Erbin介导Her2与β2-AR相互作用及参与MAPK信号通路调控的探讨

发布时间：2018-06-29 22:54

本文选题：Erbin + β-2AR　；参考：《河北医科大学》2011年硕士论文

【摘要】：目的:Erbin(ErbB2相互作用蛋白)是2000年Borg[1]等通过酵母双杂交筛选鉴定的一种新的Her2相互作用蛋白,该蛋白是LAP蛋白家族成员。LAP蛋白家族成员包括脑专一性蛋白Densin-180、线虫的Let413、果蝇的Scribble等,该家族成员N末端含有16个富含亮氨酸的重复序列LRR(leucine rich repeat)或类似LRR的结构域,C末端含1-4个PDZ结构域。LAP蛋白在维持细胞结构、调节信号转导通路、调控细胞生长发育和分化等方面发挥着重要的作用[2]。研究表明,Erbin的PDZ结构域对ErbB2在特定位点的定位或定向转移起到重要作用。Erbin是一个多功能蛋白,可与多种蛋白发生相互作用,主要为参与细胞连接和细胞基质趋化作用相关的蛋白。其在脑、心肌等组织中表达水平较高,通常定位于细胞膜及相邻细胞的连接处,参与细胞极性的形成,维持上皮细胞内环境的稳定,介导Her2受体的细胞内定位。近年来的研究发现,Erbin在多条信号通路中发挥重要的作用。事实上,Erbin的生物学功能仍远未被人们所认识。 β2肾上腺素能受体(β2-AR)是G蛋白偶联受体(GPCR)家族的成员,在人体不同器官、组织中分布广泛,心肌细胞、肾脏细胞、血管平滑肌细胞、支气管平滑肌细胞和脂肪细胞等均有表达,在心血管系统中表达水平较高,主要作用为增强心肌的收缩力、收缩速率以及血管的舒张。儿茶酚胺可通过调控人体相应组织器官的功能使机体有效应对外界刺激,β2-AR以儿茶酚胺作为内源性配体,可识别并选择性地与儿茶酚胺类物质特异性结合,通过第二信使cAMP引起胞内信号通路的活化,引起一系列生物学反应,调节、维持心脏的生理功能及血压稳态。儿茶酚胺-β2-AR系统对于维持心脏的生理功能及血压的稳态具有重要的作用,但是,儿茶酚胺的持续刺激引起经典的G蛋白偶联受体活化可导致心肌细胞损伤。原癌基因ErbB2/Her2是人表皮生长因子受体(EGFR)家族的重要成员,它是一种跨膜蛋白,属于表皮生长因子受体家族,在细胞增殖和分化中起重要作用。它具有独特的空间结构和很高的配体非依赖活性,参与细胞的分化、增殖等过程,介导MAPK信号通路的活化。Her2信号转导途径的异常不仅与肿瘤的发生、进展密切相关,也可以介导GPCR依赖的心肌功能的平衡。研究发现,心肌细胞中Her2缺失可导致心肌扩张。丝裂原活化的蛋白激酶(mitogen-activated protein kinases,MAPK)是细胞内的一类丝氨酸/苏氨酸蛋白激酶。不同的细胞外刺激信号通过各自的特异性受体传递至细胞内,触发MAPK信号通路的活化,诱导细胞产生不同的生物学效应(如细胞增殖、分化、转化及凋亡等)。新近的研究发现,在心肌细胞中β2-AR可与Her2发生相互作用,儿茶酚胺刺激可通过诱导MAPK通路活化[3],参与心肌细胞的存活,但相关作用的分子机制还不清楚。我们在前期研究中发现,Erbin、Her2及β2-AR可组成分子复合物,敲低Erbin表达可明显降低儿茶酚胺刺激诱导的MAPK信号通路的活化。这些结果提示,Erbin可能作为支架蛋白介导Her2和β2-AR的相互作用[4]。本课题通过解析Erbin介导β2-AR与Her2相互作用的分子机制,试图阐释Erbin在儿茶酚胺诱导的MAPK信号通路活化中的调控作用,相关研究内容在国内外尚未见报道,所以本研究结果可能为心血管疾病发病机制的阐明提供新的线索。方法: 1通过免疫共沉淀技术验证Erbin、Her2及β2-AR之间的相互作用。 2利用Erbin的不同突变体,通过免疫共沉淀及共聚焦显微镜观察,分析Erbin与Her2、β2-AR相互作用的功能域。 3利用Erbin突变体、敲低Erbin表达及β2-AR激动剂探讨Erbin在儿茶酚胺诱导的MAPK信号通路激活中的作用。结果: 1免疫共沉淀结果证实Erbin、β2AR及Her2可发生相互作用。 2免疫共沉淀和共聚焦显微镜结果证实Erbin PDZ结构域介导β2-AR与Her2的相互作用。 3结合转入Erbin siRNA敲低Erbin表达细胞的western blot揭示Erbin在儿茶酚胺诱导的MAPK信号通路活化中起正调控作用。结论: Erbin通过其PDZ结构域介导β-2AR与Her2相互作用;Erbin在EGF诱导的MAPK通路活化中起负调控作用;Erbin在儿茶酚胺诱导的MAPK通路活化中起正调控作用。
[Abstract]:Objective: Erbin (ErbB2 interaction protein) is a new Her2 interaction protein identified by yeast two hybrid in 2000 Borg[1] and so on. The protein is a member of the LAP protein family.LAP family members including brain specific protein Densin-180, nematode Let413, and Drosophila Scribble, and the N end of the family members contains 16 rich light. The repeating sequence of LRR (leucine rich repeat) or LRR like domains. The C terminal contains 1-4 PDZ domains.LAP proteins that play an important role in maintaining cell structure, regulating signal transduction pathways, regulating cell growth and differentiation and other aspects. The [2]. study shows that Erbin PDZ domain is located or directed at the specific location of ErbB2. Metastasis plays an important role in.Erbin, a multifunctional protein that can interact with a variety of proteins, mainly involved in cell connections and cellular matrix chemotaxis. It is highly expressed in brain, myocardium and other tissues, usually located at the junction of cell membrane and adjacent cells, and participates in the formation of cell polarity and maintenance. The environmental stability of the skin cells mediates the intracellular localization of Her2 receptors. Recent studies have shown that Erbin plays an important role in multiple signaling pathways. In fact, the biological functions of Erbin are still far from being recognized.
Beta 2 adrenergic receptor (beta 2-AR) is a member of the G protein coupled receptor (GPCR) family. It is widely distributed in different organs and tissues of the human body. Myocardial cells, renal cells, vascular smooth muscle cells, bronchial smooth muscle cells and adipocytes are expressed, and the expression level is high in the heart blood tube system. The main function is to enhance the contraction of the myocardium. Catecholamines can effectively respond to external stimuli by regulating the functions of the corresponding tissues and organs of the human body. Beta 2-AR is an endogenous ligand with catecholamine, which recognizes and selectively combines with catecholamines, and induces activation of intracellular signaling pathways through second messenger cAMP. A series of biological responses regulate the physiological functions of the heart and maintain the homeostasis of the blood pressure. The catecholamine beta 2-AR system plays an important role in maintaining the physiological function of the heart and the homeostasis of the blood pressure. However, the continuous stimulation of catecholamine induced the activation of the classical G protein coupled receptor can lead to myocardial cell damage.
Proto oncogene ErbB2/Her2 is an important member of the human epidermal growth factor receptor (EGFR) family. It is a transmembrane protein, which belongs to the epidermal growth factor receptor family. It plays an important role in cell proliferation and differentiation. It has a unique spatial structure and a very high ligand without dependence on viability, participates in cell differentiation, proliferation and other processes, and mediates MAPK The abnormalities of the activated.Her2 signal transduction pathway in the signal pathway are not only closely related to the development of the tumor, but also can mediate the balance of the function of the GPCR dependent myocardium. It is found that the absence of Her2 in cardiac myocytes can lead to myocardial dilatation.
The mitogen activated protein kinase (mitogen-activated protein kinases, MAPK) is a kind of serine / threonine protein kinase in the cell. Different extracellular stimulation signals pass through the specific receptor to the cell, triggering the activation of the MAPK signaling pathway and inducing the cell to produce different biological effects (such as cell proliferation and differentiation). Recent studies have found that beta 2-AR can interact with Her2 in cardiac myocytes. Catecholamine stimulation can activate [3] by inducing the MAPK pathway to participate in the survival of cardiac myocytes, but the molecular mechanism of the related effects is not clear. In our previous study, we found that Erbin, Her2 and beta 2-AR can form molecular complexes, knocking. Low Erbin expression can significantly reduce the activation of MAPK signaling pathway induced by catecholamine stimulation. These results suggest that Erbin may be used as a scaffold protein to mediate the interaction of Her2 and beta 2-AR [4].
By analyzing the molecular mechanism of Erbin mediated interaction between beta 2-AR and Her2, this study attempts to explain the regulatory role of Erbin in the activation of MAPK signaling pathway induced by catecholamine. The relevant research content has not yet been reported at home and abroad, so the results of this study may provide new clues for the elucidation of the pathogenesis of cardiovascular disease.
Method:
1 the interaction between Erbin, Her2 and beta 2-AR was verified by CO immunoprecipitation.
2 using Erbin mutants, the functional domains of Erbin interacting with Her2 and beta 2-AR were analyzed by immunoprecipitation and confocal microscopy.
3 to explore the role of Erbin in the activation of catecholamine induced MAPK signaling pathway by using Erbin mutants, knockdown of Erbin expression and beta 2-AR agonists.
Result:
1 co immunoprecipitation results showed that Erbin, beta 2AR and Her2 could interact.
2 co immunoprecipitation and confocal microscopy confirmed that the Erbin PDZ domain mediates the interaction between beta 2-AR and Her2.
3 combined with Western blot knockdown of Erbin expression cells in Erbin siRNA, Erbin can play a positive role in the activation of catecholamine induced MAPK signaling pathway.
Conclusion: Erbin mediates the interaction of beta -2AR and Her2 through its PDZ domain, and Erbin plays a negative regulatory role in the activation of MAPK pathway induced by EGF, and Erbin plays a positive role in the activation of the MAPK pathway induced by catecholamine.
【学位授予单位】：河北医科大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R363

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