Apelin受体与G蛋白相互作用关系的研究

发布时间：2018-04-23 11:06

本文选题：G蛋白偶联受体 + G蛋白　；参考：《曲阜师范大学》2014年硕士论文

【摘要】：G蛋白偶联受体（G protein-coupled receptor, GPCRs）是迄今发现的最大的膜整合蛋白受体超家族之一，广泛分布于人体的各个组织器官中，参与纵多的生理病理反应，是重要的药物靶点。GPCRs主要通过激活G蛋白调节下游信号分子而参与某些生理病理反应，因此受体与蛋白的相互作用是整个跨膜信号转导的关键。传统观点认为，GPCRs激活G蛋白后，Gα-GTP与Gβγ亚基相互分离，被激活的Gα亚基通过信号转导进一步调节细胞内的某些生理反应。近期研究表明， GPCRs和G蛋白存在多种偶联关系，GPCRs不仅能够选择性地激活Gα亚基，还可以与Gβγ亚基相互靠近，甚至会使G蛋白亚基构象发生重排而不分离，这为进一步研究受体与蛋白的相互作用关系提供了新思路。 Apelin受体（putative receptor protein related to AT1，APJ）是1993年O’Dowd等发现的孤儿GPCRs，广泛分布于血管系统与中枢神经系统，成为治疗缺血性心脏病、原发性高血压、心力衰竭等最具潜力的药物靶点之一。Apelin受体作为GPCRs家族中的一员，也主要是通过偶联G蛋白进而参与调节下游信号转导通路的。那么，Apelin受体会激活哪种类型的G蛋白、引起G蛋白的活化方式是怎样的以及会对信号转导产生怎样的影响，至今还不清楚。荧光共振能量转移技术（fluorescence resonance energy transfer technology, FRET）与生物发光共振能量转移技术（bioluminescence resonance energy transfer technology, BRET）是近几十年发展起来的用于研究活细胞中大分子之间相互作用的新技术。利用FRET与BRET技术能够在活细胞中实时、动态、连续地监测Apelin受体偶联G蛋白的动力学关系，这为实验的顺利开展提供了有力的研究手段。本课题旨在研究Apelin受体与G蛋白的相互作用关系。试图探明由APJ受体介导激活的G蛋白的活化机制，即Gα亚基与Gβγ亚基彼此分离亦或G蛋白构象发生重排而不分离。为Apelin受体参与的相关生理功能及多种疾病的发病机制提供实验依据，为相关药物的研发提供新的作用靶点。本实验通过构建Apelin受体与各亚型G蛋白的多种表达载体，通过瞬时转染人胚肾（human embryonic kidkey, HEK293）细胞，建立共表达Apelin受体与某一亚型G蛋白的细胞系。然后通过FRET与BRET技术研究Apelin受体与各亚基之间的相互作用关系，，即采用FRET与BRET技术在活细胞内实时、动态地监测Apelin受体介导激活的Gαi1、Gαi2、Gαi3、Gαo与Gαq蛋白的活化机制。首先利用FRET与BRET技术在HEK293T细胞中研究Apelin受体介导激活的Gαi1蛋白的活化机制，加入激动剂Apelin-13刺激后，FRET与BRET信号几乎没有变化。其次利用FRET与BRET技术在HEK293T细胞中继续探讨由Apelin-13诱导激活的Gαi2与Gαi3蛋白的活化，当加入激动剂Apelin-13刺激后，FRET与BRET数值发生明显的上升现象。进一步检测其它亚家族的G蛋白活化时是否与Gαi2、Gαi3亚基活化一致，即利用FRET与BRET技术继续探讨了Gαo、Gαq与Gβ1γ2的相互作用关系。研究发现，加入激动剂Apelin-13刺激后，FRET与BRET信号与Gαi2、Gαi3亚基相反，出现了降低的现象。通过实验充分证实了由APJ受体激活的Gαi2、Gαi3亚型的G蛋白构象发生重排而不分离，而Gαo、Gαq亚基与Gβ1γ2亚基活化时彼此分离，并发现APJ受体可能不激活Gαi1亚基或引起极少部分Gαi1亚基的活化。这为APJ受体跨膜信号转导机制的研究提供了坚实的理论基础，有助于阐明Apelin受体在相关疾病发病中的作用机理，为研发靶向于Apelin受体的相关药物提供了新的思路，具有重要的推广价值。
[Abstract]:The G protein coupling receptor (G protein-coupled receptor, GPCRs) is one of the largest membrane integrin receptor superfamilies found so far. It is widely distributed in various tissues and organs of the human body and participates in the physiological and pathological reactions in the longitudinal multi body. It is an important drug target.GPCRs to participate in some physiology mainly through the regulation of the downstream signal molecules by stimulating the active G protein. The interaction of the receptor and protein is the key to the whole transmembrane signal transduction. The traditional view is that, after GPCRs activates the G protein, the G alpha -GTP is separated from the G beta subunit, and the activated G alpha subunit further regulates some physiological responses in the cell through signal transduction. Recent studies have shown that there are many coupling between GPCRs and G protein. GPCRs can not only selectively activate G alpha subunits but also close to the G beta subunits, even make the conformation of G protein subunits rearranged and not separated. This provides a new idea for further study of the interaction between the receptor and protein.
The Apelin receptor (putative receptor protein related to AT1, APJ) is an orphan GPCRs found in 1993 O 'Dowd and so on. It is widely distributed in the vascular system and central nervous system, and is one of the most potential drug targets for the treatment of ischemic heart disease, primary hypertension and heart failure as one of the members of the family. It is by coupling G protein to regulate the downstream signal transduction pathway. Then, what type of G protein is activated by Apelin, what is the activation of G protein and how it affects signal transduction? It is not clear to date.
Fluorescence resonance energy transfer (fluorescence resonance energy transfer technology, FRET) and bioluminescence resonance energy transfer technology (bioluminescence resonance energy transfer technology, BRET) are a new technology developed in recent decades to study the interaction between large molecules in living cells. Technology can monitor the dynamic relationship of Apelin receptor coupled with G protein in real time, dynamically and continuously, which provides a powerful research method for the smooth development of the experiment.
The purpose of this study is to investigate the interaction between the Apelin receptor and the G protein. The purpose of this study is to explore the activation mechanism of the G protein activated by the APJ receptor, namely, the separation of the G alpha subunit from the G beta subunit or the rearrangement of the conformation of the G protein, and not to be separated. It provides an experimental basis for the related physiological functions of Apelin receptor and the pathogenesis of a variety of diseases. It provides new targets for the research and development of related drugs.
In this experiment, by constructing a variety of expression vectors of Apelin receptor and various subtypes of G protein, by transient transfection of human embryonic kidney (human embryonic kidkey, HEK293) cells, a cell line that CO expressed Apelin receptor and a certain subtype G protein was established. Then the interaction between Apelin receptor and subunits was studied by FRET and BRET technology. RET and BRET technology can dynamically monitor the activation of G alpha I1, G alpha I2, G alpha I3, G alpha O and the activation mechanism of G alpha protein in live cells. Secondly, the activation of G alpha I2 and G alpha I3 protein activated by Apelin-13 was further explored in HEK293T cells using FRET and BRET technology. When the activator was added to the Apelin-13 stimulation, FRET and BRET value increased obviously. The interaction between G alpha o, G alpha q and G beta 1 gamma 2 was further investigated by FRET and BRET technology. It was found that, after the stimulation of the agonist Apelin-13, the FRET and BRET signals were contrary to G alpha I2.
The experiment fully confirmed the rearrangement of the G protein conformation of the G alpha I2 activated by the APJ receptor, the rearrangement of the G protein conformation of the G alpha I3 subtype, and the separation of G alpha o, G alpha q subunit and G beta 1 gamma 2 subunit, and found that the APJ receptor may not activate the alpha subunit or the activation of a few parts of the subunit. It provides a solid theoretical basis and helps to elucidate the mechanism of Apelin receptor in the pathogenesis of related diseases. It provides a new way of thinking for developing Apelin receptor related drugs, and has important promotion value.

【学位授予单位】：曲阜师范大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R3411

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