Identification of a Heparin Binding Peptide on the Extracellular Domain of the KDR VEGF Receptor

Abstract

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.

VEGF165 requires extracellular matrix components to induce mitogenic effects and migratory response in breast cancer cells

“…Only cells that were seeded onto ®bronectin-coated membranes migrated in signi®cantly higher numbers than cells seeded on matrigel. Given that VEGF and its receptors possess regions rich in basic amino acids that confer binding to the ECM (Cardin and Weintraub, 1989;Dougher et al, 1997;Templeton, 1992), and that VEGF binds to VEGF receptors in breast cancer cells via its exon 7-encoded ECM-binding region (which is not present in VEGF 121 ) (Soker et al, 1996), it was reasonable to postulate that VEGF-induced signaling in T47D cells depends on an ECM component. Thus, the e ects of heparin and ®bronectin on these cells were considered.…”

“…In endothelial cells, VEGF binds with high-a nity to its receptors (Cohen et al, 1995; Gitay-Goren et al, 1992). In spite of its high-a nity binding, heparin Dougher et al, 1997;Gitay-Goren et al, 1992) and HSPGs (Gengrinovitch et al, 1999) Figure 7 E ect of heparin and ®bronectin on VEGF-dependent PI3K activity. Cells were seeded on PL or FN as described in Materials and methods.…”

“…The heparin homologue of HSPGs stabilizes the complex between VEGF and its receptors and increases their binding due to several basic residues on both the ligand (Templeton, 1992) and receptor (Dougher et al, 1997). Thus, this polyanion was used to test whether it would a ect VEGF-dependent mitogenic response in T47D cells.…”

“…Fibronectin (Ruoslahti, 1991) and heparin (Halper, 1990) bind to the cell surface through receptors and high a nity sites, respectively. VEGF (Cardin and Weintraub, 1989), VEGF-receptors (Dougher et al, 1997), and ®bronectin (Ruoslahti et al, 1981) possess a highly basic region that confers strong a nity for heparin. Furthermore, heparin and HSPGs enhance the binding of VEGF to its receptors (Gitay-Goren et al, 1992) and are necessary for the biological actions of b-FGF (Rapraeger et al, 1991;Yayon et al, 1991).…”

VEGF-A121a binding to Neuropilins – A concept revisited

“…36 The abundant negatively-charged HS/CS chains of endothelial-cell-surface proteoglycans bind VEGF-A165a and, as with HS/CS chains in the extracellular matrix, can differentially regulate accessibility/storage of this isoform (and other isoforms with a heparin-binding domain, with affinities depending on the exon 6–8 combinations 37 and tertiary fold). These HS/CS chains can also modify the ability of VEGF isoforms to bind to VEGFR2 and NRPs, which themselves interact with and colocalize with cell surface HSPGs (VEGFR2 was shown to directly interact with HS chains on HSPGs expressed in endothelial cells via a stretch of residues between D6-D7 of VEGFR2 38-41 ; heparin also binds to VEGFR1 42-44 , NRP1 17 and NRP2 45 independent of VEGFs).…”