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
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
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DOI：
10.1074/jbc.m512121200
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发表时间：
2006-05-12
影响因子：
4.8
通讯作者：
Zachary, IC
中科院分区：
文献类型：
--
作者：
Jia, HY;Bagherzadeh, A;Zachary, IC
神经纤毛蛋白-1（NP- 1）是内皮细胞中血管内皮生长因子- A（165）（VEGF-A（165））的受体.为了确定NP- 1在VEGF生物学功能中的作用，我们基于位于外显子7和8编码的VEGF-A165结构域中的NP- 1结合位点开发了VEGF与NP- 1结合的特异性肽拮抗剂。双环肽EG 3287有效地（Ki 1.2 μ M）和有效地（在100 μ M时抑制> 95%）抑制VEGF-A（165）与表达NP- 1的猪主动脉内皮细胞（PAE/ NP- 1）和仅表达VEGF-A的NP- 1受体的乳腺癌细胞的结合，但对与PAE/ KDR或PAE/ Flt- 1的结合没有影响。分子动力学计算、EG 3287的核磁共振结构和在培养基中的稳定性测定表明，它构成了与天然VEGF-A的相应区域非常相似的稳定亚结构域（165）.外显子8编码的C末端和三维结构对于EG 3287抑制NP- 1结合都至关重要，而N末端的修饰影响不大。尽管EG 3287对VEGF-A（165）与KDR受体的结合没有直接影响，但它抑制了共表达NP- 1的人脐静脉内皮细胞中VEGF-A（165）与KDR的交联，并抑制了对KDR和PLC-γ酪氨酸磷酸化的刺激、ERK 1/ 2的激活和前列腺素类的产生.这些发现表征了与NP- 1结合的VEGF-A（165）的第一个特异性拮抗剂，并证明NP- 1对于最佳KDR活化和细胞内信号传导是必需的。结果还确定了C-末端外显子8结构域在VEGF-A（165）与NP-1结合中的关键作用.
Neuropilin- 1 ( NP- 1) is a receptor for vascular endothelial growth factor- A(165)( VEGF- A(165)) 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 mu M) and effectively (> 95% inhibition at 100 mu M) inhibited VEGF- A(165) 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- A(165). 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- A(165) binding to KDR receptors, it inhibited cross- linking of VEGF- A(165) 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- A(165) 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- A(165) binding to NP-1.