Endocytosis of functional epidermal growth factor receptor-green fluorescent protein chimera
Endocytosis of functional epidermal growth factor receptor-green fluorescent protein chimera
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DOI：
10.1074/jbc.273.52.35000
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发表时间：
1998-12-25
影响因子：
4.8
通讯作者：
Sorkin, A
中科院分区：
文献类型：
--
作者：
Carter, RE;Sorkin, A
表皮生长因子受体（EGFR）和绿色荧光蛋白（GFP）的嵌合体已经通过将GFP融合到EGFR的羧基末端而被工程化。提供数据以证明GFP部分不影响EGFR的预期功能。EGFR-GFP响应EGF而在酪氨酸残基处磷酸化，并且能够磷酸化内源性底物并启动信号级联。EGF依赖性协会的嵌合受体与网格蛋白衔接蛋白AP-2，参与内吞作用，并与Shc衔接蛋白，结合在融合点附近，不受GFP部分。受体下调和内化的发生率与表达野生型EGFR的细胞相似。蛋白质印迹分析表明，EGFR-GFP的溶酶体降解从细胞外结构域进行，并且GFP不被优先切割。使用数字去卷积显微镜在活细胞中EGFR-GFP和德克萨斯红缀合的EGF的时间依赖性共定位证明了配体-受体复合物通过早期和多泡内体的运输，随后在内吞作用的后期阶段配体和受体分离。内吞作用早期阶段的延时光学分析揭示了EGFR-GFP在管状囊泡内体隔室中的定位。观察到这些隔室内的膜运动和融合的快速动力学。该方法和EGFR-GFP的生物化学性质的保真度证明，在配体存在或不存在的情况下酪氨酸激酶受体的运输和蛋白质相互作用的实时可视化是可行的。
A chimera of the epidermal growth factor receptor (EGFR) and green fluorescent protein (GFP) has been engineered by fusing GFP to the carboxyl terminus of EGFR. Data are provided to demonstrate that the GFP moiety does not affect the expected functioning of EGFR. EGFR-GFP becomes phosphorylated at tyrosine residues in response to EGF and is capable of phosphorylating endogenous substrates and initiating signaling cascades. EGF-dependent association of the chimeric receptor with the clathrin adaptor protein AP-2, involved in endocytosis, and with Shc adaptor protein, which binds in close proximity to the fusion point, is not affected by the GFP moiety. Receptor down-regulation and internalization occur at rates similar to those in cells expressing wild-type EGFR. Western blot analysis reveals that lysosomal degradation of EGFR-GFP proceeds from the extracellular domain and that GFP is not preferentially cleaved. Time-dependent co-localization of EGFR-GFP and Texas Red-conjugated EGF in living cells using digital deconvolution microscopy demonstrates the trafficking of ligand-receptor complexes through the early and multivesicular endosomes followed by segregation of the ligand and receptor at the late stages of endocytosis. Time-lapse optical analysis of the early stages of endocytosis reveals localization of EGFR-GFP in the tubular-vesicular endosomal compartments. Rapid dynamics of membrane movement and fusion within these compartments were observed. This approach and the fidelity of the biochemical properties of the EGFR-GFP demonstrate that real-time visualization of trafficking and protein interactions of tyrosine kinase receptors in the presence or absence of the ligand are feasible.