Role of Grb2 in EGF-stimulated EGFR internalization.
Role of Grb2 in EGF-stimulated EGFR internalization.
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发表时间：
2002-05
影响因子：
4
通讯作者：
T. Yamazaki;K. Zaal;D. Hailey;J. Presley;J. Lippincott-Schwartz;L. Samelson
中科院分区：
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作者：
T. Yamazaki;K. Zaal;D. Hailey;J. Presley;J. Lippincott-Schwartz;L. Samelson
Grb 2是一种衔接分子，其将膜受体如表皮生长因子受体（EGFR）与细胞内信号传导途径偶联。为了深入了解EGF激活后这些分子的运输途径，我们在单个活细胞中观察了Grb 2和EGFR与GFP光谱变体的融合。在nonstimulated细胞，Grb 2-YFP主要是本地化弥漫在细胞质中，而EGFR-CFP被发现在质膜上和内吞结构定位在核周区。在EGF刺激的1分钟内，Grb 2重新分布到质膜，在那里它以SH 2依赖性方式结合EGFR-CFP。然后质膜开始动态地起皱，并且发现Grb 2-YFP和EGFR-CFP在大的巨胞饮结构中一起内化。这些结构在形态上不同于常规的网格蛋白衍生的内体，并且不标记有转铁蛋白、AP-2或网格蛋白重链。这些结构不需要网格蛋白进行内化的证据来自于显示AP-180的C-末端的表达（其抑制转铁蛋白摄取）对EGF诱导的EGFR内化没有影响的实验。YFP标记的Grb 2含有抑制性突变的N-或C-SH 3结构域重新分布到质膜EGF刺激后，但巨胞饮结构含有Grb 2-YFP和EGFR-CFP没有向内易位，似乎仍然拴系到质膜。这表明Grb 2 SH 3结构域负责将含有EGFR的膜与参与这些膜内化的下游效应物偶联。在所有SH 3结构域突变体的存在下，转铁蛋白摄取不受影响，这与EGF刺激的EGFR内化途径不依赖网格蛋白一致。这些结果证明了Grb 2在与活化细胞中EGFR的巨胞饮内化途径相关的事件中的作用。
Grb2 is an adaptor molecule that couples membrane receptors such as the epidermal growth factor receptor (EGFR) to intracellular signaling pathways. To gain insight into the trafficking pathways followed by these molecules after activation by EGF, we visualized Grb2 and EGFR fused to GFP spectral variants in single live cells. In nonstimulated cells, Grb2-YFP was primarily localized diffusely in the cytoplasm, whereas EGFR-CFP was found on the plasma membrane and in endocytic structures localized in the perinuclear area. Within 1 minute of EGF stimulation, Grb2 redistributed to the plasma membrane where it bound EGFR-CFP in an SH2 dependent manner. The plasma membrane then began to dynamically ruffle, and Grb2-YFP and EGFR-CFP were found to internalize together in large macropinocytic structures. These structures were morphologically distinct from conventional, clathrin-derived endosomes and did not label with transferrin, AP-2 or clathrin heavy chain. Evidence that these structures did not require clathrin for internalization came from experiments showing that expression of the C-terminus of AP-180, which inhibited transferrin uptake, had no effect on EGF-induced internalization of EGFR. YFP-tagged Grb2 containing an inhibitory mutation in either N- or C-SH3 domain redistributed to the plasma membrane upon EGF stimulation, but the macropinocytic structures containing Grb2-YFP and EGFR-CFP did not translocate inward and appeared to remain tethered to the plasma membrane. This suggested that the Grb2 SH3 domain was responsible for coupling the membranes containing EGFR with downstream effectors involved in internalization of these membranes. Transferrin uptake was unaffected in the presence of all of the SH3 domain mutants, consistent with the EGF-stimulated EGFR internalization pathway being clathrin-independent. These results demonstrate a role for Grb2 in events associated with a macropinocytic internalization pathway for EGFR in activated cells.