Coordinated traffic of Grb2 and Ras during epidermal growth factor receptor endocytosis visualized in living cells
Coordinated traffic of Grb2 and Ras during epidermal growth factor receptor endocytosis visualized in living cells
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DOI：
10.1091/mbc.01-11-0552
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发表时间：
2002-05-01
影响因子：
3.3
通讯作者：
Sorkin, A
中科院分区：
文献类型：
--
作者：
Jiang, XJ;Sorkin, A
表皮生长因子受体(EGFR)的激活触发了多条信号通路和表皮生长因子受体复合体的快速内吞作用。为了直观地显示参与主要信号级联、RAS GTP酶激活的分子的区划，我们构建了Grb2、Shc、H-RAS和K-RAS与增强型青色荧光蛋白(CFP)或黄色荧光蛋白(YFP)的融合，并使用活细胞荧光成像显微镜结合荧光共振能量转移(FRET)技术。EGF刺激细胞后，导致Grb2-CFP和YFP-Shc在内体大量积聚，这两种接头蛋白与EGFR形成复合体。H-RAS和K-RAS融合蛋白存在于细胞膜，特别是褶皱和片状脂膜，也存在于内体中，与GTP/GDP负荷和EGF刺激无关。内体H-RAS的相对含量高于K-RAS，而K-RAS主要分布在质膜上。在EGF的应用中，Grb2和RAS通过含有Grb2或RAS的内涵体融合或通过质膜上的两种蛋白的联合内化而汇聚在同一内体中。为了研究GTP结合形式的RAS的定位，我们使用了FRET方法，该方法利用了GTP结合的CFP-RAS与c-Raf的YFP融合的RAS结合域的特异性相互作用。FRET显微镜显示，与GTP结合的RAS位于质膜上，主要位于皱纹和细胞边缘，以及含有EGFR的内体。这些数据表明，内体有可能成为通过RAS产生持久信号的场所。
Activation of the epidermal growth factor receptor (EGFR) triggers multiple signaling pathways and rapid endocytosis of the epidermal growth factor (EGF)-receptor complexes. To directly visualize the compartmentalization of molecules involved in the major signaling cascade, activation of Ras GTPase, we constructed fusions of Grb2, Shc, H-Ras, and K-Ras with enhanced cyan fluorescent protein (CFP) or yellow fluorescent protein (YFP), and used live-cell fluorescence imaging microscopy combined with the fluorescence resonance energy transfer (FRET) technique. Stimulation of cells by EGF resulted in the accumulation of large pools of Grb2-CFP and YFP-Shc in endosomes, where these two adaptor proteins formed a complex with EGFR. H-Ras and K-Ras fusion proteins were found at the plasma membrane, particularly in ruffles and lamellipodia, and also in endosomes independently of GTP/GDP loading and EGF stimulation. The relative amount of endosomal H-Ras was higher than that of K-Ras, whereas K-Ras predominated at the plasma membrane. On application of EGF, Grb2, and Ras converge in the same endosomes through the fusion of endosomes containing either Grb2 or Ras or through the joint internalization of two proteins from the plasma membrane. To examine the localization of the GTP-bound form of Ras, we used a FRET assay that exploits the specific interaction of GTP-bound CFP-Ras with the YFP-fused Ras binding domain of c-Raf. FRET microscopy revealed that GTP-bound Ras is located at the plasma membrane, mainly in ruffles and at the cell edges, as well as in endosomes containing EGFR. These data point to the potential for endosomes to serve as sites of generation for persistent signaling through Ras.