Abstract
Vascular endothelial growth factor (VEGF) directly stimulates endothelial cell proliferation and migration via tyrosine kinase receptors of the split kinase domain family. It mediates vascular growth and angiogenesis in the embryo but also in the adult in a variety of physiological and pathological conditions. The potential binding site of VEGF with its receptor was identified using cellulose-bound overlapping peptides of the extracytosolic part of the human vascular endothelial growth factor receptor II (VEGFR II). Thus, a peptide originating from the third globular domain of the VEGFR II comprising residues 247RTELNVGIDFNWEYP261 was revealed as contiguous sequence stretch, which bound 125I-VEGF165. A systematic replacement with L-amino acids within the peptide representing the putative VEGF-binding site on VEGFR II indicates Asp255 as the hydrophilic key residue for binding. The dimerized peptide (RTELNVGIDFNWEYPAS)2K inhibits VEGF165 binding with an IC50 of 0.5 microM on extracellular VEGFR II fragments and 30 microM on human umbilical vein cells. VEGF165-stimulated autophosphorylation of VEGFR II as well as proliferation and migration of microvascular endothelial cells was inhibited by the monomeric peptide RTELNVGIDFNWEYPASK at a half-maximal concentration of 3-10, 0.1, and 0.1 microM, respectively. We conclude that transduction of the VEGF165 signal can be interrupted with a peptide derived from the third Ig-like domain of VEGFR II by blockade of VEGF165 binding to its receptor.
Highlights
Angiogenesis, the formation of new blood vessels sprouting from existing ones, plays an essential role in fetal and adult life
Vascular endothelial growth factor (VEGF)-C is binding to the FLT-4 receptor (VEGFR III), which is mainly expressed in the lymphatic system, and after complete processing it binds to VEGFR II [25]
The peptide scan with cysteine containing peptides probed with soluble 125I-VEGF165 showed eight strong spots (Fig. 1A), whereas the peptide scan with the serine substitutions (Fig. 1B) displayed only two spots (124 and 125) corresponding to the peptide sequences 247RTELNVGIDFNWE259 and 249ELNVGIDFNWEYP261 of VEGFR II
Summary
Synthesis of Peptides and Cellulose-bound Peptide Libraries—Cellulose-bound and cleavable sets of peptides (PepSpotsTM and Cleavable PepSpotsTM, Jerini Bio Tools GmbH, Berlin, Germany) were automatically prepared according to standard spot synthesis protocols [40] using a spot synthesizer (Abimed GmbH, Langenfeld, Germany) as described previously [41]. After two changes of buffer the paper was blocked by incubation for 60 min in TBS/Tween 20 containing 5% milk powder at 4° C To this solution 125I-VEGF165 was added to a concentration of 0.1 C/ml. Three million cells in 50 l of PBS containing 0.1% BSA were transferred into wells of a C96 white microtiter plates (Maxisorb, Nunc) that had been coated previously with 1 g/ml anti-c-kit ϭ CD 117 antibody (Research Diagnostics Inc., Flanders, NJ) overnight at pH 9.6 and blocked with BSA. Ten thousand cells in 100 l of M199 containing 2% human serum and glutamin were given into a culture well insert with a porous filter bottom that had been washed with PBS and coated with collagen (10 g/ml) previously. Optical density of the resulting solutions was determined at 560 nm
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