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）超家族在内的跨膜受体在细胞功能的各个方面都是必不可少的，并且在许多疾病的病理中也起着关键作用。除了少数例外，由于这些受体的大量纯化和结晶困难，它们的三维超结构尚未得到证实。在这个项目中，我们正在开发一个新的系统来分析跨膜受体的三维超结构。在这个系统中，在活细胞的细胞膜上，通过一种强自聚合的突触后支架蛋白PSD-Zip45聚集靶受体，在准晶体水平上形成跨膜受体的二维微粒（Homer 1c）。为了形成高度有序的二维跨膜受体簇，我们对PSD-Zip45进行了大幅度的修饰（modified PSD-Zip45）。结果表明，该修饰后的PSD-Zip45能够在活细胞的细胞膜上形成直径为3 ~ 8 μ m的多巴胺受体大簇（典型的gpcr）。建立了高质量、大批量的多巴胺受体掸子的纯化方法。多巴胺受体掸子制备的电子显微图的傅里叶变换显示出高质量的准晶水平。该系统可应用于绝大多数跨膜受体。实际上，我们也使用我们的系统制作了LPA受体（gpcr的另一个例子）的二维大颗粒。因此，我们为分析跨膜受体的结构-功能关系提供了一个强有力的系统。出于常规用途的考虑，我们现在将尝试缩小细胞培养系统的比例，以纯化跨膜受体的二维粉尘。除了上述项目，我们进一步证明了神经元和血管细胞可塑性的分子机制，如PSD蛋白（PSD- zip45， PSD- zip70等）介导的突触动力学和不饱和溶血磷脂添加物引发的动脉粥样硬化的发生。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