Establishment of a novel analysis system for three-dimentional structure of transmembrane receptors based on neuronal and vascular cell plasticity

包括G蛋白偶联受体（GPCR）超家族的跨膜受体对于细胞功能的所有方面都是必需的，并且对于许多疾病的病理学也起关键作用。由于这些受体的大量纯化和结晶的困难，除了几个例外，它们的三维超微结构尚未得到证实。在这个项目中，我们正在开发一种新的系统，用于分析跨膜受体的三维超微结构。在该系统中，在活细胞的细胞膜中，通过经由有效的自身多聚化突触后支架蛋白PSD-Zip 45（Homer 1c）聚集靶受体，在次晶体水平上形成跨膜受体的二维簇。为了形成高度有序的二维跨膜受体簇，我们对PSD-Zip 45进行了大幅修饰（modified PSD-Zip 45）。因此，这种改进的PSD-Zip 45能够用于 ...更多信息 在活细胞的细胞膜中存在直径为3-8 μ m的m大多巴胺受体簇（GPCR的典型实例）。建立了高质量、大批量多巴胺受体粉末的纯化方法。多巴胺受体喷粉器制剂的电子显微镜照片的傅立叶变换显示，在次晶水平的高品质。该系统可应用于绝大多数跨膜受体。实际上，我们还使用我们的系统制作了LPA受体（GPCR的另一个例子）的二维大簇。因此，我们提供了一个强大的系统，分析跨膜受体的结构与功能的关系。为了便于常规应用，我们将尝试缩小细胞培养系统以纯化跨膜受体的二维簇，并进一步研究PSD蛋白介导的神经元和血管细胞可塑性的分子机制，如突触动力学（PSD-Zip 45、PSD-Zip 70和其他）和由不饱和溶血磷脂添加物引发的动脉粥样硬化的发作。少

Transmembrane receptors including a superfamily of G protein-coupled receptors (GPCRs) are essential for all aspects of cell function and also play critical roles for the pathology of a number of diseases. With several exceptions, the three-dimensional ultra-structure of these receptors has not been demonstrated due to the difficultly of their purification in large quantity and their crystallization. In this project, we were developing a novel system for analyzing the three-dimensional ultra-structure of transmembrane receptors. In this system, two-dimensional dusters of transmembrane receptors at paracrystal levels are made in the cell membrane of lived cells by clustering the target receptors through a potently self multimerizable postsynaptic scaffolding protein, PSD-Zip45 (Homer 1c). In order to form a highly ordered two-dimensional transmembrane receptor clusters, we made a drastic modification of PSD-Zip45 (modified PSD-Zip45). As a result, this modified PSD-Zip45 was able to for … More m large clusters of dopamine receptor (a typical example of GPCRs) with a diameter of 3-8 um in the cell membrane of lived cells. We then established the purification method for dopamine receptor dusters with a high quality and large quantity. The fourier transformation of electronmicrographs of dopamine receptor duster preparations revealed a high quality at paracrystal levels. This system can be applied to a vast majority of transmembrane receptors. Actually, we also made two-dimensional large dusters of LPA receptor (another example of GPCRs) using our system. Thus, we provide a powerful system for analyzing the structure-function relationship of the transmembrane receptors. For the sake of conventional use, we are now going to try the scale-down of cell culture system for purifying two-dimensional dusters of transmembrane receptors.In addition to the above project, we further demonstrated the molecular mechanisms underlying neuronal and vascular cell plasticity such as synaptic dynamics mediated by PSD proteins (PSD-Zip45, PSD-Zip70 and others) and onset of atherosclerosis triggered by unsaturated lysophosphatidic adds. Less