Characterization of a bicyclic peptide neuropilin-1 (NP-1) antagonist (EG3287) reveals importance of vascular endothelial growth factor exon 8 for NP-1 binding and role of NP-1 in KDR signaling.

Abstract

Neuropilin-1 (NP-1) is a receptor for vascular endothelial growth factor-A165 (VEGF-A165) in endothelial cells. To define the role of NP-1 in the biological functions of VEGF, we developed a specific peptide antagonist of VEGF binding to NP-1 based on the NP-1 binding site located in the exon 7- and 8-encoded VEGF-A165 domain. The bicyclic peptide, EG3287, potently (K(i) 1.2 microM) and effectively (>95% inhibition at 100 microM) inhibited VEGF-A165 binding to porcine aortic endothelial cells expressing NP-1 (PAE/NP-1) and breast carcinoma cells expressing only NP-1 receptors for VEGF-A, but had no effect on binding to PAE/KDR or PAE/Flt-1. Molecular dynamics calculations, a nuclear magnetic resonance structure of EG3287, and determination of stability in media, indicated that it constitutes a stable subdomain very similar to the corresponding region of native VEGF-A165. The C terminus encoded by exon 8 and the three-dimensional structure were both critical for EG3287 inhibition of NP-1 binding, whereas modifications at the N terminus had little effect. Although EG3287 had no direct effect on VEGF-A165 binding to KDR receptors, it inhibited cross-linking of VEGF-A165 to KDR in human umbilical vein endothelial cells co-expressing NP-1, and inhibited stimulation of KDR and PLC-gamma tyrosine phosphorylation, activation of ERKs1/2 and prostanoid production. These findings characterize the first specific antagonist of VEGF-A165 binding to NP-1 and demonstrate that NP-1 is essential for optimum KDR activation and intracellular signaling. The results also identify a key role for the C-terminal exon 8 domain in VEGF-A165 binding to NP-1.

• Authors: Haiyan Jia, Azadeh Bagherzadeh, Basil Hartzoulakis, Ashley Jarvis, Marianne Löhr, Shaheda Shaikh, Rehan Aqil, Lili Cheng, Michelle Tickner, Diego Esposito, Richard Harris, Paul C Driscoll, David L Selwood, Ian C Zachary
• Journal: The Journal of biological chemistry (J Biol Chem) Vol. 281 Issue 19 Pg. 13493-13502 (May 12 2006) ISSN: 0021-9258 [Print] United States
• PMID: 16513643 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)

Chemical References

• Peptide Fragments
• Vascular Endothelial Growth Factor A
• vascular endothelial growth factor A (138-165)
• Neuropilin-1
• Vascular Endothelial Growth Factor Receptor-2

Topics

• Amino Acid Sequence
• Animals
• Cells, Cultured
• Endothelial Cells (drug effects, metabolism)
• Humans
• Neuropilin-1 (antagonists & inhibitors, metabolism)
• Peptide Fragments (pharmacology)
• Protein Binding
• Protein Conformation
• Signal Transduction
• Swine
• Vascular Endothelial Growth Factor A (metabolism, pharmacology)
• Vascular Endothelial Growth Factor Receptor-2 (metabolism)