title: Immunoglobulin-like domain 4-mediated ligand-independent dimerization triggers VEGFR-2 activation in HUVECs and VEGFR2-positive breast cancer cells.
abstract: OBJECTIVE
  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.
authors.affiliation: 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.
authors.forename: Sheng
authors.initials: S
authors.lastname: Zhang
authors.affiliation: Sichuan Institute of Population and Family Planning, Chengdu University of Traditional Chinese Medicine, Chengdu, 610041, Sichuan, People's Republic of China.
authors.forename: Xiaoping
authors.initials: X
authors.lastname: Gao
authors.affiliation: 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.
authors.forename: Wei
authors.initials: W
authors.lastname: Fu
authors.affiliation: 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.
authors.forename: Shengwei
authors.initials: S
authors.lastname: Li
authors.affiliation: 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. yuelimin@scu.edu.cn.
authors.forename: Limin
authors.initials: L
authors.lastname: Yue
mesh_terms: D001665:Binding Sites; D001943:Breast Neoplasms / Q000235:genetics* / Q000473:pathology; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000235:genetics; D042783:Endothelial Cells / Q000378:metabolism / Q000473:pathology; D005260:Female; D061307:Human Umbilical Vein Endothelial Cells; D006801:Humans; D000070557:Immunoglobulin Domains / Q000235:genetics*; D008024:Ligands; D010766:Phosphorylation; D011485:Protein Binding / Q000235:genetics; D055503:Protein Multimerization / Q000235:genetics; D015398:Signal Transduction; D042461:Vascular Endothelial Growth Factor A / Q000737:chemistry / Q000235:genetics; D040301:Vascular Endothelial Growth Factor Receptor-2 / Q000737:chemistry / Q000235:genetics*
chemical_list: D008024:Ligands; C467484:VEGFA protein, human; D042461:Vascular Endothelial Growth Factor A; C000588866:KDR protein, human; D040301:Vascular Endothelial Growth Factor Receptor-2
keywords: Activation; Breast cancer cells; Dimerization; Domain 4; HUVECs; VEGFR-2