| Budget Amount *help |
¥49,660,000 (Direct Cost: ¥38,200,000、Indirect Cost: ¥11,460,000)
Fiscal Year 2002: ¥22,490,000 (Direct Cost: ¥17,300,000、Indirect Cost: ¥5,190,000)
Fiscal Year 2001: ¥27,170,000 (Direct Cost: ¥20,900,000、Indirect Cost: ¥6,270,000)
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| Research Abstract |
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.
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