VEGF-A 是肿瘤血管生成中的关键细胞因子，也是癌症的主要治疗靶点。 VEGF165 是 VEGF-A 的主要亚型，也是最有效的血管生成刺激剂。 VEGFR2/KDR 结构域 2 和 3 (D2D3) 与 VEGF165 的 N 末端结构域（NTD，残基 1-110）结合。由于肝素结合结构域（HBD，残基 111-165）的去除显着降低了生长因子的有丝分裂活性，因此有人提出 HBD 在 VEGF165 的有丝分裂活性中起着关键作用。在这里，我们报告 αvβ3 特异性结合分离的 VEGF165 HBD，但不结合 VEGF165 NTD。基于对接模拟和诱变，我们鉴定了 VEGF165 HBD 内 αvβ3 结合所需的几个关键氨基酸残基，即 Arg123、Arg124、Lys125、Lys140、Arg145 和 Arg149。我们发现 VEGF165 HBD 与 KDR 结构域 1 (D1) 结合，并通过诱变鉴定出 Arg123 和 Arg124 对于 KDR D1 结合至关重要，表明 KDR D1 结合位点和 αvβ3 结合位点在 HBD 中重叠。 αvβ3 和 KDR D1 结合缺陷的全长 VEGF165 突变体（R123A/R124A/K125A/K140A/R145A/R149A）未能诱导 ERK1/2 磷酸化、整合素 β3 磷酸化和 KDR 磷酸化，并且不支持内皮细胞增殖。该突变不影响 KDR D2D3 与 VEGF165 的相互作用。由于已知 β3 敲除小鼠表现出增强的 VEGF165 信号传导，因此我们提出 KDR D1 与 VEGF165 HBD 的结合以及 KDR D2D3 与 VEGF165 NTD 的结合在 VEGF165 的有效有丝分裂活性中至关重要。我们提出，KDR 和 αvβ3 与 VEGF165 HBD 之间的结合竞争赋予整合素 αvβ3 调节特性，充当 VEGF165 信号传导的负调节因子。版权所有 © 2024 Takada、Yu、Ye、Wu、Felding、Fujita 和 Takada。

VEGF-A is a key cytokine in tumor angiogenesis and a major therapeutic target for cancer. VEGF165 is the predominant isoform of VEGF-A, and it is the most potent angiogenesis stimulant. VEGFR2/KDR domains 2 and 3 (D2D3) bind to the N-terminal domain (NTD, residues 1-110) of VEGF165. Since removal of the heparin-binding domain (HBD, residues 111-165) markedly reduced the mitogenic activity of the growth factor, it has been proposed that the HBD plays a critical role in the mitogenicity of VEGF165. Here, we report that αvβ3 specifically bound to the isolated VEGF165 HBD but not to VEGF165 NTD. Based on docking simulation and mutagenesis, we identified several critical amino acid residues within the VEGF165 HBD required for αvβ3 binding, i.e., Arg123, Arg124, Lys125, Lys140, Arg145, and Arg149. We discovered that VEGF165 HBD binds to the KDR domain 1 (D1) and identified that Arg123 and Arg124 are critical for KDR D1 binding by mutagenesis, indicating that the KDR D1-binding and αvβ3-binding sites overlap in the HBD. Full-length VEGF165 mutant (R123A/R124A/K125A/K140A/R145A/R149A) defective in αvβ3 and KDR D1 binding failed to induce ERK1/2 phosphorylation, integrin β3 phosphorylation, and KDR phosphorylation and did not support proliferation of endothelial cells, although the mutation did not affect the KDR D2D3 interaction with VEGF165. Since β3-knockout mice are known to show enhanced VEGF165 signaling, we propose that the binding of KDR D1 to the VEGF165 HBD and KDR D2D3 binding to the VEGF165 NTD are critically involved in the potent mitogenicity of VEGF165. We propose that binding competition between KDR and αvβ3 to the VEGF165 HBD endows integrin αvβ3 with regulatory properties to act as a negative regulator of VEGF165 signaling.Copyright © 2024 Takada, Yu, Ye, Wu, Felding, Fujita and Takada.