G蛋白偶联受体2在细胞膜上控制蜕皮激素信号通路及调控下游效应的研究

发布时间：2017-12-27 08:23

本文关键词：G蛋白偶联受体2在细胞膜上控制蜕皮激素信号通路及调控下游效应的研究　出处：《山东大学》2017年博士论文　论文类型：学位论文

【摘要】：研究背景及国内外研究进展昆虫是世界上种类最多的生物类群,其发育大多数经历蜕皮和变态两种主要的生理过程。昆虫的生长发育主要由蜕皮激素(20E)和保幼激素(JH)控制。在昆虫幼虫阶段JH滴度较高,虫体维持其幼虫状态;在蜕皮变态期JH滴度逐渐降低,而此时20E滴度逐渐升高,最终起始昆虫的蜕皮变态。这两种激素协调控制昆虫的蜕皮与变态过程。研究这一过程的分子调控机制不仅可以为害虫防治提供科学依据,而且可以为研究人类疾病的分子机制提供参考。先前研究发现蜕皮激素主要是通过核受体ecdysone receptor(EcR)途径转导信号起始昆虫的变态。近些年,随着分子生物学技术水平的日趋提高,研究人员发现了 20E激素信号通路存在非基因组途径。许多信号通路中的蛋白质会发生快速的磷酸化修饰及移位现象。20E也可动员细胞内钙离子的释放及内流。与此同时,20E非基因组途径的膜受体也成为研究的热点。目前在许多物种中都发现了 G蛋白偶联受体(GPCR)参与动物类固醇激素的非基因组信号通路,但是具体的机制尚不清楚。钙离子是细胞内的第二信使,调控多种生理过程,包括基因转录、细胞增殖和细胞凋亡等。类固醇激素20E在昆虫变态过程中促进程序性细胞死亡,而保幼激素JH拮抗20E的功能来阻止变态的发生。20E和JH都能诱导细胞内钙离子水平的增加。然而,这两种功能相互拮抗的激素诱导钙离子水平增加的分子机制及生理结果仍不明确。在这项研究中,我们筛选到在鳞翅目昆虫棉铃虫中的ErGPCR-2作为靶标基因,研究GPCR在20E非基因组途径中的功能,以及钙离子作为第二信使在20E调控的细胞凋亡中的分子机制。丰富和完善了 20E信号途径及昆虫生长发育的理论知识。同时为害虫防治及人类疾病治疗提供靶标基因和科学依据。研究结果1.G蛋白偶联受体2在细胞膜上控制蜕皮激素信号通路在这项研究中,我们筛选到了鳞翅目昆虫棉铃虫中的一个GPCR,命名为ErGPCR-2,在细胞膜上控制类固醇激素20-羟基蜕皮酮信号通路。ErGPCR-2在棉铃虫的蜕皮和变态时期具有较高的表达。20E通过ErGPCR-2调控棉铃虫表皮细胞系中钙离子水平快速变化、有关蛋白质的磷酸化、相关基因的转录以及虫体的变态发育过程。ErGPCR-2定位于细胞膜上,在20E的诱导条件下,ErGPCR-2会被G蛋白偶联受体激酶2(GRK2)所磷酸化,磷酸化修饰后的ErGPCR-2被内吞入细胞质中。内吞入细胞质后的ErGPCR-2被蛋白酶所降解,最终脱敏20E信号通路。细胞系干扰ErGPCR-2减少了 20E的类似物[3H]ponasterone A([3H]Pon A)进入细胞的量。细胞系过表达ErGPCR-2和干扰GRK2阻止ErGPCR-2的内吞,都会增加[3H]Pon A进入细胞的量。然而,我们没有检测到ErGPCR-2蛋白直接结合于[3H]Pon A。结果表明ErGPCR-2在细胞膜上传导类固醇激素20E信号并且控制20E进入细胞。2.蜕皮激素促进钙离子动员并引起细胞凋亡20E通过GPCR诱导细胞内较强的钙离子水平升高,而JH通过受体酪氨酸激酶(RTKs)诱导较小的钙离子水平增加。钙释放激活钙通道1(Orai 1)和瞬时受体电位通道(TRPs Channel),对20E诱导快速钙离子内流是必需的。相对于JH,20E处理细胞会引起钙离子维持在长时间的高水平状态,并且提高了更多的钙通道相关基因的表达,包括Orai1和TRP通道等。20E会激活Caspase3/7和促凋亡基因的表达,而JH不能。JH可以抑制20E诱导的钙内流、Caspase3/7的激活和凋亡相关基因的表达。高水平的钙离子诱导细胞凋亡。这些结果表明,20E和JH通过不同的途径调节细胞内钙并且维持钙离子在不同的稳态水平,因此导致不同的基因表达和细胞生理反应。研究结论及科学意义1.ErGPCR-2在细胞膜上控制20E进入细胞ErGPCR-2定位于细胞膜上控制[3H]Pon A进入细胞。ErGPCR-2在细胞膜上调控20E信号通路。这一研究完善了 20E非基因组途径的科学知识,建立了基因组途径与非基因组途径的连接,为今后研究20E信号途径提供了全新的理论依据。2.蜕皮激素促进钙离子动员引起细胞凋亡20E通过GPCR动员细胞内较强的钙水平升高,而JH通过RTKs动员较弱的钙水平增加。20E处理细胞会引起钙离子在细胞质中维持在长时间的高水平状态,20E上调更多钙通道相关基因的表达。20E诱导Caspase3/7活化水平、促进细胞细胞死亡和上调促凋亡基因的表达,高水平的钙离子诱导细胞凋亡。钙离子作为细胞中重要的第二信使,在细胞增殖凋亡过程中发挥重要的功能。20E通过GPCR途径动员钙离子使其维持高水平的稳态,最终细胞中高水平的钙离子诱导了细胞凋亡。这一研究证实了钙离子在细胞凋亡中的功能,不仅为完善昆虫凋亡的分子机制做出贡献,也为癌症治疗中促进癌细胞凋亡提供科学依据。
[Abstract]:Research background and research progress at home and abroad, insects are the most diverse species in the world. Their development mostly occurs through two main physiological processes: molting and metamorphosis. The development of insect mainly by ecdysone (20E) and juvenile hormone (JH) control. In insect larvae stage, JH titer is higher, and the worm body maintains its larval state. In the molting metamorphosis stage, JH titer gradually decreases, and at this time 20E titer gradually increases, and finally starts the insect's molting metamorphosis. These two hormones coordinate the molting and metamorphosis of insects. The study of the molecular mechanism of this process not only provides a scientific basis for pest control, but also provides a reference for the study of molecular mechanisms of human diseases. Previous research found that ecdysone mainly through nuclear receptor ecdysone receptor (EcR) signaling pathway starting insect metamorphosis. In recent years, with the increasing level of molecular biology, researchers have discovered that there is a non genomic pathway in the 20E hormone signaling pathway. Many proteins in the signal pathways have rapid phosphorylation and displacement. 20E can also mobilize the release and inflow of intracellular calcium ions. At the same time, the membrane receptors of the non genomic pathway of 20E have also become a hot spot of research. G protein coupled receptors (GPCR) are involved in the non genomic signaling pathways of steroid hormones in many species, but the specific mechanisms are not yet clear. Calcium ion is the second messenger within the cell that regulates a variety of physiological processes, including gene transcription, cell proliferation and cell apoptosis. Steroid hormone 20E promotes programmed cell death during insect metamorphosis, while protective hormone JH antagonists the function of 20E to prevent the occurrence of metamorphosis. Both 20E and JH can induce the increase of intracellular calcium levels. However, the molecular mechanisms and physiological results of the increased levels of calcium ion induced by these two antagonistic hormones are still unclear. In this study, we screened ErGPCR-2 in the lepidopteran insect Helicoverpa armigera as a target gene, studied the function of GPCR in 20E non genomic pathway, and the molecular mechanism of calcium as a second messenger in 20E regulated cell apoptosis. The theoretical knowledge of the 20E signal pathway and the growth and development of insects has been enriched and perfected. At the same time, it provides the target gene and scientific basis for the pest control and the treatment of human disease. The research results of 1.G protein coupled receptor 2 in the cell membrane of control ecdysone signaling pathway in this study, we screened a GPCR cotton bollworm in, named ErGPCR-2, to control the steroid 20- HYDROXYECDYSONE signaling pathway in cell membrane. ErGPCR-2 has high expression in the period of molt and metamorphosis of Helicoverpa armigera. 20E regulates the rapid change of calcium ion level, the phosphorylation of related proteins, the transcription of related genes and the metamorphosis of worms in the cotton bollworm epidermis cell line by ErGPCR-2. ErGPCR-2 was localized on the cell membrane. Under the induction of 20E, ErGPCR-2 was phosphorylated by G protein coupled receptor kinase 2 (GRK2), and phosphorylated ErGPCR-2 was swallowed into the cytoplasm. After endocytosis of the cytoplasm, ErGPCR-2 was degraded by protease and eventually desensitized to the 20E signaling pathway. The interference of ErGPCR-2 in cell lines reduces the amount of 20E's analogues, [3H]ponasterone A ([3H]Pon A), into the cell. The overexpression of ErGPCR-2 in the cell line and the interference of GRK2 to prevent endocytosis of ErGPCR-2 will increase the amount of [3H]Pon A into the cell. However, we did not detect the direct binding of ErGPCR-2 protein to [3H]Pon A. The results showed that ErGPCR-2 uploads the steroid hormone 20E signal in the cell membrane and controls the entry of 20E into the cell. Calcium mobilization and apoptosis induced by 20E strong intracellular calcium levels increased by 2. GPCR and JH by promoting ecdysone, receptor tyrosine kinase (RTKs) levels of calcium induced smaller increase. Calcium release activates calcium channel 1 (Orai 1) and transient receptor potential channel (TRPs Channel), which is essential for 20E induced fast calcium influx. Compared with JH, 20E treated cells can cause calcium to maintain at a high level for a long time, and increase the expression of more calcium channel related genes, including Orai1 and TRP channels. 20E activates Caspase3/7 and the expression of apoptotic genes, but JH does not. JH can inhibit 20E induced calcium influx, activation of Caspase3/7 and the expression of apoptosis related genes. High levels of calcium induced apoptosis. These results indicate that 20E and JH regulate intracellular calcium through different pathways and maintain calcium at different steady-state levels, resulting in different gene expression and cellular physiological responses. Research conclusions and scientific significance 1.ErGPCR-2 control the entry of 20E into cell ErGPCR-2 on cell membrane to control the entry of [3H]Pon A into the cell membrane. ErGPCR-2 regulates the 20E signaling pathway on the cell membrane. This research has improved the scientific knowledge of 20E's non genomic approach, and established the connection between genomic pathways and non genomic pathways, providing a new theoretical basis for future research on 20E signaling pathways. Calcium mobilization induced apoptosis through 20E GPCR mobilization of intracellular calcium level of high calcium levels and promote 2. ecdysone, JH mobilized by RTKs is increased. 20E treatment cells cause calcium ions to maintain a long high level in the cytoplasm, and 20E up-regulated the expression of more calcium channel related genes. 20E induced Caspase3/7 activation level, cell death and up regulation of apoptosis gene expression, and high level of calcium ion induced apoptosis. As an important second messenger in cells, calcium ions play an important role in the process of cell proliferation and apoptosis. 20E mobilizes calcium ions by GPCR to maintain a high level of homeostasis, and at last the high level of calcium ions in the cells induce apoptosis. This study confirmed the apoptosis of calcium ions in cells
【学位授予单位】：山东大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：Q25

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