Erbin在β2-AR介导 MAPK信号通路中的调控作用及生物学功能的研究

发布时间：2018-06-14 00:29

本文选题：Erbin + Her2　；参考：《河南大学》2012年硕士论文

【摘要】：研究背景：β肾上腺素能受体（β-AR）是G蛋白偶联受体（GPCR）家族的重要成员。表达于心脏组织的β肾上腺素能受体可接受儿茶酚胺的刺激，从而将来自交感神经系统的信息转换为体内生理学功能刺激信号，从而调控心肌细胞的生物学功能，但其具体调控的机制尚未完全阐明。 Her2，原癌基因人类表皮生长因子受体2(human epidermalgrowth factor receptor-2，HER2)基因，人类表皮生长因子受体家族成员之一。近年来，通过对Her2的基因敲除小鼠的研究，Her2在心肌细胞中的功能，特别是其在心肌细胞保护方面的作用逐渐被人们所认识。而且临床上使用靶向Her2的治疗性抗体赫赛汀（Herceptin）的应用可在某些患者中引起明显的心脏毒性，亦表明Her2在心肌细胞内的作用至关重要，但是其机理尚不明确。随即，2006年， Alejandra Negro等研究发现在心肌细胞内Her2与β2-AR存在相互作用，而且他们之间的这种相互作用对于儿茶酚胺诱导的MAPK信号通路的活化及心肌细胞收缩功能的调控至关重要。研究者在文中明确指出Her2与β2-AR存的相互作用并非直接相互作用，而是可能通过某个支架分子介导的间接相互作用，具体机制不清楚。然而，2000年Borg等以HER2胞内域残基为钓饵，通过酵母双杂交的方法从cDNA文库中筛选到一种新的含有PDZ结构域的蛋白，将其命名为ErbB2相互作用蛋白（ErbB2interacting protein），简写为Erbin。Erbin的序列分析表明，，Erbin可能具有支架分子的功能，最近的相关研究也证明Erbin可作为支架蛋白介导Her2与其它受体分子发生相互作用。我们的前期研究结果提示，在心肌细胞内Erbin可能作为支架蛋白介导Her2与β2-AR之间的相互作用。随后通过一系列的研究，我们发现Erbin的PDZ结构域介导Her2与β2-AR之间的相互作用，并在β2-AR介导的MAPK信号通路活化中起到正向调控作用，本研究还首次报道了Erbin在心肌细胞中的新功能。目的：研究在细胞内，Erbin是否是作为支架蛋白介导Her2与β2-AR复合物形成的必要分子，及其揭示Erbin在心肌细胞中对β2-AR激动剂诱导的MAPK信号通路活化的调控作用，初步探索Erbin对心肌细胞生物学功能的影响。方法：利用免疫共沉淀的方法，在心肌及脑组织初步验证Erbin、β2-AR及Her2三者存在相互作用；进一步利用免疫共沉淀的方法，进行内源性，外源性检测Erbin、β2-AR及Her2三者存在相互作用；我们进行内源性敲低Erbin的表达，利用免疫共沉淀证明Erbin是介导β2-AR与Her2复合物形成的必要分子，同时通过膜，浆分离实验来证明Erbin在儿茶酚胺激素下被招募到细胞膜上，是其发挥作用的前提。利用分子生物学技术构建Erbin及其突变体，通过免疫共沉淀及免疫荧光的方法，确定Erbin介导β2-AR与Her2相互作用的关键结构域。通过SDS-PAGE和Western blot方法检测Erbin在ISO刺激β2-AR介导的MAPK信号通路中调控作用。进一步通过TUNEL染色法检测Erbin在心肌细胞内抗调亡作用，利用胰酶消化法分离与培养大鼠原代心肌细胞，通过慢病毒转染高效敲低Erbin，荧光显微镜下观察，记录心肌细胞心率的变化，初步探索Erbin在心肌细胞内潜在的生物学功能。结果：首先，我们在大鼠的心肌组织及脑组织，初步验证了Erbin，β2-AR，Her2三者之间存在相互作用；接下来我们在293T（组成性表达Erbin）细胞内，通过外源性过表达pcDNA3.0-β2-AR、pcDNA3.0-Her2两种质粒，证实Erbin、β2-AR及Her2三者之间存在相互作用；那么在心肌细胞是否也存在呢?我们研究证明在大鼠心肌细胞H9c2内源性检测结果，Erbin、β2-AR及Her2三者之间也存在相互作用。相反，如果敲低大鼠心肌细胞H9c2内Erbin的表达，是否会影响这种作用呢?我们的结果发现在大鼠心肌细胞H9c2内敲低Erbin的表达，Erbin、β2-AR及Her2三者之间相互作用被破坏。可见，Erbin是介导β2-AR与Her2两者之间相互作用的必要分子。那么，Erbin是通过什么样的机制介导了膜上蛋白分子之间的作用呢？我们的膜，浆分离实验证明Erbin在儿茶酚胺激素下被招募到细胞膜上，发挥其支架蛋白的作用。免疫共沉淀及免疫荧光的结果都证实Erbin的PDZ结构域是介导β2-AR与Her2两者之间相互作用的关键结构域。在随后的实验中，我们进一步发现Erbin在β2-AR介导的MAPK信号通路中正调控作用。此外，在我们的研究中，我们同时还发现Erbin可保护心肌细胞免受儿茶酚胺慢性刺激诱导的细胞凋亡及Erbin可能在调控心肌细胞搏动频率中发挥重要作用。结论：本研究证明Erbin PDZ结构域介导Her2与β2-AR之间的相互作用以及Erbin在β2-AR介导MAPK信号通路中起着正向调控作用；同时Erbin可保护心肌细胞免受儿茶酚胺慢性刺激诱导的细胞凋亡，并初步揭示了Erbin可能在调控心肌细胞搏动频率中发挥重要作用。
[Abstract]:Background: beta adrenergic receptor (beta -AR) is an important member of the G protein coupled receptor (GPCR) family. The beta adrenergic receptor expressed in the cardiac tissue can be stimulated by catecholamine and converts the information from the sympathetic nervous system into the physiological function stimulation signal in the body, thus regulating the biological work of the cardiac myocytes. Yes, but the mechanism of its specific regulation has not been fully elucidated.
Her2, a proto oncogene, human epidermal growth factor receptor 2 (human epidermalgrowth factor receptor-2, HER2) gene, one of the members of the human epidermal growth factor receptor family. In recent years, the function of Her2 in cardiac myocytes has been gradually developed by the study of gene knockout mice in Her2, especially in the protection of cardiac myocytes. The application of the therapeutic antibody Hessaitin (Herceptin), which is clinically targeted to Her2, can cause significant cardiac toxicity in some patients and indicates that the role of Her2 in cardiac myocytes is crucial, but the mechanism is not clear.
Then, in 2006, Alejandra Negro and other studies found that there was a interaction between Her2 and beta 2-AR in cardiac myocytes, and the interaction between them was crucial to the activation of the catecholamine induced MAPK signaling pathway and the regulation of cardiac contractile function. The researchers clearly pointed out the interaction between Her2 and the existence of beta 2-AR. Not direct interaction, but a possible indirect interaction mediated by a scaffold molecule, the specific mechanism is not clear. However, in 2000, Borg and other HER2 intracellular residues were used as bait, and a new protein containing PDZ domains was screened from the cDNA library by yeast two hybrid method and named as ErbB2 interaction protein (E). RbB2interacting protein), the sequence analysis of Erbin.Erbin shows that Erbin may have the function of scaffold molecules. Recent related studies have also shown that Erbin can mediate the interaction between Her2 and other receptor molecules as a scaffold protein.
Our previous results suggest that Erbin may act as a scaffold protein to mediate the interaction between Her2 and beta 2-AR in cardiac myocytes. Then, through a series of studies, we found that the PDZ domain of Erbin mediates the interaction between Her2 and beta 2-AR and plays a positive role in the activation of the MAPK signaling pathway mediated by beta 2-AR. This study also reported for the first time the new functions of Erbin in cardiac myocytes.
Objective: To investigate whether Erbin is a necessary molecule to mediate the formation of Her2 and beta 2-AR complex as a scaffold protein, and to reveal the regulatory effect of Erbin on the activation of MAPK signaling pathway induced by beta 2-AR agonist in cardiac myocytes, and to explore the effect of Erbin on the biological function of cardiac myocytes.
Methods: the interaction between Erbin, beta 2-AR and Her2 three was preliminarily verified by immunoprecipitation in the myocardium and brain tissue, and the endogenous and exogenous detection of Erbin, beta 2-AR and Her2 were carried out by immunoprecipitation method, and the expression of endogenous knockout Erbin and immunoprecipitation were carried out. It is proved that Erbin is a necessary molecule to mediate the formation of the complex of beta 2-AR and Her2. At the same time, it is proved that Erbin is recruited on the cell membrane under the catecholamine hormone by membrane and slurry separation experiment. It is the premise of its function. Using molecular biology technology, Erbin and its mutants are constructed, and Erbin is determined by the method of immunoprecipitation and immunofluorescence. The key domain of interaction between beta 2-AR and Her2 is mediated by SDS-PAGE and Western blot methods to detect the regulation of Erbin in ISO stimulated MAPK signaling pathway mediated by beta 2-AR. Further, TUNEL staining method is used to detect the anti regulating action of Erbin in cardiac myocytes, and trypsin digestion method is used to isolate and cultivate primary cardiomyocytes in rats. The virus transfected efficiently knocked down Erbin and observed the changes of heart rate under fluorescence microscope. The potential biological function of Erbin in cardiac myocytes was preliminarily explored.
Results: first, we preliminarily verified the interaction between Erbin, beta 2-AR, and Her2 three in the myocardium and brain tissue of rats; then we confirmed the interaction between Erbin, beta 2-AR and Her2 three in 293T (constituent expression Erbin) cells by overexpressing pcDNA3.0- beta 2-AR, pcDNA3.0-Her2 two plasmids. Is it possible to exist in cardiac myocytes? We have shown that there is also interaction between Erbin, beta 2-AR, and Her2 three in rat cardiac myocytes. On the contrary, if the expression of Erbin in H9c2 of rat cardiac myocytes in the low rat heart can affect this use, we found that H9c2 in rat cardiac myocytes is found to be H9c2 The interaction between Erbin, Erbin, beta 2-AR and Her2 three is destroyed. So, Erbin is a necessary molecule to mediate the interaction between beta 2-AR and Her2. Then, what mechanism does Erbin mediate the role of protein molecules on the membrane? Our membrane, pulp separation experiment shows that Erbin is under catecholamine. The results of immuno coprecipitation and immunofluorescence have confirmed that the PDZ domain of Erbin is a key domain that mediates the interaction between beta 2-AR and Her2. In the subsequent experiment, we further found that Erbin is positive in the MAPK signaling pathway mediated by beta 2-AR. In our study, we also found that Erbin protects cardiomyocytes from apoptosis induced by catecholamine induced chronic stimulation and that Erbin may play an important role in regulating the frequency of cardiac myocyte pulsation.
Conclusion: This study shows that the Erbin PDZ domain mediates the interaction between Her2 and beta 2-AR, and Erbin plays a positive role in the beta 2-AR mediated MAPK signaling pathway, and Erbin protects cardiomyocytes from the apoptosis induced by catecholamine induced chronic stimulation, and preliminarily reveals that Erbin may regulate the pulsation frequency of cardiac myocytes. The rate plays an important role.
【学位授予单位】：河南大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R363

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