Wang Yi, Pennock Steven, Chen Xinmei, Wang Zhixiang
Department of Cell Biology and Signal Transduction Research Group, Faculty of Medicine and Dentistry, University of Alberta, Edmonton, Alberta, T6G 2H7, Canada.
Sci STKE. 2002 Dec 3;2002(161):pl17. doi: 10.1126/stke.2002.161.pl17.
Despite intensive efforts to understand cell signaling from endosomes, there is no direct evidence demonstrating that endosomal signaling is sufficient to activate signal transduction pathways or that endosomal signaling can produce biological responses. The lack of breakthrough is due in part to the inability to generate endosomal signals in isolation from plasma membrane signals. In this Protocol, we describe a system in which epidermal growth factor (EGF) receptor (EGFR) is specifically activated when it is endocytosed into endosomes. We treated cells with EGF in the presence of AG1478, a specific EGFR tyrosine kinase inhibitor, and monensin, which blocks recycling of EGFR. This treatment led to the internalization of nonactivated EGF-EGFR complex into endosomes. The endosome-associated EGFR was then activated by removing AG1478 and monensin. During this procedure, we did not observe any detectable surface EGFR phosphorylation. We also achieved specific activation of endosome-associated EGFR without using monensin. Specific activation of endosome-associated EGFR provides a unique tool to study endosomal signaling of EGFR. This method may also be applied to other receptor tyrosine kinases to study whether they, too, can signal from endosomes.
尽管人们为了解内体的细胞信号传导付出了巨大努力，但尚无直接证据表明内体信号传导足以激活信号转导途径，或者内体信号传导能够产生生物学反应。缺乏突破部分归因于无法在与质膜信号隔离的情况下产生内体信号。在本实验方案中，我们描述了一种系统，其中表皮生长因子（EGF）受体（EGFR）在被内吞入内体时会被特异性激活。我们在AG1478（一种特异性EGFR酪氨酸激酶抑制剂）和莫能菌素存在的情况下用EGF处理细胞，莫能菌素会阻断EGFR的再循环。这种处理导致未活化的EGF-EGFR复合物内化到内体中。然后通过去除AG1478和莫能菌素激活与内体相关的EGFR。在此过程中，我们未观察到任何可检测到的表面EGFR磷酸化。我们还在不使用莫能菌素的情况下实现了与内体相关的EGFR的特异性激活。与内体相关的EGFR的特异性激活为研究EGFR的内体信号传导提供了一种独特的工具。该方法也可应用于其他受体酪氨酸激酶，以研究它们是否也能从内体发出信号。
