G蛋白βγ亚基影响G蛋白偶联受体磷酸化的作用研究

发布时间：2018-06-06 02:22

本文选题：G蛋白βγ亚基 + G蛋白偶联受体　；参考：《吉林大学》2009年硕士论文

【摘要】： 本实验研究了G蛋白βγ亚基(Gβγ)对G蛋白偶联受体激酶-2(GRK2)介导的G蛋白偶联受体,如毒蕈碱乙酰胆碱m2受体(mAChR2)和β2肾上腺素能受体(β2-AR)磷酸化的影响,证明G蛋白βγ亚基在调节G蛋白偶联受体激酶活性中有着重要作用。探讨G蛋白βγ亚基如何影响mAChR2和β2-AR受体磷酸化,引发涉及受体磷酸化的级联反应。揭示在G蛋白信号通路的作用中,Gβγ亚基复合物通过直接激活某些胞内靶分子可能存在新的调控分子机制。本实验首先利用亲和层析方法从大鼠脑中纯化了mAChR2;再通过DEAE-Sepharose FF、Sephacryl S-300 HR和Phenyl-Sepharose CL-4B三个层析柱从猪脑中分离纯化了G蛋白;纯化的G蛋白与GTP作用后,使用一次Phenyl-Sepharose CL-4B柱层析,成功分离了G蛋白α亚基与G蛋白βγ亚基。将纯化的G蛋白βγ亚基、G蛋白偶联受体激酶-2,[γ-p32]标记的ATP分别与mAChR2,β2-AR共同保温,聚丙烯酰胺凝胶电泳,凝胶片干燥后放射性自显影检测mAChR2磷酸化结果。之后将干燥凝胶片中放射性标记的磷酸化mAChR2蛋白带剪下,同位素液体闪烁计数器计数。实验结果显示:G蛋白βγ亚基在没有激动剂存在的情况下明显增强了mAChR2的磷酸化,也增强了β2-AR的磷酸化;氨甲酰胆碱明显增强mAChR2的磷酸化,而阿托品或肝素(GRK2抑制剂)完全阻断了mAChR2的磷酸化;mAChR2的磷酸化是依赖激活剂如氨甲酰胆碱作用发生的,这种依赖关系呈明显的剂量关系;特布他林增强β2-AR的磷酸化,而肝素完全阻断了β2-AR的磷酸化;这些结果证实氨甲酰胆碱对mAChR2,特布他林对β2-AR磷酸化的增强作用是选择性的作用结果。而G蛋白βγ亚基同时增强mAChR2和β2-AR的磷酸化的结果提示,G蛋白βγ亚基是通过上调GRK2活性增强mAChR2和β2-AR的磷酸化。我们的研究发现G蛋白βγ亚基对受体激酶GRK2有着重要的调节作用。说明Gβγ同Gα一样均可引起效应蛋白的激活,在细胞信号转导中起同样重要作用,共同介导一系列的生物学效应。
[Abstract]:The effects of G protein 尾纬 subunit G 尾纬) 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 尾纬 subunit plays an important role in regulating the activity of G protein coupled receptor kinase. To investigate how G protein 尾纬 subunit affects phosphorylation of mAChR2 and 尾 2-AR receptors and triggers cascade reactions involving receptor phosphorylation. It is suggested that the G 尾纬 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 伪 subunits and G protein 尾纬 subunits were successfully isolated. The purified G protein 尾纬 subunit, G protein coupled receptor kinase 2, [纬 -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 尾纬 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 尾纬 subunit enhanced the phosphorylation of mAChR2 and 尾 2-AR at the same time, suggesting that G protein 尾纬 subunit enhanced the phosphorylation of mAChR2 and 尾 2-AR by up-regulating the activity of GRK2. We found that G-protein 尾-纬 subunit plays an important role in regulating receptor kinase GRK2. It is concluded that G 尾纬, like G 伪, can induce the activation of effector proteins and play an equally important role in cell signal transduction and mediate a series of biological effects.
【学位授予单位】：吉林大学
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R341

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