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

Authors

  • Chris D. Willis,

    1. Department of Pathology, Anatomy and Cell Biology, and the Cancer Cell Biology and Signaling, Program, Kimmel Cancer Center, Thomas Jefferson University, Philadelphia, PA, USA
    Current affiliation:
    1. Thomson Reuters, IP & Science, Philadelphia, PA, USA
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  • Chiara Poluzzi,

    1. Department of Pathology, Anatomy and Cell Biology, and the Cancer Cell Biology and Signaling, Program, Kimmel Cancer Center, Thomas Jefferson University, Philadelphia, PA, USA
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  • Maurizio Mongiat,

    1. Department of Molecular Oncology and Translational Research, National Cancer Institute, Aviano, Italy
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  • Renato V. Iozzo

    Corresponding author
    • Department of Pathology, Anatomy and Cell Biology, and the Cancer Cell Biology and Signaling, Program, Kimmel Cancer Center, Thomas Jefferson University, Philadelphia, PA, USA
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  • C.D.W. and C.P. contributed equally to this work

Correspondence

R. V. Iozzo, Department of Pathology, Anatomy and Cell Biology, Thomas Jefferson University, 1020 Locust Street, Suite 336 JAH, Philadelphia, PA 19107, USA

Fax: +1 215 923 7969

Tel: +1 215 503 2208

E-mail: iozzo@KimmelCancerCenter.org

Abstract

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 (VEGFR2) and α2β1 integrin, resulting in dual receptor antagonism. Treatment of endothelial cells with endorepellin inhibits transcription of VEGFA, the natural ligand for VEGFR2, attenuating the pro-survival and migratory activities of VEGFA/VEGFR2 signaling cascade. Here, we investigated the specific binding site of endorepellin within the ectodomain of VEGFR2. Full-length endorepellin was not capable of displacing VEGFA binding from VEGFR2 and LG3 domain alone did not bind VEGFR2. This suggested different binding mechanisms of the extracellular Ig domains of VEGFR2. Therefore, we hypothesized that endorepellin would bind through its proximal LG1/2 domains to VEGFR2 in a different region than VEGFA. Indeed, we found that LG1/2 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 VEGFR2. Moreover, we found that LG1/2 blocked the rapid VEGFA activation of VEGFR2 at Tyr1175 in endothelial cells. In contrast, LG1/2 did not result in actin cytoskeletal disassembly in endothelial cells whereas LG3 alone did induce cytoskeletal collapse. However, LG1/2 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 abstract

[Structured digital abstract was added on 3 May 2013 after original online publication]

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