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Endorepellin laminin‐like globular 1/2 domains bind Ig3–5 of vascular endothelial growth factor ( VEGF ) receptor 2 and block pro‐angiogenic signaling by VEGFA in endothelial cells
Author(s) -
Willis Chris D.,
Poluzzi Chiara,
Mongiat Maurizio,
Iozzo Renato V.
Publication year - 2013
Publication title -
the febs journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.981
H-Index - 204
eISSN - 1742-4658
pISSN - 1742-464X
DOI - 10.1111/febs.12164
Subject(s) - vascular endothelial growth factor a , microbiology and biotechnology , kinase insert domain receptor , angiogenesis , biology , integrin , receptor , vascular endothelial growth factor c , chemistry , vascular endothelial growth factor , cancer research , biochemistry , vegf receptors
Endorepellin, a processed fragment of perlecan protein core, possesses anti‐angiogenic activity by antagonizing endothelial cells. Endorepellin contains three laminin G‐like ( LG ) domains and binds simultaneously to vascular endothelial growth factor receptor 2 ( VEGFR 2) and α2β1 integrin, resulting in dual receptor antagonism. Treatment of endothelial cells with endorepellin inhibits transcription of VEGFA , the natural ligand for VEGFR 2, attenuating the pro‐survival and migratory activities of VEGFA / VEGFR 2 signaling cascade. Here, we investigated the specific binding site of endorepellin within the ectodomain of VEGFR 2. Full‐length endorepellin was not capable of displacing VEGFA binding from VEGFR 2 and LG 3 domain alone did not bind VEGFR 2. This suggested different binding mechanisms of the extracellular Ig domains of VEGFR 2. Therefore, we hypothesized that endorepellin would bind through its proximal LG 1/2 domains to VEGFR 2 in a different region than VEGFA . Indeed, we found that LG 1/2 did not bind Ig1–3, but did bind with high affinity to Ig3–5, distal to the known VEGFA binding site, i.e. Ig2–3. These results support a role for endorepellin as an allosteric inhibitor of VEGFR 2. Moreover, we found that LG 1/2 blocked the rapid VEGFA activation of VEGFR 2 at Tyr1175 in endothelial cells. In contrast, LG 1/2 did not result in actin cytoskeletal disassembly in endothelial cells whereas LG 3 alone did induce cytoskeletal collapse. However, LG 1/2 did inhibit VEGFA ‐dependent endothelial migration through fibrillar collagen I. These studies provide a mechanistic understanding of how the different LG domains of endorepellin signal in endothelial cells while serving as a template for protein design of receptor tyrosine kinase antagonists. Structured digital abstractIg1-3 of VEGFR2 binds to VEGFA by filter binding ( View interaction ) Ig3-5 of VEGFR2 binds to Endorepellin LG1/2 by enzyme linked immunosorbent assay ( View interaction ) Ig1-3 of VEGFR2 binds to Endorepellin LG1/2 by filter binding (View Interaction: 1 , 2 ) Endorepellin LG1/2 binds to Ig3-5 of VEGFR2 by anti bait coimmunoprecipitation ( View interaction ) [Structured digital abstract was added on 3 May 2013 after original online publication]