發(fā)布時(shí)間：2018-06-06 02:22

  本文選題：G蛋白βγ亞基 + G蛋白偶聯(lián)受體�。� 參考：《吉林大學(xué)》2009年碩士論文

【摘要】： 本實(shí)驗(yàn)研究了G蛋白βγ亞基(Gβγ)對(duì)G蛋白偶聯(lián)受體激酶-2(GRK2)介導(dǎo)的G蛋白偶聯(lián)受體,如毒蕈堿乙酰膽堿m2受體(mAChR2)和β2腎上腺素能受體(β2-AR)磷酸化的影響,證明G蛋白βγ亞基在調(diào)節(jié)G蛋白偶聯(lián)受體激酶活性中有著重要作用。探討G蛋白βγ亞基如何影響mAChR2和β2-AR受體磷酸化,引發(fā)涉及受體磷酸化的級(jí)聯(lián)反應(yīng)。揭示在G蛋白信號(hào)通路的作用中,Gβγ亞基復(fù)合物通過直接激活某些胞內(nèi)靶分子可能存在新的調(diào)控分子機(jī)制。 本實(shí)驗(yàn)首先利用親和層析方法從大鼠腦中純化了mAChR2;再通過DEAE-Sepharose FF、Sephacryl S-300 HR和Phenyl-Sepharose CL-4B三個(gè)層析柱從豬腦中分離純化了G蛋白;純化的G蛋白與GTP作用后,使用一次Phenyl-Sepharose CL-4B柱層析,成功分離了G蛋白α亞基與G蛋白βγ亞基。將純化的G蛋白βγ亞基、G蛋白偶聯(lián)受體激酶-2,[γ-p32]標(biāo)記的ATP分別與mAChR2,β2-AR共同保溫,聚丙烯酰胺凝膠電泳,凝膠片干燥后放射性自顯影檢測mAChR2磷酸化結(jié)果。之后將干燥凝膠片中放射性標(biāo)記的磷酸化mAChR2蛋白帶剪下,同位素液體閃爍計(jì)數(shù)器計(jì)數(shù)。 實(shí)驗(yàn)結(jié)果顯示:G蛋白βγ亞基在沒有激動(dòng)劑存在的情況下明顯增強(qiáng)了mAChR2的磷酸化,也增強(qiáng)了β2-AR的磷酸化;氨甲酰膽堿明顯增強(qiáng)mAChR2的磷酸化,而阿托品或肝素(GRK2抑制劑)完全阻斷了mAChR2的磷酸化;mAChR2的磷酸化是依賴激活劑如氨甲酰膽堿作用發(fā)生的,這種依賴關(guān)系呈明顯的劑量關(guān)系;特布他林增強(qiáng)β2-AR的磷酸化,而肝素完全阻斷了β2-AR的磷酸化;這些結(jié)果證實(shí)氨甲酰膽堿對(duì)mAChR2,特布他林對(duì)β2-AR磷酸化的增強(qiáng)作用是選擇性的作用結(jié)果。而G蛋白βγ亞基同時(shí)增強(qiáng)mAChR2和β2-AR的磷酸化的結(jié)果提示,G蛋白βγ亞基是通過上調(diào)GRK2活性增強(qiáng)mAChR2和β2-AR的磷酸化。 我們的研究發(fā)現(xiàn)G蛋白βγ亞基對(duì)受體激酶GRK2有著重要的調(diào)節(jié)作用。說明Gβγ同Gα一樣均可引起效應(yīng)蛋白的激活,在細(xì)胞信號(hào)轉(zhuǎn)導(dǎo)中起同樣重要作用,共同介導(dǎo)一系列的生物學(xué)效應(yīng)。
[Abstract]:The effects of G protein 尾 緯 subunit G 尾 緯) on the phosphorylation of G protein-coupled receptor kinase-2mAChR2 and 尾 2-adrenergic receptor (尾 2-ARR) mediated by G protein coupled receptor kinase 2 (GRK2), such as muscarinic acetylcholine m2 receptor (mAChR2) and 尾 2 adrenergic receptor (尾 2 ARR), were studied. It is demonstrated that G protein 尾 緯 subunit plays an important role in regulating the activity of G protein coupled receptor kinase. To investigate how G protein 尾 緯 subunit affects phosphorylation of mAChR2 and 尾 2-AR receptors and triggers cascade reactions involving receptor phosphorylation. It is suggested that the G 尾 緯 subunit complex may have a new regulatory molecular mechanism by directly activating some intracellular target molecules in the G protein signaling pathway. In this experiment, mAChR2 was purified from rat brain by affinity chromatography, then G protein was purified from porcine brain by DEAE-Sepharose FFF Sephacryl S-300 HR and Phenyl-Sepharose CL-4B. After the purified G protein was treated with GTP, a Phenyl-Sepharose CL-4B column chromatography was used. G protein 偽 subunits and G protein 尾 緯 subunits were successfully isolated. The purified G protein 尾 緯 subunit, G protein coupled receptor kinase 2, [緯 -p32] labeled ATP were incubated with mAChR2, 尾 2-AR, polyacrylamide gel electrophoresis and autoradiography respectively. The results of mAChR2 phosphorylation were detected by autoradiography after drying. The radiolabeled phosphorylated mAChR2 protein band in the dried gel was then clipped and the isotope liquid scintillation counter was counted. The results showed that G protein 尾 緯 subunit significantly enhanced mAChR2 phosphorylation and 尾 2-AR phosphorylation without agonist, and carbamylcholine significantly increased mAChR2 phosphorylation. The phosphorylation of mAChR2 in mAChR2 was completely blocked by atropine or heparin antagonist, which was dependent on activator such as carbamylcholine in a dose-dependent manner, and terbutaline enhanced the phosphorylation of 尾 2-AR. Heparin completely blocked the phosphorylation of 尾 2-AR, which confirmed that the enhancement of mAChR2 and terbutaline phosphorylation of 尾 2-AR by carbamylcholine was a selective result. The results of G protein 尾 緯 subunit enhanced the phosphorylation of mAChR2 and 尾 2-AR at the same time, suggesting that G protein 尾 緯 subunit enhanced the phosphorylation of mAChR2 and 尾 2-AR by up-regulating the activity of GRK2. We found that G-protein 尾-緯 subunit plays an important role in regulating receptor kinase GRK2. It is concluded that G 尾 緯, like G 偽, can induce the activation of effector proteins and play an equally important role in cell signal transduction and mediate a series of biological effects.
【學(xué)位授予單位】：吉林大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2009
【分類號(hào)】：R341

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