【摘要】：GABA_B受體是中樞神經(jīng)系統(tǒng)中主要的抑制性神經(jīng)遞質(zhì)??氨基丁酸(GABA)的代謝型受體,在神經(jīng)元的突觸前和突觸后都有廣泛的表達(dá)。同時(shí),它也是GPCR C家族成員之一,與Gi/o型G蛋白相互偶聯(lián),介導(dǎo)緩慢而持久的神經(jīng)突觸活動(dòng)。GABA_B受體功能的減弱或亢進(jìn)會(huì)導(dǎo)致多種中樞神經(jīng)系統(tǒng)疾病,如癲癇,痙攣,焦慮,抑郁,疼痛,藥物成癮,認(rèn)知損傷等,是重要的藥物靶點(diǎn)。 最近的研究表明,GABA_B受體的激活具有神經(jīng)保護(hù)的功能,但是,其作用的具體分子機(jī)制還并不清楚。在本課題研究的第一部分,我們以低鉀誘導(dǎo)的小腦顆粒神經(jīng)元的凋亡作為細(xì)胞模型,確認(rèn)了GABA_B受體的神經(jīng)保護(hù)作用。進(jìn)一步,我們發(fā)現(xiàn)GABA_B受體的激活介導(dǎo)了PI3K/Akt信號(hào)通路,并且這條信號(hào)通路在GABA_B受體介導(dǎo)的神經(jīng)保護(hù)作用中起到了重要的作用。進(jìn)而,我們有意思的發(fā)現(xiàn)GABA_B受體的激活可以引起IGF-1受體活性的增強(qiáng)。用IGF-1受體的抑制劑抑制受體活性或用RNA干擾降低IGF-1受體表達(dá)后,GABA_B受體介導(dǎo)的Akt的激活被抑制了。這表明GABA_B受體可以轉(zhuǎn)激活I(lǐng)GF-1受體并進(jìn)一步激活了Akt信號(hào)通路。同時(shí),通過(guò)免疫共沉淀和配體競(jìng)爭(zhēng)實(shí)驗(yàn),我們發(fā)現(xiàn)GABA_B受體可以與IGF-1受體相互作用并且兩者之間的轉(zhuǎn)激活效應(yīng)是配體非依賴的。這個(gè)研究一方面闡明了GABA_B受體神經(jīng)保護(hù)功能的分子機(jī)制,暗示GABA_B受體在神經(jīng)退行性疾病中潛在藥理學(xué)功能;另一方面,這首次發(fā)現(xiàn)了GABA_B受體對(duì)RTK的轉(zhuǎn)激活作用,也進(jìn)一步表明了GPCR與RTK之間信號(hào)交聯(lián)的重要意義。 新的蛋白質(zhì)與蛋白質(zhì)相互作用研究手段的建立使得傳統(tǒng)的GPCR與G蛋白相互作用的基于“碰撞”的模型受到了挑戰(zhàn)。在GPCR A家族成員的研究表明受體與G蛋白之間可以形成穩(wěn)定的復(fù)合物,并且受體的激活并不導(dǎo)致G蛋白三聚體的解離。但是,對(duì)GPCR C家族成員與G蛋白的相互作用的研究還沒(méi)有報(bào)道。在第二部分的研究中,我們用BRET和TR-FRET的方法研究了GABA_B受體與G蛋白的相互作用,以及受體激活前后受體與G蛋白,G蛋白G?和G??亞基之間相互作用的動(dòng)態(tài)變化。我們的結(jié)果暗示G蛋白可以和GABA_B受體預(yù)結(jié)合,并且受體激活后會(huì)使得G蛋白與GABA_B受體相互作用的減弱,同時(shí)也導(dǎo)致了G(?)和G(?)(?)之間相互作用的變化。這些結(jié)果一方面證實(shí)了G蛋白可以受體預(yù)結(jié)合并伴隨著受體的激活呈現(xiàn)動(dòng)態(tài)的變化,另一方面也為建立基于BRET技術(shù)的GABA_B受體的新的藥物篩選細(xì)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.
【學(xué)位授予單位】：華中科技大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2011
【分類號(hào)】：R341

【共引文獻(xiàn)】

相關(guān)期刊論文 前3條

1 張楠楠;梁錦鋒;宋淑亮;吉愛(ài)國(guó);;神經(jīng)發(fā)育毒性動(dòng)物實(shí)驗(yàn)替代方法研究進(jìn)展[J];中國(guó)藥理學(xué)與毒理學(xué)雜志;2012年01期

2 銀巍;黃奕俊;皮榮標(biāo);蘇興文;趙靈芝;邱鵬新;顏光美;;應(yīng)用mRNA差異顯示技術(shù)篩選大鼠小腦顆粒神經(jīng)元凋亡模型中差異表達(dá)基因[J];中國(guó)病理生理雜志;2007年11期

3 陰正勤,余濤,陳莉;斜視性弱視貓發(fā)育過(guò)程中視皮層神經(jīng)元NMDA-R1表達(dá)的免疫組織化學(xué)電鏡觀察[J];中華眼科雜志;2002年08期

相關(guān)博士學(xué)位論文 前3條

1 許嬋娟;GABA_B受體神經(jīng)保護(hù)功能的分子機(jī)制研究及其與G蛋白動(dòng)態(tài)相互作用研究[D];華中科技大學(xué);2011年

2 陶艷梅;細(xì)胞內(nèi)鉀離子穩(wěn)態(tài)參與神經(jīng)細(xì)胞凋亡機(jī)制的研究[D];中國(guó)科學(xué)院研究生院（上海生命科學(xué)研究院）;2005年

3 丁學(xué)鋒;Cpne5基因在腦發(fā)育中的功能初步研究[D];中國(guó)人民解放軍軍事醫(yī)學(xué)科學(xué)院;2010年

相關(guān)碩士學(xué)位論文 前1條

1 孫R，

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