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活细胞荧光成像显示,在胞吞作用期间,Grb2与表皮生长因子受体结合的化学计量比很高且保持稳定。

Live-cell fluorescence imaging reveals high stoichiometry of Grb2 binding to the EGF receptor sustained during endocytosis.

作者信息

Fortian Arola, Sorkin Alexander

机构信息

Department of Cell Biology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261, USA.

出版信息

J Cell Sci. 2014 Jan 15;127(Pt 2):432-44. doi: 10.1242/jcs.137786. Epub 2013 Nov 20.

Abstract

Activation of epidermal growth factor (EGF) receptor (EGFR) leads to its interaction with Grb2, a dual-function adapter mediating both signaling through Ras and receptor endocytosis. We used time-lapse three-dimensional imaging by spinning disk confocal microscopy to analyze trafficking of EGFR and Grb2 in living HeLa cells stimulated with low, physiological concentrations of EGFR ligands. Endogenous Grb2 was replaced in these cells by Grb2 fused to yellow fluorescent protein (YFP). After transient residence in the plasma membrane, Rhodamine-conjugated EGF (EGF-Rh) and Grb2-YFP were rapidly internalized and accumulated in endosomes. Quantitative image analysis revealed that on average two Grb2-YFP molecules were colocalized with one EGF-Rh in cells stimulated with 2 ng/ml EGF-Rh, and the excess of Grb2-YFP over EGF-Rh was even higher when a receptor-saturating concentration of EGF-Rh was used. Therefore, we hypothesize that a single EGFR molecule can be simultaneously associated with functionally distinct Grb2 interaction partners during and after endocytosis. Continuous presence of Grb2-YFP in endosomes was also observed when EGFR was activated by transforming growth factor-α and amphiregulin, suggesting that endosomal EGFRs remain ligand occupied and signaling competent, despite the fact that these growth factors are thought to dissociate from the receptor at acidic pH. The prolonged localization and activity of EGFR-Grb2 complexes in endosomes correlated with the sustained activation of extracellular stimulus-regulated kinase 1/2, suggesting that endosomal EGFRs contribute significantly to this signaling pathway. We propose that endosomal EGFRs function to extend signaling in time and space to compensate for rapid downregulation of surface EGFRs in cells with low receptor expression levels.

摘要

表皮生长因子(EGF)受体(EGFR)的激活导致其与Grb2相互作用,Grb2是一种双功能衔接蛋白,介导通过Ras的信号传导和受体的内吞作用。我们使用转盘共聚焦显微镜进行延时三维成像,以分析在生理浓度的低剂量EGF配体刺激下,活的HeLa细胞中EGFR和Grb2的运输情况。在这些细胞中,内源性Grb2被融合了黄色荧光蛋白(YFP)的Grb2所取代。在质膜短暂停留后,罗丹明偶联的EGF(EGF-Rh)和Grb2-YFP迅速内化并积聚在内体中。定量图像分析显示,在用2 ng/ml EGF-Rh刺激的细胞中,平均两个Grb2-YFP分子与一个EGF-Rh共定位,当使用受体饱和浓度的EGF-Rh时,Grb2-YFP相对于EGF-Rh的过量甚至更高。因此,我们推测单个EGFR分子在胞吞作用期间和之后可同时与功能不同的Grb2相互作用伙伴相关联。当EGFR被转化生长因子-α和双调蛋白激活时,也观察到内体中持续存在Grb2-YFP,这表明内体中的EGFRs仍然被配体占据并具有信号传导能力,尽管这些生长因子被认为在酸性pH下会与受体解离。EGFR-Grb2复合物在内体中的延长定位和活性与细胞外信号调节激酶1/2的持续激活相关,这表明内体中的EGFRs对该信号通路有显著贡献。我们提出,内体中的EGFRs发挥作用,在时间和空间上扩展信号传导,以补偿低受体表达水平细胞中表面EGFRs的快速下调。

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