發(fā)布時間：2018-04-23 11:06

  本文選題：G蛋白偶聯(lián)受體 + G蛋白　； 參考：《曲阜師范大學(xué)》2014年碩士論文

【摘要】：G蛋白偶聯(lián)受體（G protein-coupled receptor, GPCRs）是迄今發(fā)現(xiàn)的最大的膜整合蛋白受體超家族之一，廣泛分布于人體的各個組織器官中，參與縱多的生理病理反應(yīng)，是重要的藥物靶點。GPCRs主要通過激活G蛋白調(diào)節(jié)下游信號分子而參與某些生理病理反應(yīng)，因此受體與蛋白的相互作用是整個跨膜信號轉(zhuǎn)導(dǎo)的關(guān)鍵。傳統(tǒng)觀點認(rèn)為，GPCRs激活G蛋白后，Gα-GTP與Gβγ亞基相互分離，被激活的Gα亞基通過信號轉(zhuǎn)導(dǎo)進一步調(diào)節(jié)細(xì)胞內(nèi)的某些生理反應(yīng)。近期研究表明， GPCRs和G蛋白存在多種偶聯(lián)關(guān)系，GPCRs不僅能夠選擇性地激活Gα亞基，還可以與Gβγ亞基相互靠近，甚至?xí)笹蛋白亞基構(gòu)象發(fā)生重排而不分離，這為進一步研究受體與蛋白的相互作用關(guān)系提供了新思路。 Apelin受體（putative receptor protein related to AT1，APJ）是1993年O’Dowd等發(fā)現(xiàn)的孤兒GPCRs，廣泛分布于血管系統(tǒng)與中樞神經(jīng)系統(tǒng)，成為治療缺血性心臟病、原發(fā)性高血壓、心力衰竭等最具潛力的藥物靶點之一。Apelin受體作為GPCRs家族中的一員，也主要是通過偶聯(lián)G蛋白進而參與調(diào)節(jié)下游信號轉(zhuǎn)導(dǎo)通路的。那么，Apelin受體會激活哪種類型的G蛋白、引起G蛋白的活化方式是怎樣的以及會對信號轉(zhuǎn)導(dǎo)產(chǎn)生怎樣的影響，至今還不清楚。 熒光共振能量轉(zhuǎn)移技術(shù)（fluorescence resonance energy transfer technology, FRET）與生物發(fā)光共振能量轉(zhuǎn)移技術(shù)（bioluminescence resonance energy transfer technology, BRET）是近幾十年發(fā)展起來的用于研究活細(xì)胞中大分子之間相互作用的新技術(shù)。利用FRET與BRET技術(shù)能夠在活細(xì)胞中實時、動態(tài)、連續(xù)地監(jiān)測Apelin受體偶聯(lián)G蛋白的動力學(xué)關(guān)系，這為實驗的順利開展提供了有力的研究手段。 本課題旨在研究Apelin受體與G蛋白的相互作用關(guān)系。試圖探明由APJ受體介導(dǎo)激活的G蛋白的活化機制，即Gα亞基與Gβγ亞基彼此分離亦或G蛋白構(gòu)象發(fā)生重排而不分離。為Apelin受體參與的相關(guān)生理功能及多種疾病的發(fā)病機制提供實驗依據(jù)，為相關(guān)藥物的研發(fā)提供新的作用靶點。 本實驗通過構(gòu)建Apelin受體與各亞型G蛋白的多種表達(dá)載體，通過瞬時轉(zhuǎn)染人胚腎（human embryonic kidkey, HEK293）細(xì)胞，建立共表達(dá)Apelin受體與某一亞型G蛋白的細(xì)胞系。然后通過FRET與BRET技術(shù)研究Apelin受體與各亞基之間的相互作用關(guān)系，，即采用FRET與BRET技術(shù)在活細(xì)胞內(nèi)實時、動態(tài)地監(jiān)測Apelin受體介導(dǎo)激活的Gαi1、Gαi2、Gαi3、Gαo與Gαq蛋白的活化機制。首先利用FRET與BRET技術(shù)在HEK293T細(xì)胞中研究Apelin受體介導(dǎo)激活的Gαi1蛋白的活化機制，加入激動劑Apelin-13刺激后，F(xiàn)RET與BRET信號幾乎沒有變化。其次利用FRET與BRET技術(shù)在HEK293T細(xì)胞中繼續(xù)探討由Apelin-13誘導(dǎo)激活的Gαi2與Gαi3蛋白的活化，當(dāng)加入激動劑Apelin-13刺激后，F(xiàn)RET與BRET數(shù)值發(fā)生明顯的上升現(xiàn)象。進一步檢測其它亞家族的G蛋白活化時是否與Gαi2、Gαi3亞基活化一致，即利用FRET與BRET技術(shù)繼續(xù)探討了Gαo、Gαq與Gβ1γ2的相互作用關(guān)系。研究發(fā)現(xiàn)，加入激動劑Apelin-13刺激后，F(xiàn)RET與BRET信號與Gαi2、Gαi3亞基相反，出現(xiàn)了降低的現(xiàn)象。 通過實驗充分證實了由APJ受體激活的Gαi2、Gαi3亞型的G蛋白構(gòu)象發(fā)生重排而不分離，而Gαo、Gαq亞基與Gβ1γ2亞基活化時彼此分離，并發(fā)現(xiàn)APJ受體可能不激活Gαi1亞基或引起極少部分Gαi1亞基的活化。這為APJ受體跨膜信號轉(zhuǎn)導(dǎo)機制的研究提供了堅實的理論基礎(chǔ)，有助于闡明Apelin受體在相關(guān)疾病發(fā)病中的作用機理，為研發(fā)靶向于Apelin受體的相關(guān)藥物提供了新的思路，具有重要的推廣價值。
[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.

【學(xué)位授予單位】：曲阜師范大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2014
【分類號】：R3411

【參考文獻】

相關(guān)期刊論文 前2條

1 李雅林;白波;陳京;劉有旺;;Apelin/APJ系統(tǒng)研究進展[J];第四軍醫(yī)大學(xué)學(xué)報;2009年20期

2 王瓏瓏;黃e

本文編號：1791685