Trafficking of TrkA-green fluorescent protein chimerae during nerve growth factor-induced differentiation
Trafficking of TrkA-green fluorescent protein chimerae during nerve growth factor-induced differentiation
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DOI：
10.1074/jbc.m202401200
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发表时间：
2003-03-07
影响因子：
4.8
通讯作者：
Rudkin, BB
中科院分区：
文献类型：
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作者：
Jullien, J;Guili, V;Rudkin, BB
通过与TrkA的羧基端同相表达GFP，构建神经生长因子（NGF）受体、TrkA和绿色荧光蛋白（GFP）的嵌合体。TrkA-GFP响应于NGF而在酪氨酸残基上磷酸化，并且能够启动信号级联，导致PC 12 nnr 5细胞中延长的MAPK活化和分化。TrkA构建体，在羧基末端结构域中逐渐截短，制备成GFP嵌合体，以鉴定受体胞内结构域的哪一部分参与其运输。免疫荧光观察显示TrkA-GFP主要存在于细胞表面膜皱褶和内体中。生化分析表明，TrkA的胞质结构域不是受体正确成熟和细胞表面易位所必需的。使用针对TrkA的细胞外结构域的抗体（RTA）作为配体以刺激TrkA的内化和磷酸化。用抗磷酸化TrkA抗体进行的共定位研究支持这种复合物在从细胞表面传播信号传导中的作用，导致TrkA在缺乏受体-配体复合物的内体区域中活化。共聚焦时间推移分析表明，TrkA-GFP嵌合体显示出细胞表面和内部位置之间的高度动态的贩运。TrkA阳性囊泡估计为0.46 +/- 0.09 mum/s顺行和0.48 +/- 0.07 mum/s逆行移动。这种方法和TrkA-GFP的生物化学性质的保真度证明了在配体存在或不存在的情况下酪氨酸激酶受体的运输的实时可视化是可行的。
A chimera of the nerve growth factor (NGF) receptor, TrkA, and green fluorescent protein (GFP) was engineered by expressing GFP in phase with the carboxyl terminus of TrkA. TrkA-GFP becomes phosphorylated on tyrosine residues in response to NGF and is capable of initiating signaling cascades leading to prolonged MAPK activation and differentiation in PC12 nnr5 cells. TrkA constructs, progressively truncated in the carboxyl-terminal domain, were prepared as GFP chimerae in order to identify which part of the receptor intracellular domain is involved in its trafficking. Immunofluorescence observations show that TrkA-GFP is found mainly in cell surface membrane ruffles and in endosomes. Biochemical analysis indicated that the cytoplasmic domain of TrkA is not necessary for correct maturation and cell surface translocation of the receptor. An antibody against the extracellular domain of TrkA (RTA) was used as ligand to stimulate internalization and phosphorylation of TrkA. Co-localization studies with anti-phosphorylated TrkA antibodies support a role for such complexes in the propagation of signaling from the cell surface, resulting in the activation of TrkA in areas of the endosome devoid of receptor-ligand complexes. Confocal time-lapse analysis reveals that the TrkA-GFP chimera shows highly dynamic trafficking between the cell surface and internal locations. TrkA-positive vesicles were estimated to move 0.46 +/- 0.09 mum/s anterograde and 0.48 +/- 0.07 mum/s retrograde. This approach and the fidelity of the biochemical properties of the TrkA-GFP demonstrate that real-time visualization of trafficking of tyrosine kinase receptors in the presence or absence of the ligand is feasible.