Internalization of the epidermal growth factor receptor: role in signalling
Internalization of the epidermal growth factor receptor: role in signalling
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DOI：
10.1042/bst0290480
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发表时间：
2001-08-01
影响因子：
3.9
通讯作者：
Sorkin, A
中科院分区：
文献类型：
--
作者：
Sorkin, A
激活的表皮生长因子 (EGF) 受体 (EGFR) 与 Grb2（生长因子受体结合蛋白 2）的 Src 同源 2 (SH2) 结构域的相互作用通过 Ras 和丝裂原激活蛋白激酶启动信号传导，Grb2 可以直接或通过另一种包含 SH2 的蛋白 Shc 结合 EGFR。配体激活 EGFR 还会引发 EGF 受体复合物的快速内吞作用。为了分析活细胞中 EGFR 与 SH2 结构域相互作用的空间和时间调节，我们将成像显微镜与一种改进的荧光共振能量转移 (FRET) 测量方法相结合，使用与青色荧光蛋白 (CFP) 融合的 EGFR 与与黄色荧光蛋白 (YFP) 融合的 Grb2 或 Shc 配对。 EGF 刺激导致 Grb2-YFP 和 YFP-Shc 募集到含有 EGFR-CFP 的细胞区室中，并且 FRET 信号大幅增加。特别是，FRET 测量表明，活化的 EGFR-CFP 与膜皱褶和内体中的 YFP-Shc 和 Grb2-YFP 相互作用。这些结果表明，通过 EGFR 的信号传导可以发生在内体区室中。此外，与之前的生化研究相比，FRET实验表明，在EGF刺激后，大量的Grb2和Shc在很长一段时间内与EGFR相关。
The interaction of the activated epidermal growth factor (EGF) receptor (EGFR) with the Src homology 2 (SH2) domain of Grb2 (growth-factor-receptor-bound protein 2) initiates signalling through Ras and mitogen-activated protein kinase, Grb2 can bind EGFR directly or through another SH2-containing protein, Shc. Activation of EGFRs by ligand also triggers rapid endocytosis of EGF-receptor complexes. To analyse the spatial and temporal regulation of EGFR interactions with SH2 domains in living cells, we have combined imaging microscopy with a modified method of measuring fluorescence resonance energy transfer (FRET) on a pixel-by-pixel basis using EGFR fused to cyan fluorescent protein (CFP) in pair with Grb2 or Shc fused to yellow fluorescent protein (YFP). Stimulation by EGF resulted in the recruitment of Grb2-YFP and YFP-Shc to cellular compartments that contained EGFR-CFP, and a large increase in the FRET signal. In particular, FRET measurements indicated that activated EGFR-CFP interacted with YFP-Shc and Grb2-YFP in membrane ruffles and endosomes. These results demonstrate that signalling via EGFRs can occur in the endosomal compartment. Moreover, in contrast with previous biochemical studies, FRET experiments show that a large pool of Grb2 and Shc is associated with EGFRs for a prolonged period after EGF stimulation.