Breast Cancer Research and Treatment

, Volume 163, Issue 3, pp 423–434 | Cite as

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

  • Sheng Zhang
  • Xiaoping Gao
  • Wei Fu
  • Shengwei Li
  • Limin YueEmail author
Preclinical study



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.


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.


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.


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


VEGFR-2 Domain 4 Dimerization Activation HUVECs Breast cancer cells 



Vascular endothelial growth factor


Vascular endothelial growth factor receptor-2


Kinase insert domain receptor


Receptor tyrosine kinase


Extracellular signal-regulated kinase


Phospholipase Cγ


Platelet-derived growth factor receptor


Epidermal growth factor receptor


Fibroblast growth factor receptor


Stem cell factor receptor


Endothelial cell medium


Enzyme-linked immunosorbent assay


Compliance with ethical standards

Conflict of Interest

All authors declare no actual, potential, or perceived conflict of interest that would prejudice the impartiality of the article.

Ethical approval

This article does not contain any studies with human participants or animals performed by any of the authors.

Supplementary material

10549_2017_4189_MOESM1_ESM.docx (19 kb)
Supplementary material 1 (DOCX 19 kb)
10549_2017_4189_MOESM2_ESM.docx (315 kb)
Supplementary material 2 (DOCX 314 kb)


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Copyright information

© Springer Science+Business Media New York 2017

Authors and Affiliations

  • Sheng Zhang
    • 1
  • Xiaoping Gao
    • 2
  • Wei Fu
    • 1
  • Shengwei Li
    • 3
  • Limin Yue
    • 1
    Email author
  1. 1.Department of Physiology, West China School of Preclinical and Forensic MedicineSichuan UniversityChengduPeople’s Republic of China
  2. 2.Sichuan Institute of Population and Family PlanningChengdu University of Traditional Chinese MedicineChengduPeople’s Republic of China
  3. 3.Key Laboratory of Bio-resources and Eco-environment (Ministry of Education), College of Life SciencesSichuan UniversityChengduPeople’s Republic of China

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