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Immunoglobulin-like domain 4-mediated ligand-independent dimerization triggers VEGFR-2 activation in HUVECs and VEGFR2-positive breast cancer cells

  • Preclinical study
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Abstract

Purpose

The extracellular region (EC) of the vascular endothelial growth factor (VEGF) receptor-2 (VEGFR-2) contains seven immunoglobulin-like (Ig-like) domains that are required for specific ligand binding and receptor dimerization. Studies of domain 4–7 deletions and substitutions provided insights into the interaction between receptors in the absence of VEGF. In this study, we investigated the effect of domain 4 in ligand-independent VEGFR-2 dimerization and activation in human vascular endothelial cells and human breast cancer cells.

Methods

To confirm the role of domain 4 in ligand-independent receptor dimerization and activation, two VEGFR-2 fragments with and without domain 4, KFP1 and KFP2, were generated by recombinant DNA technology. We measured the affinity of KFP1 and KFP2 with VEGFR-2, and the roles of KFP1 and FKP2 in dimerization and phosphorylation of VEGFR-2. We also evaluated the effect of KFP1 and FKP2 on cell proliferation and migration in HUVECs and in human breast cancer cells.

Results

We showed that KFP1 did not affect the interaction of VEGFR-2 and VEGF but bound VEGFR-2 in the absence of VEGF. Furthermore, cross-linking and cross-linking immunoblotting demonstrated that KFP1 could form a complex with VEGFR-2, which resulted in VEGFR-2 dimerization in the absence of VEGF. Importantly, we found that the KDR fragment with domain 4 induced phosphorylation of VEGFR-2, as well as phosphorylation of downstream receptor kinases in HUVECs and VEGFR-2-positive breast cancer cells. Consistent with these results, this ligand-independent activation of VEGFR-2 also promoted downstream signaling and cell proliferation and migration.

Conclusions

The domain 4 of VEGFR-2 plays an important role in the interaction between VEGFR receptors in the absence of VEGF.

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Abbreviations

VEGF:

Vascular endothelial growth factor

VEGFR-2:

Vascular endothelial growth factor receptor-2

KDR:

Kinase insert domain receptor

RTK:

Receptor tyrosine kinase

ERK:

Extracellular signal-regulated kinase

PLCγ:

Phospholipase Cγ

PDGFR:

Platelet-derived growth factor receptor

EGFR:

Epidermal growth factor receptor

FGFR:

Fibroblast growth factor receptor

SCFR:

Stem cell factor receptor

ECM:

Endothelial cell medium

ELISA:

Enzyme-linked immunosorbent assay

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Authors and Affiliations

  1. Department of Physiology, West China School of Preclinical and Forensic Medicine, Sichuan University, No. 17, Section 3 Renmin South Road, Chengdu, 610064, Sichuan, People’s Republic of China

    Sheng Zhang, Wei Fu & Limin Yue

  2. Sichuan Institute of Population and Family Planning, Chengdu University of Traditional Chinese Medicine, Chengdu, 610041, Sichuan, People’s Republic of China

    Xiaoping Gao

  3. Key Laboratory of Bio-resources and Eco-environment (Ministry of Education), College of Life Sciences, Sichuan University, Chengdu, 610064, Sichuan, People’s Republic of China

    Shengwei Li

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  1. Sheng Zhang
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  3. Wei Fu
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  4. Shengwei Li
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Correspondence to Limin Yue.

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Zhang, S., Gao, X., Fu, W. et al. Immunoglobulin-like domain 4-mediated ligand-independent dimerization triggers VEGFR-2 activation in HUVECs and VEGFR2-positive breast cancer cells. Breast Cancer Res Treat 163, 423–434 (2017). https://doi.org/10.1007/s10549-017-4189-5

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