Study for IP_3 - detecting system of IP_3 receptor

肌醇1，4，5-三磷酸(IP_3)受体(IP_3Rs)是细胞内钙离子储备上的IP_3门控钙通道。我们鉴定了一种新的蛋白，命名为IRBIT(IP_3R(内质网)结合蛋白)，它与I型IP_3R(IP_3R1)相互作用，并在IP_3与IP_3R1结合时释放。IRBIT是用IP_3洗脱的粗大鼠脑微粒体的高盐抽提物，用IP_3R1的N-末端胞浆区(残基1-2217)的亲和层析柱纯化而成的。IRBIT由530个氨基酸组成，在C端有一个与S同型半胱氨酸腺苷水解酶同源的结构域，在N端有104个氨基酸残基，含有多个潜在的磷酸化位点。体外结合实验表明，IRBIT的N-末端区域是相互作用所必需的，IP3R1的IRBIT结合区被定位到IP_3结合核心。IP_3从IP_3R1上解离Irbit，其EC_(50)为~0.5 mm，是其他肌醇多聚磷酸盐的50倍。此外，碱性磷酸酶处理取消了这种相互作用，表明这种相互作用受IP_3和磷酸化的双重调节。免疫组织化学研究和免疫共沉淀分析表明，这种相互作用在生理背景下是相关的。这些结果表明，Irbit是从激活的IP_3R中释放出来的，这增加了Irbit作为IP_3R下游信号分子的可能性。

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.