GABA_B受体神经保功能的分子机制研究及其与G蛋白动态相作用研究
发布时间：2019-05-14 20:24
【摘要】：GABA_B受体是中枢神经系统中主要的抑制性神经递质??氨基丁酸(GABA)的代谢型受体,在神经元的突触前和突触后都有广泛的表达。同时,它也是GPCR C家族成员之一,与Gi/o型G蛋白相互偶联,介导缓慢而持久的神经突触活动。GABA_B受体功能的减弱或亢进会导致多种中枢神经系统疾病,如癫痫,痉挛,焦虑,抑郁,疼痛,药物成瘾,认知损伤等,是重要的药物靶点。 最近的研究表明,GABA_B受体的激活具有神经保护的功能,但是,其作用的具体分子机制还并不清楚。在本课题研究的第一部分,我们以低钾诱导的小脑颗粒神经元的凋亡作为细胞模型,确认了GABA_B受体的神经保护作用。进一步,我们发现GABA_B受体的激活介导了PI3K/Akt信号通路,并且这条信号通路在GABA_B受体介导的神经保护作用中起到了重要的作用。进而,我们有意思的发现GABA_B受体的激活可以引起IGF-1受体活性的增强。用IGF-1受体的抑制剂抑制受体活性或用RNA干扰降低IGF-1受体表达后,GABA_B受体介导的Akt的激活被抑制了。这表明GABA_B受体可以转激活IGF-1受体并进一步激活了Akt信号通路。同时,通过免疫共沉淀和配体竞争实验,我们发现GABA_B受体可以与IGF-1受体相互作用并且两者之间的转激活效应是配体非依赖的。这个研究一方面阐明了GABA_B受体神经保护功能的分子机制,暗示GABA_B受体在神经退行性疾病中潜在药理学功能;另一方面,这首次发现了GABA_B受体对RTK的转激活作用,也进一步表明了GPCR与RTK之间信号交联的重要意义。 新的蛋白质与蛋白质相互作用研究手段的建立使得传统的GPCR与G蛋白相互作用的基于“碰撞”的模型受到了挑战。在GPCR A家族成员的研究表明受体与G蛋白之间可以形成稳定的复合物,并且受体的激活并不导致G蛋白三聚体的解离。但是,对GPCR C家族成员与G蛋白的相互作用的研究还没有报道。在第二部分的研究中,我们用BRET和TR-FRET的方法研究了GABA_B受体与G蛋白的相互作用,以及受体激活前后受体与G蛋白,G蛋白G?和G??亚基之间相互作用的动态变化。我们的结果暗示G蛋白可以和GABA_B受体预结合,并且受体激活后会使得G蛋白与GABA_B受体相互作用的减弱,同时也导致了G(?)和G(?)(?)之间相互作用的变化。这些结果一方面证实了G蛋白可以受体预结合并伴随着受体的激活呈现动态的变化,另一方面也为建立基于BRET技术的GABA_B受体的新的药物筛选细胞模型提供思路。
[Abstract]:GABA_B receptor is the main inhibitory neurotransmitter in the central nervous system. The metabolic receptor of aminobutyric acid (GABA) is widely expressed in both presynaptic and postsynaptic neurons. At the same time, it is also a member of the GPCR C family, coupled with Gi/ o G protein, mediating slow and persistent synaptic activity. The weakening or hyperactivity of GABA_B receptor can lead to a variety of central nervous system diseases, such as seizures and spasms. Anxiety, depression, pain, drug addiction, cognitive impairment and so on are important drug targets. Recent studies have shown that the activation of GABA_B receptor has neuroprotective function, but the specific molecular mechanism of its action is not clear. In the first part of this study, we used the apoptosis of cerebellar granule neurons induced by low potassium as a cell model to confirm the neuroprotective effect of GABA_B receptor. Furthermore, we found that the activation of GABA_B receptor mediates the PI3K/Akt signaling pathway, and this signaling pathway plays an important role in the neuroprotective effect mediated by GABA_B receptor. Furthermore, it is interesting to find that the activation of GABA_B receptor can increase the activity of IGF-1 receptor. After inhibition of receptor activity by inhibitor of IGF-1 receptor or decrease of expression of IGF-1 receptor by RNA interference, the activation of Akt mediated by GABA_B receptor was inhibited. This suggests that GABA_B receptor can activate IGF-1 receptor and further activate Akt signaling pathway. At the same time, through immunoprecipitation and ligand competition test, we found that GABA_B receptor can interact with IGF-1 receptor and the transactivation effect between them is ligand independent. On the one hand, this study clarifies the molecular mechanism of neuroprotective function of GABA_B receptor, suggesting the potential pharmacological function of GABA_B receptor in neurodegenerative diseases. On the other hand, the transactivation of RTK by GABA_B receptor was found for the first time, which further indicated the significance of signal cross-linking between GPCR and RTK. The establishment of a new method for the study of protein-protein interaction has challenged the traditional "collision" model of the interaction between GPCR and G protein. Studies on members of the GPCR A family have shown that a stable complex can be formed between the receptor and G protein, and the activation of the receptor does not lead to the dissociation of G protein trimer. However, the interaction between GPCR C family members and G protein has not been reported. In the second part, we studied the interaction between GABA_B receptor and G protein by BRET and TR-FRET, and the interaction between GABA_B receptor and G protein before and after receptor activation. And G? The dynamic change of the interaction between subunits. Our results suggest that G protein can prebind to GABA_B receptor, and the activation of G protein can weaken the interaction between G protein and GABA_B receptor, and also lead to G (?) And G (?) A change in the interaction between them. On the one hand, these results confirm that G protein can prebind with receptor activation, and on the other hand, it also provides an idea for the establishment of a new drug screening cell model of GABA_B receptor based on BRET technology.
【学位授予单位】：华中科技大学
【学位级别】：博士
【学位授予年份】：2011
【分类号】：R341
[Abstract]:GABA_B receptor is the main inhibitory neurotransmitter in the central nervous system. The metabolic receptor of aminobutyric acid (GABA) is widely expressed in both presynaptic and postsynaptic neurons. At the same time, it is also a member of the GPCR C family, coupled with Gi/ o G protein, mediating slow and persistent synaptic activity. The weakening or hyperactivity of GABA_B receptor can lead to a variety of central nervous system diseases, such as seizures and spasms. Anxiety, depression, pain, drug addiction, cognitive impairment and so on are important drug targets. Recent studies have shown that the activation of GABA_B receptor has neuroprotective function, but the specific molecular mechanism of its action is not clear. In the first part of this study, we used the apoptosis of cerebellar granule neurons induced by low potassium as a cell model to confirm the neuroprotective effect of GABA_B receptor. Furthermore, we found that the activation of GABA_B receptor mediates the PI3K/Akt signaling pathway, and this signaling pathway plays an important role in the neuroprotective effect mediated by GABA_B receptor. Furthermore, it is interesting to find that the activation of GABA_B receptor can increase the activity of IGF-1 receptor. After inhibition of receptor activity by inhibitor of IGF-1 receptor or decrease of expression of IGF-1 receptor by RNA interference, the activation of Akt mediated by GABA_B receptor was inhibited. This suggests that GABA_B receptor can activate IGF-1 receptor and further activate Akt signaling pathway. At the same time, through immunoprecipitation and ligand competition test, we found that GABA_B receptor can interact with IGF-1 receptor and the transactivation effect between them is ligand independent. On the one hand, this study clarifies the molecular mechanism of neuroprotective function of GABA_B receptor, suggesting the potential pharmacological function of GABA_B receptor in neurodegenerative diseases. On the other hand, the transactivation of RTK by GABA_B receptor was found for the first time, which further indicated the significance of signal cross-linking between GPCR and RTK. The establishment of a new method for the study of protein-protein interaction has challenged the traditional "collision" model of the interaction between GPCR and G protein. Studies on members of the GPCR A family have shown that a stable complex can be formed between the receptor and G protein, and the activation of the receptor does not lead to the dissociation of G protein trimer. However, the interaction between GPCR C family members and G protein has not been reported. In the second part, we studied the interaction between GABA_B receptor and G protein by BRET and TR-FRET, and the interaction between GABA_B receptor and G protein before and after receptor activation. And G? The dynamic change of the interaction between subunits. Our results suggest that G protein can prebind to GABA_B receptor, and the activation of G protein can weaken the interaction between G protein and GABA_B receptor, and also lead to G (?) And G (?) A change in the interaction between them. On the one hand, these results confirm that G protein can prebind with receptor activation, and on the other hand, it also provides an idea for the establishment of a new drug screening cell model of GABA_B receptor based on BRET technology.
【学位授予单位】：华中科技大学
【学位级别】：博士
【学位授予年份】：2011
【分类号】：R341
【共引文献】
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2 银巍;黄奕俊;皮荣标;苏兴文;赵灵芝;邱鹏新;颜光美;;应用mRNA差异显示技术筛选大鼠小脑颗粒神经元凋亡模型中差异表达基因[J];中国病理生理杂志;2007年11期
3 阴正勤,余涛,陈莉;斜视性弱视猫发育过程中视皮层神经元NMDA-R1表达的免疫组织化学电镜观察[J];中华眼科杂志;2002年08期
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2 陶艳梅;细胞内钾离子稳态参与神经细胞凋亡机制的研究[D];中国科学院研究生院（上海生命科学研究院）;2005年
3 丁学锋;Cpne5基因在脑发育中的功能初步研究[D];中国人民解放军军事医学科学院;2010年
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