University of Leicester, Department of Cardiovascular Sciences, RKCSB, PO Box 65, Leicester LE2 7LX, UK.
Cell Signal. 2010 Mar;22(3):527-32. doi: 10.1016/j.cellsig.2009.11.007.
Angiopoietin-1 (Ang1) and Ang2 are ligands for the receptor tyrosine kinase Tie2. Structural data suggest that the two ligands bind Tie2 similarly. However, in endothelial cells Ang1 activates Tie2 whereas Ang2 can act as an apparent antagonist. In addition, each ligand exhibits distinct kinetics of release following binding. These observations suggest that additional factors influence function and binding of angiopoietins with receptors in the cellular context. Previous work has shown that Ang1 binding and activation of Tie2 are inhibited by Tie1, a related receptor that complexes with Tie2 in cells. In this study we have investigated binding of Ang1 and Ang2 to Tie2 in endothelial cells. In contrast to Ang1, binding of Ang2 to Tie2 was found to be not affected by Tie1. Neither PMA-induced Tie1 ectodomain cleavage nor suppression of Tie1 expression by siRNA affected the ability of Ang2 to bind Tie2. Analysis of the level of Tie1 co-immunoprecipitating with angiopoietin-bound Tie2 demonstrated that Ang2 can bind Tie2 in Tie2:Tie1 complexes whereas Ang1 preferentially binds non-complexed Tie2. Stimulation of Tie1 ectodomain cleavage did not increase the agonist activity of Ang2 for Tie2. Similarly, the Tie2-agonist activity of Ang2 was not affected by siRNA suppression of Tie1 expression. Consistent with previous reports, loss of Tie1 ectodomain enhanced the agonist activity of Ang1 for Tie2. Importantly, Ang2 was still able to antagonize the elevated Ang1-activation of Tie2 that occurs on Tie1 ectodomain loss. Together these data demonstrate that Ang1 and Ang2 bind differently to Tie2 at the cell surface and this is controlled by Tie1. This differential regulation of angiopoietin binding allows control of Tie2 activation response to Ang1 without affecting Ang2 agonist activity and maintains the ability of Ang2 to antagonize even the enhanced Ang1 activation of Tie2 that occurs on loss of Tie1 ectodomain. This provides a mechanism by which signalling through Tie2 can be modified by stimuli in the cellular microenvironment.
血管生成素 1（Ang1）和 Ang2 是受体酪氨酸激酶 Tie2 的配体。结构数据表明，这两种配体以相似的方式结合 Tie2。然而，在内皮细胞中，Ang1 激活 Tie2，而 Ang2 可以作为明显的拮抗剂。此外，在结合后，每种配体的释放动力学都不同。这些观察结果表明，在细胞环境中，其他因素会影响配体与受体的功能和结合。先前的工作表明，Ang1 结合和 Tie2 的激活受 Tie1 抑制，Tie1 是一种与细胞中的 Tie2 形成复合物的相关受体。在这项研究中，我们研究了 Ang1 和 Ang2 与内皮细胞中 Tie2 的结合。与 Ang1 相反，发现 Ang2 与 Tie2 的结合不受 Tie1 影响。PMA 诱导的 Tie1 胞外结构域裂解或 siRNA 抑制 Tie1 表达均不影响 Ang2 结合 Tie2 的能力。分析与血管生成素结合的 Tie2 共免疫沉淀的 Tie1 水平表明，Ang2 可以与 Tie2:Tie1 复合物结合，而 Ang1 则优先与非复合物结合的 Tie2 结合。刺激 Tie1 胞外结构域裂解并未增加 Ang2 对 Tie2 的激动剂活性。同样，siRNA 抑制 Tie1 表达也不影响 Ang2 对 Tie2 的激动剂活性。与先前的报道一致，缺失 Tie1 胞外结构域增强了 Ang1 对 Tie2 的激动剂活性。重要的是，即使在 Tie1 胞外结构域缺失导致 Ang1 激活增加的情况下，Ang2 仍能够拮抗 Tie2。这些数据表明，Ang1 和 Ang2 在细胞表面以不同的方式结合 Tie2，这由 Tie1 控制。这种对血管生成素结合的差异调节允许控制 Tie2 对 Ang1 的激活反应，而不影响 Ang2 的激动剂活性，并维持 Ang2 拮抗即使在 Tie1 胞外结构域缺失导致的 Ang1 激活增加的能力。这为细胞微环境中的刺激可以修饰 Tie2 信号提供了一种机制。
