IRBIT, a Novel Inositol 1,4,5-Trisphosphate (IP3) Receptor-binding Protein, Is Released from the IP3 Receptor upon IP3 Binding to the Receptor

The inositol 1,4,5-trisphosphate (IP 3 ) receptors (IP 3 Rs) are IP 3 -gated Ca 2+ channels on intracellular Ca 2+ stores. Herein, we report a novel protein, termed IRBIT ( I P 3 R b inding protein released with i nositol 1,4,5- t risphosphate), which interacts with type 1 IP 3 R (IP 3 R1) and was released upon IP 3 binding to IP 3 R1. 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 3 R1 (residues 1–2217). IRBIT, consisting of 530 amino acids, has 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 IP 3 R1 was mapped to the IP 3 binding core. IP 3 dissociated IRBIT from IP 3 R1 with an EC 50 of ∼0.5 μ m , 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 3 R, raising the possibility that IRBIT acts as a signaling molecule downstream from IP 3 R.