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Comparative studies on the moleular architecture of insitol-trisphosphate receptor and ryanodine receptor and their intracellular distribution

肌醇三磷酸受体与兰尼定受体分子结构及其细胞内分布的比较研究

基本信息

批准号：
04454123
负责人：
KATAYAMA Eisaku
金额：
$ 4.35万
依托单位：
The University of Tokyo
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (B)
财政年份：
1992
资助国家：
日本
起止时间：
1992 至 1993
项目状态：
已结题

项目摘要

Both inositol-1,4,5-trisphosphate receptor (IP_3R) and ryanodine-receptor (RyaR) form ligand-gated Ca^<2+> -channel and play a crucial role in intracellular signal transduction. Though the total amino-acid sequence was already determined for both, histological studies on these receptors still remain insufficient and nothing substantial has been done on their three-dimensional distribution in cells or tissues. On the structure side, the molecular architecture of tetrameric Rya-R particle has been extensively studied either by negative staining or cryo-electron microscopy, leading to the reconstruction of its three-dimensional structure. Despite many efforts similarly done for IP_3-R,however, nobody has successfully obtained, so far, the concrete information on the molecular architecture of that receptor. The author has been applying his expertised technique of quick-freeze deep-etch replica electron microscopy to the studies of various kinds of protein architecture. With this technique, one obtain very conrasty images of macromolecules in solution or in cells/tissues with very high time-and spacial-resolution, with nicely preserved three-dimensional organization. Thus, we could succesfully obtain the images of IP_3R in situ in the dendrites of cerebellar Purkinje cells. We compared them with those of RyaR and were surprized to find that the physical size of IP_3R is by far smaller than the value calculated from the ratio of molecular weights of two receptors and the actual size of RyaR.We also found that tetrameric IP_3R forms a very regular two-dimensional molecular array in situ in the cell, under certain anoxemic conditions. Further studies must be done to understand the physiological role of such array. It is also of great importance to determine the higher-resolution structure of the receptor molecules to define the sites of interacton with variuos ligands.

三磷酸肌醇受体（IP_3R）和鱼雁碱受体（RyaR）都是配体门控的Ca^2+通道，在细胞内信号转导中起重要作用。虽然已经确定了两者的总氨基酸序列，但对这些受体的组织学研究仍然不足，并且对其在细胞或组织中的三维分布没有进行实质性的研究。在结构方面，四聚体Rya-R颗粒的分子结构已经通过负染色或冷冻电子显微镜进行了广泛的研究，导致其三维结构的重建。然而，尽管对IP_3-R做了许多类似的努力，但到目前为止，还没有人成功地获得该受体分子结构的具体信息。作者一直在应用他的速冻深蚀复型电子显微镜技术研究各种蛋白质的结构。利用这种技术，人们可以获得溶液或细胞/组织中大分子的非常conrasty的图像，具有非常高的时间和空间分辨率，并且很好地保留了三维组织。因此，我们成功地获得了IP_3R在小脑浦肯野细胞树突上的原位图像。我们将它们与RyaR进行了比较，发现IP_3R的物理尺寸远小于由两种受体分子量之比和RyaR的实际尺寸计算的值，并发现在一定的缺氧条件下，四聚体IP_3R在细胞内原位形成非常规则的二维分子阵列。必须做进一步的研究来了解这种阵列的生理作用。确定受体分子的高分辨结构，确定与各种配体相互作用的位点也是非常重要的。