Expression of a neuronal nicotinic acetylcholine receptor in insect and mammalian host cell systems
Expression of a neuronal nicotinic acetylcholine receptor in insect and mammalian host cell systems
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DOI：
10.1023/a:1007512121082
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发表时间：
2000-01-01
影响因子：
4.4
通讯作者：
Barrantes, FJ
中科院分区：
文献类型：
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作者：
Aztiria, EM;Sogayar, MC;Barrantes, FJ
对哺乳动物和昆虫不同的体细胞系统在转录和翻译水平上表达、折叠和组装α7型神经元乙酰胆碱受体(AChR)的能力进行了测试。为此，我们使用了来源于神经脊的克隆细胞系，如来自大鼠肾上腺嗜铬细胞瘤的PC12细胞，来自大鼠垂体瘤的GH3细胞，以及非神经细胞，如来自NIH瑞士小鼠胚胎的NIH-3T3成纤维细胞和来自草地贪夜蛾卵巢组织的Sf9细胞。用Northern印迹法分析转染或未转染的PC12、GH3或3T3细胞以及重组AcNPV感染和模拟感染的Sf9细胞的总RNA。内源性表达α7 AChR的PC12细胞及其所有异源的α7转基因克隆，显示出可变但通常高数量的单个转录本。GH3和NIH-3T3转基因克隆和重组AcNPV感染的Sf9细胞表达不同水平的α7-mRNA，只有一个转录本与28S大鼠rRNA共迁移。根据[I-125]银环蛇毒素结合能力的测量，只有神经脊来源的细胞系似乎在功能上表达α7 AChR。结果表明，α7的异源表达不是在转录水平上调节的，而是在翻译后水平上调节的，并且并不是所有的宿主细胞系统都表达正确的翻译后修饰所需的细胞因子，从而导致成熟和功能的α7 AChR。此外，结果表明，严格控制的表达机制与这个古老的胆碱能序列平行进化。
Different mammalian and insect somatic host cell systems were tested in their ability to express, fold, and assemble alpha 7-type neuronal acetylcholine receptor (AChR) both at the transcriptional and translational level. For this purpose we employed clonal cell lines derived from the neural crest, such as PC12 cells from a rat adrenal pheochromocytoma, and GH3 cells isolated from a rat pituitary tumor, as well as non-neuronal cells such as NIH-3T3 fibroblasts from embryonic NIH Swiss mouse and Sf9 cells from ovary tissue of the Spodoptera frugiperda butterfly. Total RNA, isolated from either transfected or non-transfected PC12, GH3 or 3T3 cells, or recombinant AcNPV-infected and mock-infected Sf9 cells was analyzed by Northern blot. PC12 cells, which endogenously express alpha 7 AChR, and all its heterologous alpha 7-transfectant clones, exhibited variable but generally high amounts of a single transcript. GH3 and NIH-3T3 transfectant clones and recombinant AcNPV-infected Sf9 cells expressed variable levels of alpha 7-mRNA, with a single transcript that co-migrated with the 28S rat rRNA. Only the neural crest-derived cell lines appeared to functionally express the alpha 7 AChR, as measured by their [I-125]alpha-bungarotoxin binding ability. The results suggest that heterologous expression of alpha 7 is regulated not at the transcriptional, but at the postranslational level and that not all host cell systems appear to express the cellular factors needed for the correct postranslational modifications leading to mature and functional alpha 7 AChR. Furthermore, the results suggest that tightly controlled expression mechanisms have evolved in parallel with this ancient cholinergic sequence.