EphA2 receptor activation by monomeric Ephrin-A1 on supported membranes.
EphA2 receptor activation by monomeric Ephrin-A1 on supported membranes.
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DOI：
10.1016/j.bpj.2011.10.039
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发表时间：
2011-12
影响因子：
3.4
通讯作者：
Qian Xu;Wan-Chen Lin;Rebecca S. Petit;J. Groves
中科院分区：
文献类型：
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作者：
Qian Xu;Wan-Chen Lin;Rebecca S. Petit;J. Groves
受体酪氨酸激酶EphA2与其糖基磷脂酰肌醇（GPI）连接的ephrin-A1配体以近肽构型相互作用。可溶性ephrin-A1蛋白，没有GPI膜连接物，不能激活EphA2。然而，聚簇前ephrin-A1蛋白在溶液中是有活性的，并且经常被用来触发EphA2受体。尽管这种方法已经对EphA2信号传导产生了深入的了解，但聚簇前配体绕过了自然受体聚簇过程，从而掩盖了聚簇作为信号调节机制的任何作用。在这里，我们展示了表达epha2的细胞，其融合蛋白是单体ephrin-A1 （mEA1）和增强的单体黄色荧光蛋白，该蛋白通过镍-十组氨酸锚点连接到支持的脂质双分子层。通过荧光成像、相关光谱和光子计数直方图分析，mEA1分别具有均匀分散、横向移动和单体的特性。通过EphA2的磷酸化和降解检测，以这种方式呈现的Ephrin-A1激活了MDA-MB-231人乳腺癌细胞表面的EphA2。在空间突变实验中，底物上的纳米图案限制了mEA1在支持的脂质双分子层中的运动，揭示了该信号通路的空间机械调节，这与最近报道的使用合成交联ephrin-A1二聚体的观察结果一致。
The receptor tyrosine kinase EphA2 interacts with its glycosylphosphatidylinositol (GPI)-linked ephrin-A1 ligand in a juxtacrine configuration. The soluble ephrin-A1 protein, without its GPI membrane linker, fails to activate EphA2. However, preclustered ephrin-A1 protein is active in solution and has been frequently used to trigger the EphA2 receptor. Although this approach has yielded insights into EphA2 signaling, preclustered ligands bypass natural receptor clustering processes and thus mask any role of clustering as a signal regulatory mechanism. Here, we present EphA2-expressing cells with a fusion protein of monomeric ephrin-A1 (mEA1) and enhanced monomeric yellow fluorescent protein that is linked to a supported lipid bilayer via a nickel-decahistidine anchor. The mEA1 is homogeneously dispersed, laterally mobile, and monomeric as measured by fluorescence imaging, correlation spectroscopy, and photon counting histogram analysis, respectively. Ephrin-A1 presented in this manner activates EphA2 on the surface of MDA-MB-231 human breast cancer cells, as measured by EphA2 phosphorylation and degradation. Spatial mutation experiments in which nanopatterns on the underlying substrate restrict mEA1 movement in the supported lipid bilayer reveal spatio-mechanical regulation of this signaling pathway, consistent with recently reported observations using a synthetically cross-linked ephrin-A1 dimer.