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Ligand Binding Domains of human Vascular Endothelial Growth Factor Receptor
Li Ma1, Xiao-Ning Wang, Zhi-Qing Zhang
1Institute of Molecular Immunology, The First Military Medical University, Guangzhou 510515, China. hmzmary@hotmail.com
Abstract:
Four cDNA clones, encoding truncated Flt-1 mutants consisting of loop 2, 1-2, 2-3 and 1-3, were amplified by PCR from human placenta cDNA library and the corresponding gene fragments were named Flt-1(2) Flt-1(1-2) Flt-1(2-3) and Flt-1(1-3). In order to detect which part of the extracellular domain was involved in ligand binding, the interaction between Flt-1 mutants and hVEGF(165) was studied with yeast two-hybrid system. The data presented here suggest that both Flt-1(2-3) and Flt-1(1-3) were able to bind hVEGF(165). Two recombinant expression plasmids, pPIC9K/Flt-1(1-3) and pPIC9K/Flt-1(2-3), were constructed and transformed into Pichia pastoris strain GS115, respectively. After 4 days of methanol induction, the amount of the expressed Flt-1s reached 60% of total proteins in supernatant by SDS-PAGE. Recombinant proteins were purified with CM-Sepharose Fast Flow and Sephacryl S-100 chromatography. Biological activities analysis proved that 1-3 loop had slightly better biological activity than 2-3 loop in VEGF(165) binding and in inhibition of HUVEC proliferation stimulated by hVEGF(165).
Insights
The Flt-1 (fms-like tyrosine kinase 1) extracellular domain
Area of Science:
- Molecular Biology
- Biochemistry
- Cell Biology
Background:
- Vascular Endothelial Growth Factor (VEGF) signaling is crucial for angiogenesis.
- Flt-1 (fms-like tyrosine kinase 1) is a receptor tyrosine kinase that binds VEGF and modulates its activity.
- Understanding the specific domains of Flt-1 involved in VEGF binding is essential for therapeutic development.
Purpose of the Study:
- To identify the specific extracellular domains of Flt-1 responsible for binding human VEGF (hVEGF).
- To generate and characterize recombinant Flt-1 mutants for functional studies.
- To compare the biological activity of different Flt-1 extracellular domain fragments in VEGF binding and inhibition of HUVEC proliferation.
Main Methods:
- Polymerase chain reaction (PCR) amplification of Flt-1 extracellular domain mutants (Flt-1(2), Flt-1(1-2), Flt-1(2-3), Flt-1(1-3)).
- Yeast two-hybrid system to assess the interaction between Flt-1 mutants and hVEGF(165).
- Recombinant protein expression in Pichia pastoris, purification using chromatography, and SDS-PAGE analysis.
- Biological activity assays, including VEGF(165) binding and inhibition of VEGF-stimulated HUVEC proliferation.
Main Results:
- Flt-1(2-3) and Flt-1(1-3) mutants demonstrated binding to hVEGF(165) in the yeast two-hybrid system.
- Recombinant Flt-1(1-3) and Flt-1(2-3) proteins were successfully expressed in Pichia pastoris and purified.
- The 1-3 loop of Flt-1 exhibited slightly higher biological activity in VEGF(165) binding and inhibition of HUVEC proliferation compared to the 2-3 loop.
Conclusions:
- The extracellular domain fragments Flt-1(2-3) and Flt-1(1-3) are involved in hVEGF(165) binding.
- The Flt-1 1-3 loop possesses potent biological activity in mediating VEGF binding and inhibiting endothelial cell proliferation.
- These findings provide insights into the structure-function relationship of Flt-1 and its interaction with VEGF.