and FLT1. To compare the interaction of VEGF with each receptor, cell lines which express individual receptor subtypes were identified using Northern blot hybridization. Bovine aortic endothelial (ABAE) cells and WM35 melanoma cells were found to express KDR, while FLT1 was primarily expressed on SK-MEL-37. Both receptor subtypes were detected on another melanoma cell line (WM9). Heparin augmented VEGF binding to KDR-expressing cells (ABAE and WM35), but inhibited VEGF binding to FLT1-expressing cells (SK-MEL-37 and WM9). The concentration of heparin required for half maximal stimulation of VEGF binding to KDR-expressing cells (500 ng/ml) was 25 times greater than that required for half maximal inhibition of binding to FLT1-expressing cells (20 ng/ml). In WM9 cells, the effect of heparin was bimodal; low concentration inhibited, while higher concentrations stimulated binding of 125I-VEGF. Placenta growth factor (PIGF-1) is a recently described growth factor structurally similar to VEGF. PIGF-1 had a negligible or no effect on 125I-VEGF binding to KDR-expressing cells (ABAE, WM35), but did complete for binding to FLT1-expressing cells (SK-MEL-37 and WM9). Addition of heparin had no effect on its ability to compete for binding with 125I-VEGF. The data indicate differential regulation of the two VEGF receptors by heparin and extended specificity of FLT1 receptor, but not KDR, for binding PIGF-1 growth factor.

Identification of a heparin binding peptide on the extracellular domain of the KDR VEGF receptor

Vascular endothelial growth factor (VEGF), a potent and specific activator of endothelial cells, is expressed as multiple homodimeric forms resulting from alternative RNA splicing. VEGF121 does not bind heparin while the other three isoforms do, and it has been documented that the binding of VEGF165 to its receptor is dependent upon cell surface heparin sulfate proteoglycans. Little is known about the biochemical mechanism that allows for heparin regulation of growth factor binding. For example, it is not clear whether heparin interactions with growth factor or with cell surface receptors or both are essential for VEGF binding to its receptor. In this manuscript we provide results which are consistent with the hypothesis that an interaction between heparin and a site on the KDR receptor subtype is essential for VEGF165 binding. First, we demonstrate that expression of KDR into a CHO cell line deficient in heparan sulfate biosynthesis does not allow VEGF165 binding unless heparin is exogenously added during the binding assay. Secondly, we show that a ten amino acid synthetic peptide, corresponding to a sequence from the extracellular domain of the KDR, both inhibits VEGF165 binding to the receptor and also binds heparin with high avidity. Third, affinity purification of heparin molecules on a KDR-derived peptide affinity column, together with capillary electrophoresis and polyacrylamide electrophoresis analysis, was used to show that the KDR-derived peptide interacts with a specific subset of polysaccharide chains contained in the unfractionated heparin. Taken together, these results are consistent with the hypothesis that interactions between cell surface heparan sulfate proteoglycans and the VEGF receptor contribute to allowing maximal VEGF binding.

Biological properties of VEGF/VPF receptors.

Several experimental findings have emphasized the significant role played by Vascular Endothelial Growth Factor/Vascular Permeability Factor (VEGF/VPF) in tumor angiogenesis. For example, VEGF/VPF actions are predominantly restricted to endothelial cells [1], and the expression of VEGF/ VPF is enhanced in tumor cells that lack oxygen and glucose [2]. In addition, inhibiting the interaction of VEGF/VPF with endothelial cells slows the growth of several tumors in vivo [3, 4]. The purpose of this review is to summarize what is known concerning one aspect of the biology of VEGF/VPF, the interaction of the growth factor with its cell surface receptors.

Characterization of a Soluble Vascular Endothelial Growth Factor Receptor-Immunoglobulin Chimera

To investigate the interaction between vascular endothelial growth factor (VEGF) and its receptor, we have constructed a chimeric protein consisting of the extracellular ligand-binding domain of the human VEGF receptor subtype KDR fused to a human IgG1 Fc domain (KDR-Fc). KDR-Fc was expressed in human 293 kidney epithelial cells as a 300-kDa secreted, dimeric glycoprotein that bound 125I-VEGF165 with high affinity (Kd = 150 pM). Unlike the full length cellular receptor, KDR-Fc did not require heparin for 125I-VEGF165 binding, although heparin did stimulate 125I-VEGF165 binding approximately 50 to 100%. Similar results were observed for KDR-Fc expressed in yeast cells. Since yeast do not synthesize heparan sulfate proteoglycans, we conclude that cellular heparan sulfates do not account for the lack of a heparin requirement for 125I-VEGF165 binding to KDR-Fc. The polycationic protein protamine, which inhibits (IC50 = 1 microgram/ml) 125I-VEGF165 binding to bovine aortic endothelial cells and other KDR-expressing cells by blocking heparin interactions, had no effect on the heparin independent component of 125I-VEGF165 binding to KDR-Fc. Protamine does inhibit (IC50 = 1 microgram/ml) the heparin dependent component of 125I-VEGF165 binding to KDR-Fc. KDR-Fc bound VEGF121 with the same affinity as VEGF165. Heparin had no effect on 125I-VEGF121 binding to KDR-Fc, indicating that heparin interaction with the 44 amino acids contained in VEGF165 but not VEGF121 allow for maximal VEGF165 binding. Deletion analysis of KDR-Fc demonstrated that the determinants required for high affinity VEGF binding are located in the three aminoterminal Ig-domains of the protein. Heparin had no effect on 125I-VEGF165 binding to the three Ig-domain receptor, suggesting that there are heparin binding determinants located in KDR Ig-domains 4 to 7.

Basic FGF Treatment of Endothelial Cells Down-regulates the 85-KDa TGFβ Receptor Subtype and Decreases the Growth Inhibitory Response to TGF-β1

AbstractTransforming growth factor-beta 1 (TGFβ1) and basic fibroblast growth factor (bFGF) are known to be potent inhibitors and stimulators, respectively, of endothelial cell growth in vitro. In the present study we examined the effect of bFGF on endothelial cell growth inhibitory activity of TGFβ1 and on the binding of (l25I)-TGFβl to these cells. The concentration of TGFβ1 required for half-maximal inhibition of endothelial cell growth was increased in a dose-dependent manner by bFGF (a 20–100 fold increase at 1 ng/ml of bFGF). A 24 h-pretreatment of cells with bFGF resulted in abolition of the TGFβ1 inhibitory effect on DNA synthesis. Moreover, the binding of (l25I)-TGFβl to the endothelial cell surface was decreased in a concentration-dependent and time-dependent manner after a preincubation of these cells with bFGF. Analysis of the binding parameters showed that bFGF decreased by two-fold the number of TGFβ receptors (to approximately 6000 receptors per cell). Cross-linking experiments with disucci...