Study for IP_3 - detecting system of IP_3 receptor

IP_3的研究——IP_3受体检测系统

基本信息

批准号：
13357001
负责人：
MIKOSHIBA Katsuhiko
金额：
$ 31.78万
依托单位：
The University of Tokyo
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (A)
财政年份：
2001
资助国家：
日本
起止时间：
2001 至 2002
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-13357001/
关键词：
IP_3 receptor calcium calcium signaling カルシウムシグナリング IP_3 IP_3スポンジ

项目摘要

The inositol 1,4,5-trisphosphate (IP_3) receptors (IP_3Rs) are IP_3-gated Ca^<2+> channels on intracellular Ca^<2+> stores. We identified a novel protein, termed IRBIT (IP_3R(endoplasmic reticulum) binding protein released with inositol 1,4,5-trisphosphate), which interacts with type 1 IP_3R(IP_3R1) and was released upon IP_3 binding to IP_3R1. IRBIT was purified from a high salt extract of crude rat brain microsomes with IP_3 elution using an affinity column with the huge immobilized N-terminal cytoplasmic region of IP_3R1 (residues 1-2217). IRBIT, consisting of 530 amino acids, had a domain homologous to S-adenosylhomocysteine hydrolase in the C-terminal and, in the N-terminal, a 104 amino acid appendage containing multiple potential phosphorylation sites. In vitro binding experiments showed the N-terminal region of IRBIT to be essential for interaction and the IRBIT binding region of IP3R1 was mapped to the IP_3-binding core. IP_3 dissociated IRBIT from IP_3R1 with an EC_<50> of 〜0.5 mM, i.e. it was 50 times more potent than other inositol polyphosphates. Moreover, alkaline phosphatase treatment abolished the interaction, suggesting that the interaction was dualistically regulated by IP_3 and phosphorylation. Immunohistochemical studies and co-immunoprecipitation assays showed the relevance of the interaction in a physiological context. These results suggest that IRBIT is released from activated IP_3R, raising the possibility that IRBIT acts as a signaling molecule downstream from IP_3R.

肌醇1,4,5-三磷酸（IP_3）受体（IP_3RS）是IP_3门控<2+>在细胞内Ca^<2+>商店上的通道。我们确定了一种新型蛋白质，称为IRBIT（IP_3R（内质网）结合蛋白与肌醇1,4,5-三磷酸释放的蛋白质，该蛋白质与1型IP_3R（IP_3R1）相互作用，并以IP_3结合到IP_3R1而释放。使用IP_3R1的巨大固定的N末端细胞质区域（残基1-2217）的亲和力柱从具有IP_3洗脱的粗大鼠脑微粒体的高盐提取物中纯化IRBIT。 IRIT由530个氨基酸组成，具有与S-腺基质类半胱氨酸水解酶在C末端的域同源，在N端，具有104个氨基酸附属物，其中包含多个潜在的磷酸化位点。体外结合实验表明，IRBIT的N末端区域对于相互作用至关重要，IP3R1的IRBIT结合区域映射到IP_3结合核心。 IP_3从IP_3R1中分离出IP_3R1的EC_ <50>为〜0.5 mm，即它的潜力是其他肌醇多磷酸盐的50倍。此外，酒精磷酸酶处理消除了这种相互作用，表明这种相互作用是由IP_3和磷酸化对双重论调节的。免疫组织化学研究和共免疫沉淀测定法显示了在物理背景下相互作用的相关性。这些结果表明，IRBIT是从激活的IP_3R中释放出来的，从而提高了IRBIT充当IP_3R下游的信号分子的可能性。