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

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.