The Discovery of the IP3 Receptor
The Discovery of the IP3 Receptor
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IP3受体的发现
DOI：
10.1166/msr.2012.1009
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发表时间：
2012
期刊：
影响因子：
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通讯作者：
Mikoshiba K.
中科院分区：
文献类型：
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作者：
Hatanaka N;Tokuno H;Nambu A;Takada M;神永拓,安藤雄太,大月智史,小田中浩平,中村仁彦;明和政子;Mikoshiba K.
众所周知，细胞内Ca2+释放是由信使IP3触发的，但IP3的作用方式仍然是一个谜。假设IP3受体将IP3信号转换为多个信号。通过比较正常小鼠和突变小鼠，我们使用了一种独特的方法来鉴定IP3受体的候选蛋白。缺乏Ca2+峰值的突变小鼠缺乏P400蛋白，我们假设P400是IP3受体。使用特异性单克隆抗体，IP3结合活性可以与P400一起呈剂量依赖性免疫沉淀，表明P400与IP3受体/IP3结合蛋白有关。然后，我们采用免疫筛选方法，利用表达文库和我们生成的特异性鉴定P400的单克隆抗体筛选来克隆IP3受体。通过这种方法，我们确定了IP3受体的整个cDNA序列。单克隆抗体免疫金技术在内质网上定位。纯化的IP3受体被发现作为一个Ca2+通道，当纳入脂质双分子层。此外，我们发现了一个新的信使，并将其命名为IRBIT （IP3R结合蛋白与肌醇1,4,5-三磷酸释放），因为它可以从IP3受体释放。这是一种与IP3受体的IP3结合核心结构域相互作用的IP3受体的伪配体。IRBIT通过结合各种离子转运体（如胰型NBC1 （pNBC1）和CFTR）来调节酸碱平衡。我们发现IP3受体结合了许多分子。低温电子显微镜显示，IP3受体内部有一个具有多孔表面积的空位，这便于IP3受体与许多蛋白质结合。IP3受体作为信号中枢，与各种分子形成大分子复合物。IP3受体作为信号中枢的功能是将一个简单的IP3信号转化为多种多样的多重信号的关键特征。
It was known that intracellular Ca2+ release was triggered by the messenger IP3, but the mode of action of IP3 remained a mystery. The IP3 receptor was hypothesized to convert the IP3 signal to multiple signals. We used a unique approach to identify a candidate protein for the IP3 receptor by comparing normal and mutant mice. The mutant mice, which lacked Ca2+ spikes, were deficient in the protein P400 and we hypothesized that P400 was the IP3 receptor. Using specific monoclonal antibodies, IP3 binding activity could be immunoprecipitated along with P400, in a dose-dependent manner, suggesting that P400 is related to the IP3 receptor/IP3 binding protein. We then employed an immunoscreening approach to clone the IP3 receptor by using an expression library and screening with the monoclonal antibodies we had generated for the specific identification of P400. With this approach we determined the entire cDNA sequence for the IP3 receptor. The immunogold technique using monoclonal antibodies showed its localization on the endoplasmic reticulum. The purified IP3 receptor was found to work as a Ca2+ channel when incorporated into a lipid bilayer. In addition, we discovered a new messenger and named it IRBIT (IP3R binding protein released with inositol 1,4,5-trisphosphate) because it can be released from the IP3 receptor. This is a pseudo-ligand of the IP3 receptor that interacts with the IP3-binding core domain of the IP3 receptor. IRBIT regulates the acid-base balance by binding to various ion transporters, such as pancreas-type NBC1 (pNBC1) and CFTR. We found that the IP3 receptor binds many molecules. Cryo-electron microscopy shows a balloon-like structure, which has a vacancy inside the IP3 receptor with a multi-porous surface area, which is convenient for the IP3 receptor to associate with many proteins. The IP3 receptor serves as a signaling hub, which forms a macromolecular complex with various molecules. The function of IP3 receptor as a signaling hub is the key feature of converting a simple IP3 signal to the variety of multiple signals.