A FRET-based probe for epidermal growth factor receptor bound non-covalently to a pair of synthetic amphipathic helixes
A FRET-based probe for epidermal growth factor receptor bound non-covalently to a pair of synthetic amphipathic helixes
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DOI：
10.1016/j.yexcr.2005.02.026
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发表时间：
2005-07-01
影响因子：
3.7
通讯作者：
Matsuda, M
中科院分区：
文献类型：
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作者：
Itoh, RE;Kurokawa, K;Matsuda, M
表皮生长因子(EGF)受体在多种细胞功能中起着关键作用，如增殖、分化和迁移。为了监测活细胞中表皮生长因子受体(EGFR)的活性，我们基于荧光共振能量转移(FRET)的原理开发了一种EGFR活性的探针。此前，我们开发了一种名为Picchu(CrkII嵌合单元的磷酸化指示剂)的探针，它检测CrkII接头蛋白的酪氨酸磷酸化。我们使用一对合成的两亲性螺旋WinZipA2和WinZipB1将Picchu非共价结合到EGFR的羧基-Tenning上。使用这种名为Picchu-Z的改良探针，跟踪EGF刺激的Cos7细胞中EGFR的活性。我们发现，Picchu-Z探针的高水平酪氨酸磷酸化在内吞作用后保持不变，直到EGFR移位到核周。这些发现与先前报道的“信号内体”模型是一致的。此外，通过用EGF脉冲刺激和用AG1478急性阻断EGFR活性，表明在EGF存在的情况下，Picchu-Z探针的磷酸化，可能也包括EGFR的磷酸化，在磷酸化和去磷酸化状态之间经历了快速平衡(tau(1/2)和lt；2min)。(C)2005 Elsevier Inc.保留所有权利。
Epidermal growth factor (EGF) receptor plays a pivotal role in a variety of cellular functions, such as proliferation, differentiation, and migration. To monitor the EGF receptor (EGFR) activity in living cells, we developed a probe for EGFR activity based on the principle of fluorescence resonance energy transfer (FRET). Previously, we developed a probe designated as Picchu (Phosphorylation indicator of the CrkII chimeric unit), which detects the tyrosine phosphorylation of the CrkII adaptor protein. We used a pair of synthetic amphipathic helixes, WinZipA2 and WinZipB1, to bind Picchu non-covalently to the carboxyl-tenninus of the EGFR. Using this modified probe named Picchu-Z, the activity of EGFR was followed in EGF-stimulated Cos7 cells. We found that a high level of tyrosine phosphorylation of Picchu-Z probe remained after endocytosis until the point when the EGFR was translocated to the perinuclear region. These findings are in agreement with the previously reported "signaling endosome" model. Furthermore, by pulse stimulation with EGF and by acute ablation of EGFR activity with AG1478, it was suggested that the phosphorylation of Picchu-Z probe, and probably the phosphorylation of EGFR also, underwent a rapid equilibriurn (tau(1/2) < 2 min) between the phosphorylated and dephosphorylated states in the presence of EGF. (c) 2005 Elsevier Inc. All rights reserved.