多种细胞体系G_q蛋白偶联受体和IP3受体偶联的研究

发布时间：2018-09-19 20:12

【摘要】： G蛋白偶联受体(GPCR)是目前已发现受体中种类最多的一类。这些受体在结构上有很大的相似性,由一条肽链形成,跨膜七次,N端在胞外,C端在胞内。这些受体与效应器间都通过G蛋白介导。G蛋白种类较多,但所有类型G蛋白都由3个不同的亚单位即α、β、γ所组成,α亚单位具有特异的GTP结合位点,有GTP酶活性。根据G蛋白的α亚单位的结构,可将其分为6个亚家族:Gs,Gi/o,Gq,Gt,Gg和G12[1]。由Gq蛋白偶联受体介导的磷脂酶C (PLC)信号系统是一种广泛存在的信号转导机制。Gαq蛋白激活后首先激活PLCβ,进而水解胞膜PIP2,水解生成两种第二信使IP3与DAG,进而调控诸多细胞功能,例如离子通道功能、细胞周期、腺体分泌,等等。 IP3受体广泛存在于细胞内质网膜上,它是一种能引发内钙释放的通道蛋白,能与IP3结合,引发细胞内钙释放,导致内钙水平的升高,从而启动细胞内钙信号系统,通过依赖钙离子的钙调素等酶类活性的变化来调控一系列的生理过程。到目前为止,共有四种IP3受体被发现,其中,Ⅰ型受体广泛存在于神经组织中。细胞内钙的释放在细胞信号转导过程中具有重要的作用。另外,钙信号的异常也参与了诸多病理过程中。 M电流是在1980年由Brown和Admas在牛蛙的颈上交感神经节被首次发现,它是一种慢激活,非失活的电压依赖性外向钾离子电流,因其可被毒蕈碱受体强烈抑制而得名。自1998年以来人们逐渐认识到其分子基础是KCNQ家族(KCNQ2/3)的钾离子通道。M电流与神经系统的兴奋性密切相关。自电流被发现到现在20多年的时间里,人们对其调节机制做了大量深入的研究,目前已发现许多于Gq蛋白偶联的受体激活后均能引起M电流的抑制,并且膜磷脂酰肌醇4,5二磷酸(PIP2)参与了膜受体的信号转导。已证实Gq蛋白偶联受体激活后,都会引起位于胞膜内侧的PIP2水解,产生IP3和DAG。已有的研究结果证实M1受体的激活引起M电流的抑制是由于PIP2的水解所为,而另外一种受体BK2受体激活后虽然也产生IP3和DAG,但它激活后所引起的M电流抑制是通过钙离子介导,由钙调素来实现的。同样是利用PLC信号通路的M1受体和BK2受体为何会分别利用PIP2和钙离子来实现对M电流的调控?因此,目前对于这一问题有人提出了用微域结构来解释这种信号传导的特异性,他们认为BK2受体与IP3受体处于同一个微域结构内,因此BK2受体激活后所产生的IP3能以较大浓度作用于附近的IP3受体,进而引起细胞内钙释放;而M受体和IP3受体却没有形成相应的微域结构,因此其激活后所产生的IP3在局部不能形成使IP3受体激活的有效浓度,从而不能引起细胞内钙的释放。上述实验结论是建立在以大鼠颈上交感神经节为研究的基础上而得出的。众所周知,磷脂酶C信号通路是一种广泛存在于众多生命形式中的信号转导机制。因此,这种信号转导的差异性在其他的神经细胞是否存在还不得而知。这一问题的阐明,将对深入了解磷脂酶C信号通路的多样性具有至关重要的意义。本小组前期的研究结果表明:在不同的神经细胞中,M1,BK2,H1,AT1受体激活后虽然都可以水解PIP2,产生IP3和DAG,但是引发内钙释放的作用之间存在明显不同,在颈上神经节细胞内BK2,H1,AT1受体激活后均可以引起内钙的升高,而M1受体则不能。在海马神经细胞内,BK2,H1,M1受体激动后引起内钙升高,而AT1受体则不能。在HEK293细胞内,过表达M1和BK2受体引起的内钙释放与神经细胞又有所不同,二者均可以引起内钙升高。在爪蟾卵母细胞过表达M1和BK2受体也均可以引起内钙的升高。本实验以大鼠颈上交感神经节细胞、海马神经元、HEK293细胞、爪蟾卵母细胞为研究对象,利用免疫共沉淀技术观察不同Gq蛋白偶联受体和位于内质网膜上IP3受体之间的关系,探究是否存在一种信号微域以及其特点,从而为Gq蛋白偶联受体的信号通路的多样性寻找生化依据。目的:利用免疫共沉淀技术,在两种不同类型的神经细胞、HEK293细胞以及爪蟾卵母细胞,观察不同Gq蛋白偶联受体与内质网IP3受体之间的关系。方法:采用免疫共沉淀(coimmunoprecipitation),SDS-PAGE,Western blot等方法观察在颈上交感神经节和海马神经元中上述四种Gq蛋白偶联受体与内质网IP3受体之间的关系。在HEK293细胞中,利用脂质体转染的方法外源性表达M1、H1、AT1、BK2受体,观察这四种受体与IP3受体的关系。在爪蟾卵母细胞中,利用体外转录的RNA在卵母细胞外源性表达M1和BK2受体,观察这两种受体与IP3受体之间的关系。结果:⑴在颈上神经节和海马神经元中,均可检测到M1、H1、AT1、BK2受体的表达。(2)在颈上神经节和海马神经元中,均可检测到IP3受体的表达。(3)免疫共沉淀的结果表明,在颈上交感神经节中,H1、AT1、BK2受体均可以和IP3受体共沉淀,未发现M1和IP3受体共沉淀。在海马神经元,AT1受体可以和IP3受体共沉淀,未发现M1、H1、BK2受体和IP3受体共沉淀。(4)在分别外源性表达M1、H1、AT1、BK2受体的HEK293细胞中,均可以分别检测到四种受体的表达。(5)在外源性表达这四种受体的HEK293细胞中,均可以检测到IP3受体的表达。(6)在过表达这四种受体的细胞中,M1、BK2、AT1与IP3受体共沉淀,而H1受体不能与IP3受体共沉淀。(7)在过表达M1和BK2受体的爪蟾卵母细胞中,均可以检测到这两种受体的过表达。(8)在过表达M1和BK2受体的爪蟾卵母细胞中,均可以检测到IP3受体的表达。(9)在爪蟾卵母细胞中,这两种受体均可以和IP3受体共沉淀。结论:(1)在大鼠颈上交感神经节细胞中中,M1受体和H1、AT1、BK2受体与IP3受体之间的关系不同,其中,H1、AT1、BK2受体与IP3受体相偶连,而M1和IP3受体之间不存在偶连关系。在海马神经元中,AT1和IP3受体相偶连,M1、BK2、H1受体和IP3受体之间不存在偶连关系。(2)在HEK293细胞中,M1、H1、AT1、BK2受体与IP3受体之间存在偶连关系,与神经组织存在明显不同。(3)在爪蟾卵母细胞中,M1和BK2受体与IP3受体之间存在偶联关系,与HEK293细胞中的结果一致。(4)不同组织细胞中不同GPCR与IP3受体偶联有较大差异,其细胞生理学意义有待进一步阐明。
[Abstract]:G-protein-coupled receptors (GPCRs) are the most widely recognized class of receptors. These receptors are structurally similar, formed by a peptide chain, transmembrane seven times, N-terminus extracellular, C-terminus intracellular. These receptors and effectors are mediated by G proteins. There are many G proteins, but all types of G proteins are composed of three distinct subunits. According to the structure of the alpha subunit of G protein, it can be divided into six subfamilies: Gs, Gi/o, G q, Gt, Gg and G12 [1]. Phospholipase C (PLC) signaling system mediated by G Q protein-coupled receptors is a widespread signal transduction mechanism. First, PLC beta is activated, then the membrane PIP2 is hydrolyzed to produce two second messengers IP3 and DAG, which regulate many cellular functions, such as ion channel function, cell cycle, gland secretion, and so on.
IP3 receptor is a kind of channel protein which can induce intracellular calcium release. It can bind to IP3 and induce intracellular calcium release, which leads to the elevation of intracellular calcium level. Up to now, four IP3 receptors have been found, of which type I receptors are ubiquitous in nerve tissues. Intracellular calcium release plays an important role in cell signal transduction. In addition, abnormal calcium signaling is also involved in many pathological processes.
M current was first discovered by Brown and Admas in the superior cervical sympathetic ganglion of bullfrog in 1980. It is a slow-activated, non-inactivated voltage-dependent outward potassium current, named for its strong inhibition by muscarinic receptors. Since 1998, it has been gradually recognized that its molecular basis is the potassium channel of the KCNQ family (KCNQ2/3). Current is closely related to the excitability of the nervous system. Since the discovery of current, a great deal of research has been done on its regulation mechanism. It has been found that many Gq protein-coupled receptors can induce M-current inhibition after activation, and membrane phosphatidylinositol 4,5 diphosphate (PIP2) is involved in the letter of membrane receptors. It has been proved that the activation of Gq protein-coupled receptor can induce the hydrolysis of PIP2 and the production of IP3 and DAG on the inner surface of the cell membrane. Previous studies have shown that the inhibition of M current caused by the activation of M1 receptor is due to the hydrolysis of PIP2, while the activation of another receptor, BK2 receptor, can also produce IP3 and DAG, but the activation of the receptor causes the M current. Current inhibition is mediated by calcium ions and is achieved by calmodulin. Why do M1 receptors and BK2 receptors, which also use the PLC signaling pathway, use PIP2 and calcium ions to regulate M current respectively? Therefore, some people have proposed to explain the specificity of this signal transduction by using the micro-domain structure. They believe that BK2 receptors are affected. The body is in the same microdomain as IP3 receptor, so the IP3 produced by the activation of BK2 receptor can act on the nearby IP3 receptor at a higher concentration, and then induce intracellular calcium release; while the M receptor and IP3 receptor do not form the corresponding microdomain structure, so the IP3 produced by the activation of BK2 receptor can not form the IP3 receptor activated locally. These results are based on the study of the rat superior cervical sympathetic ganglion. It is well known that the phospholipase C signaling pathway is a signal transduction mechanism that exists widely in many forms of life. Therefore, the differences in this signal transduction are among other gods. It is not known whether the cells exist or not. The clarification of this question will be of great significance to the further understanding of the diversity of phospholipase C signaling pathways.
Previous studies by our team have shown that in different nerve cells, M1, BK2, H1, AT1 receptors can hydrolyze PIP2 and produce IP3 and DAG after activation, but there are significant differences in the role of intracellular calcium release. In the superior cervical ganglion cells, BK2, H1, AT1 receptors can activate the increase of intracellular calcium, while M1 receptors can not. In hippocampal neurons, the activation of BK2, H1 and M1 receptors results in elevation of intracellular calcium, whereas AT1 receptors do not. In HEK293 cells, the release of intracellular calcium caused by overexpression of M1 and BK2 receptors is different from that of neurons, both of which can cause elevation of intracellular calcium. Overexpression of M1 and BK2 receptors in Xenopus oocytes can also cause elevation of intracellular calcium. The relationship between different Gq protein-coupled receptors and IP3 receptors located on the endoplasmic reticulum was observed by immunoprecipitation technique in rat superior cervical sympathetic ganglion cells, hippocampal neurons, HEK293 cells and Xenopus oocytes, and whether there was a signal microdomain and its characteristics were explored in order to provide Gq protein-coupled receptors. The diversity of signal pathways is to find biochemical evidence.
AIM: To investigate the relationship between different Gq protein-coupled receptors and endoplasmic reticulum IP3 receptors in two different types of nerve cells, HEK293 cells and Xenopus oocytes by immunoprecipitation technique.
Methods: The relationship between these four Gq protein-coupled receptors and endoplasmic reticulum IP3 receptors in superior cervical sympathetic ganglion and hippocampal neurons was observed by immunoprecipitation, SDS-PAGE and Western blot. In Xenopus oocytes, M1 and BK2 receptors were exogenously expressed in Xenopus oocytes using in vitro transcripted RNA. The relationship between these two receptors and IP3 receptors was observed.
Results: (1) The expression of M1, H1, AT1 and BK2 receptors was detected in both superior cervical ganglion and hippocampal neurons. (2) The expression of IP3 receptors was detected in both superior cervical ganglion and hippocampal neurons. (3) The results of immunoprecipitation showed that H1, AT1, BK2 receptors could co-precipitate with IP3 receptors in superior cervical sympathetic ganglion, but no M1 and IP3 receptors were found. Receptor coprecipitation. In hippocampal neurons, AT1 receptor can coprecipitate with IP3 receptor, but no coprecipitation of M1, H1, BK2 receptor and IP3 receptor was found. (4) In HEK293 cells expressing exogenous M1, H1, AT1 and BK2 receptors respectively, the expression of four receptors can be detected. (5) In HEK293 cells expressing these four receptors, the expression of these receptors can be detected. To the expression of IP3 receptors. (6) M1, BK2, AT1 and IP3 receptors co-precipitated in cells overexpressing these four receptors, while H1 receptors could not co-precipitate with IP3 receptors. (7) Overexpression of these two receptors was detected in Xenopus oocytes overexpressing M1 and BK2 receptors. (8) Both M1 and BK2 receptors could be detected in Xenopus oocytes overexpressing Xenopus oocytes. The expression of IP3 receptor was detected. (9) In Xenopus oocytes, both receptors could coprecipitate with IP3 receptor.
CONCLUSIONS: (1) The relationship between M1 receptor, H1, AT1, BK2 receptor and IP3 receptor is different in rat superior cervical sympathetic ganglion cells. H1, AT1, BK2 receptor and IP3 receptor are coupled, but there is no coupling between M1 and IP3 receptor. In hippocampal neurons, there is no coupling between AT1 and IP3 receptor, M1, BK2, H1 receptor and IP3 receptor. (2) In HEK293 cells, M1, H1, AT1, BK2 receptors were associated with IP3 receptors, which were significantly different from nerve tissues. (3) In Xenopus oocytes, there was a coupling relationship between M1 and BK2 receptors and IP3 receptors, which was consistent with the results of HEK293 cells. (4) GPCR and IP3 receptors were coupled differently in different tissues and cells. Its cellular physiological significance needs to be further elucidated.
【学位授予单位】：河北医科大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R33

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