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 (IP(3)R binding protein released with inositol 1,4,5-trisphosphate), 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 approximately 0.5 microm, 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.

The hydrolysis of phosphatidylinositol 4,5-bisphosphate in response to cell surface receptor activation leads to the production of an intracellular second messenger, inositol 1,4,5trisphosphate (IP 3 ). 1 IP 3 mediates the release of Ca 2ϩ from intracellular Ca 2ϩ storage organelles, mainly the endoplasmic reticulum, by binding to its receptor (IP 3 R). In these IP 3 /Ca 2ϩ signaling cascades, IP 3 R works as a signal converter from IP 3 to Ca 2ϩ (1)(2)(3).
IP 3 R is a tetrameric intracellular IP 3 -gated Ca 2ϩ release channel (3,4). There are three distinct types of IP 3 R in mammals (5)(6)(7). Type 1 IP 3 R (IP 3 R1) is highly expressed in the central nervous system, particularly in the cerebellum (8,9). Mouse IP 3 R1 is composed of 2749 amino acids (5), and is divided into three functionally distinct regions: the IP 3 -binding domain near the N terminus, the channel-forming domain with six membrane-spanning regions close to the C terminus, and the regulatory domain separating the two regions (10,11). Deletion mutagenesis analysis of the IP 3 -binding domain has shown that residues 226 -578 of IP 3 R1 are close to the minimum for specific and high affinity ligand binding, thus assigned to the IP 3 binding core (12). The precise gating mechanism of IP 3 R triggered by IP 3 remains unclear, but IP 3 binding induces a substantial but as yet undefined conformational change, which may cause channel opening (10). Besides this channel opening, such IP 3 -induced conformational change has been assumed to be responsible for degradation of IP 3 R (13,14).
The increase in the cytoplasmic Ca 2ϩ concentration resulting from IP 3 R activation regulates the activities of thousands of downstream targets that play key roles in many aspects of cellular processes, including fertilization, development, proliferation, secretion, and synaptic plasticity (1,2,15). To control such a vast array of cell functions, Ca 2ϩ signals need to be precisely regulated in terms of space, time and amplitude (2,15). Such a complex regulation of Ca 2ϩ signals has been partly attributed to the diversity of IP 3 R isoform expression, assembly of heterotetrameric complexes of IP 3 R isoforms, subcellular distributions of IP 3 R, and regulation of IP 3 R by Ca 2ϩ itself, ATP, and phosphorylation (3,4,16). IP 3 R channels are also regulated by their interacting proteins (4,17), including calmodulin (18,19), FKBP12 (Refs. 20 -22, but also see Refs. 23 and 24), calcineurin (Refs. 21 and 25, but also see Refs. 23 and 24), ankyrin (26 -28), sigma-1 receptor (28), chromogranins A and B (29 -31), IRAG (32), Fyn (33), and BANK (34). Moreover, a family termed CaBP has been shown to interact with IP 3 R in a Ca 2ϩ -dependent manner, and to directly activate IP 3 R in the absence of IP 3 (35). IP 3 R has also been demonstrated to be physically coupled to its upstream or downstream signaling molecules by protein-protein interactions. For example, IP 3 R is coupled with group 1 metabotropic glutamate receptors (mGluRs) via the Homer family of proteins (36) and with B 2 bradykinin receptors (B 2 Rs) by an unknown mechanism (37). Activations of mGluRs and B 2 Rs lead to the production of IP 3 in proximity to IP 3 R, the result being efficient and specific signal propagation. Another example is Trp3, a candidate for plasma membrane Ca 2ϩ channels regulated by intracellular Ca 2ϩ store depletion (capacitative calcium entry channels). IP 3 R has been shown to interact with Trp3 directly, and to activate it via a conformational coupling mechanism (38,39). These proteinprotein interactions are supposed to regulate the IP 3 /Ca 2ϩ signaling pathway and contribute to the specificity of intracellular Ca 2ϩ dynamics.
To gain further insights into regulation of the IP 3 /Ca 2ϩ signaling pathway, we searched for IP 3 R-binding proteins. In particular, we focused on molecules that interact with IP 3 R in a manner regulated by IP 3 , because such molecules may recognize the conformational change in IP 3 R induced by IP 3 binding, and/or may function as novel upstream or downstream signaling molecules of IP 3 R. For this purpose, we used an affinity column conjugated with the N-terminal 2217 amino acid residues of IP 3 R1 containing most of the large cytoplasmic region of the receptor molecule. By eluting bound proteins with IP 3 from this affinity column, we identified a novel IP 3 R-binding protein, IRBIT (IP 3 R binding protein released with inositol 1,4,5-trisphosphate). IRBIT bound to IP 3 R1 in vitro and in vivo, and co-localized intensively with IP 3 R1. Moreover, IRBIT was released from IP 3 R1 at a physiological concentration of IP 3 . On the basis of these results, we consider herein the role of IRBIT in IP 3 /Ca 2ϩ signaling.
Purification and Partial Amino Acid Sequencing of IRBIT-Adult rat cerebella (ϳ5 g) were homogenized in 45 ml of homogenizer buffer (10 mM Hepes (pH 7.4), 320 mM sucrose, 2 mM EDTA, 1 mM 2-mercaptoethanol, and protease inhibitors) with a glass-Teflon homogenizer (950 rpm, 10 strokes), and the homogenate was centrifuged at 1,000 ϫ g for 10 min. The supernatant (S1 fraction) was centrifuged at 100,000 ϫ g for 60 min to obtain the cytosolic fraction (the supernatant) and the crude microsome (the pellet). The crude microsome was homogenized in 25 ml of homogenizer buffer containing 500 mM NaCl with a glass-Teflon homogenizer (1,200 rpm, 10 strokes), incubated on ice for 15 min, and centrifuged at 100,000 ϫ g for 60 min to obtain the high salt extract (the supernatant) and the stripped-crude microsome (the pellet). The high salt extract was diluted five times with 10 mM Hepes (pH 7.4), 2 mM EDTA, 1 mM 2-mercaptoethanol, 0.01% Brij 35, and protease inhibitors. The diluted high salt extract was precleared with glutathione-Sepharose and loaded onto a GST-EL affinity column equilibrated with binding buffer (10 mM Hepes (pH 7.4), 100 mM NaCl, 2 mM EDTA, and 1 mM 2-mercaptoethanol). The GST column was used as a control. The columns were washed with 20 column volumes of binding buffer, and bound proteins were eluted with binding buffer containing 50 M IP 3 (Dojindo) and 0.05% Brij 35. The eluted material was concentrated, separated by SDS-polyacrylamide gel electrophoresis (PAGE) on a 10% gel, and stained with Coomassie Brilliant Blue. The 60-kDa protein band was excised from the gel and digested with lysyl endopeptidase (Wako) essentially according to the previously described method (41). The polypeptides were separated by a C 18 reversed-phase column (RPC C2/C18 SC 2.1/10, Amersham Biosciences) connected on a SMART system (Amersham Biosciences). The amino acid sequence of each peptide was determined by 494 procise protein sequencer (Applied Biosystems). Two peptide sequences, N-YSFMATVTK-C and N-QIQ-FADDMQEFTK-C were obtained.
cDNA Cloning of IRBIT-BLAST searches of two peptide sequences derived from the 60-kDa protein against the non-redundant data base revealed that these sequences match the sequence of a human cDNA deposited in a patent (GenBank TM accession number CAC09285). Based on the data bases of mouse expressed sequence tags (accession number AW229870 and BE282170) homologous to this cDNA, primers (5Ј-ATGTCGATGCCTGACGCGATGC-3Ј and 5Ј-GCGTGGTTCATGTG-GACTGGTC-3Ј) were synthesized. cDNA of IRBIT was amplified by polymerase chain reaction (PCR) using mouse cerebellum oligo(dT)primed, first-strand cDNA as a template. PCR product was cloned into pBluescript II KS(ϩ) (Stratagene) and sequenced. Sequences of three independent clones were confirmed.
Production of Affinity-purified Anti-IRBIT Antibody-A Japanese white rabbit was immunized with purified IRBIT-(1-104)-His 6 by subcutaneous injection with the complete Freund's adjuvant at 14-day intervals. The anti-IRBIT antisera was affinity-purified by passing serum from the immunized rabbit over a GST-IRBIT-(1-104) column covalently coupled with cyanogen bromide-activated Sepharose 4B (Amersham Biosciences), and specific antibodies bound to the column were eluted with 100 mM glycine-HCl (pH 2.5).
Subcellular Fractionation and Immunoblotting-Cerebrum, cerebellum, heart, lung, liver, kidney, thymus, spleen, testis, and ovary were dissected from the adult mouse and S1 fraction were obtained essentially as described above. The cytosol, the crude microsome, the high salt extract, and the stripped-crude microsome of mouse cerebellum were obtained essentially as described above. Proteins with the amount indicated were subjected to 10% SDS-PAGE and transferred onto polyvinylidene difluoride membrane by electroblotting. After blocking, membranes were immunoblotted with anti-IRBIT antibody (1 g/ml) for 1 h at room temperature, followed by horseradish peroxidase-conjugated donkey anti-rabbit IgG (Amersham Biosciences). Immunoreactive bands were visualized with the enhanced chemiluminescence detection system (Amersham Biosciences).
In Vitro Binding Experiments-Mouse cerebellar cytosolic fraction was diluted two times with 10 mM Hepes (pH 7.4), 200 mM NaCl, 2 mM EDTA, 1 mM 2-mercaptoethanol, and 0.02% Triton X-100. The high salt extract was diluted five times with 10 mM Hepes (pH 7.4), 2 mM EDTA, 1 mM 2-mercaptoethanol, and 0.01% Triton X-100. Diluted fractions (the final NaCl concentration of both fractions was 100 mM) were incubated with 20 g of GST-EL or GST for 2 h at 4°C. After adding 10 l of glutathione-Sepharose and another 2-h incubation, the resins were washed five times with wash buffer (10 mM Hepes, pH 7.4, 100 mM NaCl, 2 mM EDTA, 1 mM 2-mercaptoethanol, and 0.01% Triton X-100), and bound proteins were eluted with 20 mM glutathione. Eluted proteins were analyzed by Western blotting with anti-IRBIT antibody.
For dephosphorylation, the diluted high salt extract was incubated with or without bacterial alkaline phosphatase (Toyobo) in the presence of 2 mM MgCl 2 for 30 min at 37°C after which 5 mM EDTA was added, and the sample was processed for pull-down assay as described above.
For the determination of the IRBIT binding region and the critical amino acids of IP 3 R1, the diluted high salt extract were processed for pull-down assay with 100 pmol of GST, GST-EL, GST-Ia, GST-Iab, GST-IbIIa, GST-IIab, GST-IIbIIIa, GST-IIIab, GST-IV, GST-IV-Va, K508A, or R441Q as described above and analyzed by Western blotting with anti-IRBIT antibody.
For the determination of the IP 3 R1-interacting region of IRBIT, COS-7 cells expressing GFP-tagged full-length IRBIT or its truncated mutants were lysed in lysis buffer (10 mM Hepes pH 7.4, 100 mM NaCl, 2 mM EDTA, 1 mM 2-mercaptoethanol, 0.5% Nonidet P-40, and protease inhibitors) for 30 min at 4°C, followed by centrifugation (100,000 ϫ g, 30 min). The supernatants were processed for pull-down assay with GST-EL or GST as described above, and bound proteins were subjected to immunoblot analysis with anti-GFP antibody (Medical & Biological Laboratories).
Indirect Immunofluorescence and Confocal Microscopy-Transfected COS-7 cells grown on glass coverslips were washed once in phosphatebuffered saline (PBS), fixed in 4% formaldehyde in PBS for 15 min, permeabilized in 0.1% Triton X-100 in PBS for 5 min, and blocked in PBS containing 2% normal goat serum for 60 min at room temperature. For washing out cytosolic proteins, transfected cells were washed once in PBS, permeabilized in ice-cold permeabilization buffer (80 mM PIPES, pH 7.2, 1 mM MgCl 2 , 1 mM EGTA, and 4% polyethylene glycol) containing 0.1% saponin for 10 min on ice, and washed twice with ice-cold permeabilization buffer before fixation. Cells were then stained with rabbit anti-IRBIT antibody (1 g/ml for 60 min at room temperature) and rat anti-IP 3 R1 antibody 18A10 (44) overnight at 4°C. Following four 5-min PBS washes, Alexa 488-conjugated goat anti-rabbit IgG and Alexa 594-conjugated goat anti-rat IgG (Molecular Probes) were applied for 45 min at 37°C. Following four 5-min PBS washes, the coverslips were mounted with Vectashield (Vector Laboratories) and observed under IX-70 confocal fluorescence microscopy (Olympus) with a ϫ60 objective.
Immunoprecipitation-Immunoprecipitation was performed as described (45) with modifications. Adult mouse cerebellum was homogenized in 10 volumes of 4 mM Hepes (pH 7.4), 320 mM sucrose, and protease inhibitors with a glass-Teflon homogenizer. The homogenate was centrifuged at 800 ϫ g for 10 min, and the supernatant was subjected to another centrifugation at 9000 ϫ g for 15 min. The supernatant from the second centrifugation was solubilized in 1% sodium deoxycholate at 36°C for 30 min, followed by adding 0.1 volume of 1% Triton X-100 in 50 mM Tris-HCl (pH 9.0), and the preparation was centrifuged at 100,000 ϫ g for 10 min. The supernatant was incubated with 5 l of protein G-Sepharose 4 fast flow (Amersham Biosciences) for 2 h at 4°C to clarify nonspecific binding to the protein G beads. At the same time, 3 g of rabbit anti-IRBIT antibody, control rabbit IgG, rat anti-IP 3 R1 antibody 10A6 (46), control rat IgG, mouse anti-IP 3 R2 antibody KM1083 (47), or control mouse IgG was preincubated with 5 l of protein G beads for 2 h, and the protein G-antibody complex was spun down at 3,000 rpm for 2 min. The clarified supernatant was then added to the antibody-bound protein G beads, and the mixture was incubated for 2 h at 4°C. Beads were washed five times with 10 mM Hepes (pH 7.4), 100 mM NaCl, and 0.5% Triton X-100 and analyzed by Western blotting with anti-IRBIT antibody, mouse anti-IP 3 R1 antibody KM1112 (47), KM1083, or mouse anti-IP 3 R3 antibody KM1082 (47).

Purification and cDNA Cloning of a Novel IP 3 R-interacting
Protein-To identify IP 3 R-interacting molecules, we used a GST fusion protein of the N-terminal 2217 amino acids of mouse IP 3 R1 (GST-EL). This region is the large cytoplasmic portion of IP 3 R1 containing the IP 3 binding domain and regulatory domain (10,11). GST-EL or GST was expressed using a baculovirus/Sf9 cell system and conjugated to glutathione-Sepharose. The extract with a high salt buffer (containing 500 mM NaCl) from crude rat cerebellar microsomes, which was thought to be enriched with peripherally membrane-bound proteins, was loaded onto a glutathione-Sepharose affinity column on which GST-EL or GST was immobilized. To detect proteins that were dissociated from IP 3 R in the presence of IP 3 , the proteins bound to the affinity columns were eluted by addition of 50 M IP 3 . A protein with a mass of about 60 kDa was detected in the 50 M IP 3 -eluate from the GST-EL column (Fig.  1A), but not from the GST column (data not shown). Two peptide sequences derived from the 60-kDa protein were determined. BLAST searches of non-redundant databases revealed that these two sequences matched the sequence of a human cDNA deposited in a patent. On the basis of sequence information on mouse expressed sequence tags homologous to this cDNA, the cDNA of the 60-kDa protein was obtained by reverse transcriptase-PCR from the mouse cerebellum. The predicted amino acid sequence of the cloned cDNA revealed a protein composed of 530 amino acid residues (Fig. 1B), with a calculated molecular mass of 58.9 kDa, which was close to its apparent molecular mass of 60 kDa estimated by SDS-PAGE (Fig.  1A). We designated the 60-kDa protein IRBIT.
Homology analysis of the deduced amino acid sequence of IRBIT revealed the C-terminal region (residues 105-530) to be homologous (51% identical, 74% similar) to the methylation pathway enzyme S-adenosylhomocysteine hydrolase (EC 3.3.1.1.) (48) (Fig. 1, C and D). An appendage of the N-terminal region (residues 1-104) of IRBIT had no homology with reported proteins and contained a serine-rich region (residues 62-103) (Fig. 1, B and D). Motif searches of the IRBIT sequence revealed the presence of a putative coiled-coil motif (residues 111-138) and a putative NAD ϩ binding region (residues 314 -344) (Fig. 1, B and D). There were 17 potential phosphorylation sites for protein kinases such as casein kinase II, PKC, PKA/ PKG, and tyrosine kinases, out of which seven sites were concentrated in the N-terminal region (Fig. 1B). Neither putative membrane-spanning regions nor signal sequences were found. Recently, a mRNA expressed in dendritic cells was cloned from a human cDNA library, and it was named DCAL (49), but its physiological function was not addressed. The 100% identical amino acid sequences of IRBIT and DCAL indicate that IRBIT is a mouse homologue of human DCAL.
Although IRBIT was homologous with S-adenosylhomocysteine hydrolase, which catalyzes the reversible hydrolysis of S-adenosylhomocysteine to adenosine and homocysteine, recombinant IRBIT expressed in bacteria had no enzyme activi-ties in the hydrolysis direction, nor had any effects on the enzyme activity of S-adenosylhomocysteine hydrolase (data not shown).
Tissue Distribution and Subcellular Localization of IR-BIT-We generated an affinity-purified antibody against the N-terminal region of the IRBIT (Fig. 1B, boxed). To confirm the specificity of this antibody, we transfected the cDNA of IRBIT into COS-7 cells, and the whole cells lysates obtained were analyzed by immunoblotting with the anti-IRBIT antibody. As shown in Fig. 2A, the anti-IRBIT antibody recognized only a single protein with a size of ϳ60 kDa. The molecular mass of the exogenously expressed IRBIT (Fig. 2A, lane 1) was the same as that of the endogenous protein in COS-7 (Fig. 2A, lane  3), confirming that the cDNA clone encodes the full-length IRBIT protein. We examined the expression of IRBIT in several mouse tissues by immunoblot analysis with this anti-IRBIT antibody. IRBIT was detected ubiquitously, with the highest expressions in the cerebrum and cerebellum (Fig. 2B).
Next, we investigated the subcellular distribution of IRBIT by fractionation of the mouse cerebellum. IRBIT was present in both the cytosolic and the crude microsome fraction (Fig. 2C,  lanes 2 and 3, respectively). The crude microsome fraction was further separated into a peripherally membrane-bound fraction (the fraction from which IRBIT was originally purified) and a stripped-membrane fraction, with the aforementioned high salt buffer. As shown in Fig. 2C, IRBIT in the crude microsome fraction was partially extracted with the high salt buffer (Fig. 2C, lane 4). In contrast, IP 3 R1, which is an integral membrane protein of the endoplasmic reticulum, was not extracted (Fig. 2C, lower panel). These results indicate IRBIT to be both a cytosolic and a peripherally membrane-bound protein.
IRBIT in the High Salt Extract Interacted with IP 3 R1 and the N-terminal Region of IRBIT Was Essential for Interaction-IRBIT was present in both the cytosolic and the peripherally membrane-bound fraction of the mouse cerebellum (Fig. 2C). We investigated whether IRBIT in these fractions interacted with IP 3 R1 in vitro employing GST pull-down techniques. The cytosol and high salt extracts from crude mouse cerebellar microsomes were incubated with GST-EL or GST, and binding of IRBIT to the recombinant proteins was analyzed by immunoblotting with anti-IRBIT antibody. As shown in Fig. 3A, IRBIT in the high salt extract interacted with GST-EL (Fig. 3A,  lane 6), but not with GST (Fig. 3A, lane 5). In contrast, IRBIT in the cytosolic fraction did not interact with GST-EL (Fig. 3A,  lane 3). The same result was obtained when both fractions were dialyzed against the same buffer, indicating that the difference was due neither to a difference in buffer composition nor to excluded small molecules (data not shown). We speculated that the difference might be attributable to a post-translational modification of IRBIT such as phosphorylation. To test this possibility, we treated the high salt extract with alkaline phosphatase, a nonspecific phosphatase, followed by incubation with GST-EL or GST. As shown in Fig. 3B, IRBIT in the high salt extract no longer interacted with GST-EL after phosphatase treatment (Fig. 3B, lane 6). This result raises the possibility that phosphorylation of IRBIT may be necessary for association with IP 3 R1, although the possibility that phosphorylation of other proteins may regulate the interaction between IRBIT and IP 3 R1 cannot be excluded.
To determine the region of IRBIT responsible for the interaction with IP 3 R1, GST pull-down experiments were carried out using GFP-tagged deletion mutants of IRBIT (Fig. 4A). As shown in Fig. 4B, both GFP-IRBIT and GFP-IRBIT-(1-277) bound to GST-EL efficiently (Fig. 4B, lanes 3 and 6, respectively). Although GFP-IRBIT-(1-104) interacted with GST-EL, the interaction was much weaker than those of GFP-IRBIT and  2 and 5). Bound proteins were pulled down with glutathione-Sepharose, eluted with glutathione, and analyzed by Western blotting using anti-IRBIT antibody (upper panel). GST-EL and GST pulled down with glutathione-Sepharose were visualized by staining with Coomassie Brilliant Blue (lower panel). B, the high salt extract of crude mouse cerebellar microsomes was incubated without (lanes 1-3) or with (lanes 4 -6) alkaline phosphatase prior to pull down with GST-EL (lanes 3 and 6) or GST (lanes 2 and 5). IRBIT binding was analyzed as in A.  2 and 3), and the whole cell lysates were analyzed by Western blotting with anti-IRBIT antibody. In lane 3, 10ϫ amounts of the lysate were loaded as compared with those in lanes 1 and 2. B, tissue distribution of IRBIT. S1 fractions (2 g of total protein) of adult mouse tissues were analyzed by Western blotting with anti-IRBIT antibody. C, subcellular fractionation of the mouse cerebellum. S1 fraction (lane 1) of mouse cerebella was centrifuged at 100,000 ϫ g to obtain the cytosolic fraction (lane 2) and the crude microsomes (lane 3). The crude microsomes were extracted with the high salt buffer containing 500 mM NaCl and centrifuged at 100,000 ϫ g to obtain the peripherally membrane-bound fraction (lane 4) and the stripped-crude microsomes (lane 5). Upper, each fraction (1 g of total protein) was analyzed by Western blotting with anti-IRBIT antibody. Lower, each fraction (0.2 g of total protein) was analyzed by Western blotting with anti-IP 3 R1 antibody.
GFP-IRBIT-(1-277) (Fig. 4B, compare lanes 7 and 9 with lanes  1 and 3 and lanes 4 and 6). In contrast, GFP-IRBIT-(105-530), which lacked the N-terminal region, and GFP alone did not interact with GST-EL (Fig. 4B, lanes 12 and 15, respectively). These results demonstrate the N-terminal region of IRBIT to be essential for the interaction with IP 3 R1, and the following ϳ170 amino acids containing a coiled-coil structure might be important for stabilizing the interaction.
IRBIT Co-localized with IP 3 R1 on the Endoplasmic Reticulum in Transfected COS-7 Cells-To test whether IRBIT interacts with IP 3 R1 in intact cells, IRBIT and IP 3 R1 were coexpressed in COS-7 cells, and their distribution was analyzed by confocal immunofluorescence microscopy. IRBIT was diffusely distributed in the cytoplasm, with no immunoreactivity in the nucleus (Fig. 5A). Because IRBIT was shown to be present in both the cytosolic and the crude microsome fraction by biochemical fractionation (Fig. 2C), we attempted to visualize only the membrane-bound population of IRBIT. For this purpose, we permeabilized plasma membranes of transfected COS-7 cells with saponin and washed out cytosolic IRBIT prior to fixation. As shown in Fig. 5B, in cells treated with saponin, localization of IRBIT on the reticular structure was revealed (Fig. 5B, left panels). The immunoreactivity of IRBIT extensively overlapped with that of IP 3 R1 (Fig. 5B, middle panels, and merged image right panels). The staining pattern of IP 3 R1 was not altered by permeabilization with saponin (data not shown). Since IRBIT expressed alone showed a coarse distribution instead (data not shown), these results indicate that IRBIT co-expressed with IP 3 R1 localized on the endoplasmic reticulum via the interaction with IP 3 R1. IP 3 R1 was expressed in COS-7 cells to a trace level, whereas type 2 IP 3 R (IP 3 R2) and type 3 IP 3 R (IP 3 R3) were predominantly expressed (50, 51). These endogenous IP 3 Rs showed again a coarse, not a reticular, distribution in COS-7 cells both in a previous report and in our hands (Ref. 52 and data not shown, respectively). Furthermore, a complex of IRBIT and endogenous IP 3 R2/IP 3 R3 were revealed by co-immunoprecipitation assay (data not shown). Taken together, these findings support our idea that IRBIT interacted not only with IP 3 R1 but also with IP 3 R2 and IP 3 R3 (see below).
When we transfected IP 3 R1 and GFP-IRBIT instead of IR-BIT and observed the fluorescence of GFP, essentially the same results were obtained (Fig. 5, C and D). To confirm the specificity of co-localization, we transfected GFP-IRBIT-(105-530), which did not interact with GST-EL because of the lack of the N-terminal region (Fig. 4), with IP 3 R1 into COS-7 cells. In contrast to GFP-IRBIT, GFP-IRBIT-(105-530) was distributed in the nucleus as well as the cytosol (Fig. 5E). IRBIT does not harbor predicted nuclear localization signals, and the reason GFP-IRBIT-(105-530) localized in the nucleus is unclear at present. When the cytosolic population was washed out by permeabilization, GFP-IRBIT-(105-530) localized only in the nucleus and did not co-localize with IP 3 R1 (Fig. 5F). This observation is consistent with biochemical results indicating the N-terminal region of IRBIT to be necessary for binding to IP 3 R1 (Fig. 4B).
IRBIT Interacted with IP 3 R in Vivo-To demonstrate an in vivo association between IRBIT and IP 3 R in native tissues, we performed co-immunoprecipitation experiments using mouse cerebellum. Cerebellar lysates were immunoprecipitated with anti-IRBIT antibody, and the immunoprecipitates were analyzed by immunoblotting with anti-IP 3 R1, anti-IP 3 R2, or anti-IP 3 R3 antibody. All three IP 3 R isoforms were co-immunoprecipitated by anti-IRBIT antibody, but not control IgG (Fig. 6A). In the reciprocal experiments, immunoprecipitation of IP 3 R1 or IP 3 R2 resulted in the co-precipitation of IRBIT (Fig. 6, B and  C). IRBIT was not detected in the anti-IP 3 R3 precipitates, probably due to the inefficiency of immunoprecipitation with anti-IP 3 R3 antibody (data not shown). When we performed immunoprecipitation assay using lysates of COS-7 cells transfected with IRBIT and IP 3 R3, in which most IP 3 R3 forms homotetramers (51), IRBIT was shown to interact with IP 3 R3 (data not shown). As for IP 3 R2, essentially the same result was obtained (data not shown). These results confirm IRBIT interacted with all IP 3 R isoforms in vivo.
Physiological Concentration of IP 3 Selectively Dissociated IR-BIT from IP 3 R1-IRBIT was originally identified in the GST-EL column eluate with 50 M IP 3 (Fig. 1A), suggesting that IP 3 disrupted the interaction between IRBIT and IP 3 R1. However, 50 M is a relatively high concentration compared with the physiological range of IP 3 , which was estimated to be a few micromolar after stimulation (53). Thus, we examined the dose-dependence of IP 3 with which IRBIT was dissociated from GST-EL, and its selectivity against other related inositol polyphosphates. IRBIT in the high salt extract of crude mouse cerebellar microsomes was pulled down with GST-EL, and eluted with 0.1-10 M IP 3 , IP 2 , IP 4 , IP 6 , or ATP. As shown in Fig. 7A, IP 3 dissociated IRBIT from GST-EL most efficiently in a dose-dependent manner (Fig. 7Aa, lower panel). We confirmed GST-EL to be undetectable in the IP 3 eluates (Fig. 7Aa,  upper panel), even with longer exposure (data not shown). The EC 50 (the concentration required for half-maximal dissociation of IRBIT from GST-EL) was ϳ0.5 M, which was within the physiological IP 3 concentration range (53) (Fig. 7B). IP 3 dissociated IRBIT from GST-EL about 50 times more efficiently than other inositol polyphosphates (Fig. 7, Ab-d and B). ATP, which has three phosphate groups like IP 3 , did not dissociate IRBIT from GST-EL even at 10 M (Fig. 7, Ae and B). These  13-15). The lysates of COS-7 cells expressing each construct (input; I) were incubated with GST-EL (E) or GST (G). Bound proteins were pulled down with glutathione-Sepharose, eluted with glutathione, and subjected to immunoblot analysis with anti-GFP antibody. results indicate that IRBIT was dissociated from IP 3 R1 selectively within the physiological concentration range of IP 3 .
IRBIT Interacted with the IP 3 Binding Region of IP 3 R1 and Lys-508 of IP 3 R1 Was Essential for Interactions with Both IRBIT and IP 3 -We investigated which region, the IP 3 binding region or the regulatory region, of IP 3 R1 was necessary for the interaction with IRBIT, using eight deletion mutants of IP 3 R1 constructed as GST fusion proteins based on the domain structure of IP 3 R1 (54) (Fig. 8A). As shown in Fig. 8B, GST-IbIIa (residues 224 -604), which contains the IP 3 binding core region (residues 226 -578) (12) bound to IRBIT to the same extent as GST-EL. In contrast, other GST fusion proteins, including GST-Iab and GST-IIab, did not interact with IRBIT. Next, we performed a site-directed mutagenesis analysis to determine the IP 3 R1 amino acids important for the interaction with IR-BIT. Lys-508 of IP 3 R1 was a critical amino acid residue for IP 3 binding (12), and substitution of Lys-508 of GST-IbIIa with alanine (K508A) resulted in an enormous loss of IP 3 binding

FIG. 6. IRBIT associated with IP 3 R in vivo.
A, cerebellar lysates were immunoprecipitated with anti-IRBIT or control antibody. The immunoprecipitates were subjected to SDS-PAGE followed by Western blotting with anti-IP 3 R1, anti-IP 3 R2, anti-IP 3 R3, or anti-IRBIT antibody. B, cerebellar lysates were immunoprecipitated with anti-IP 3 R1 or control antibody. The immunoprecipitates were subjected to Western blotting with anti-IRBIT or anti-IP 3 R1 antibody. C, cerebellar lysates were immunoprecipitated with anti-IP 3 R2 or control antibody. The immunoprecipitates were subjected to Western blotting with anti-IRBIT or anti-IP 3 R2 antibody. Arrowheads indicate immunoglobulin heavy chains. affinity (42). Conversely, R441Q, in which Arg-441 of GST-IbIIa was substituted for Gln, had higher IP 3 affinity than GST-IbIIa (42). GST pull-down assays using these recombinant proteins showed that IRBIT bound to GST-IbIIa and R441Q to the same extent, but not to K508A (Fig. 8C). Taken together, these results indicate that IRBIT binds to the IP 3 -binding region of IP 3 R1 and that Lys-508 of IP 3 R1 is required for the interaction with IRBIT as well as IP 3 , supporting the observation that IP 3 disrupts the interaction between IRBIT and IP 3 R1. DISCUSSION We screened IP 3 R1-binding proteins released from IP 3 R1 in the presence of IP 3 and identified a novel protein, IRBIT, from a high salt extract of crude cerebellar microsomes. IRBIT interacted with IP 3 R1 in vitro and in vivo, and co-localized extensively with IP 3 R1 in the endoplasmic reticulum in transfected cells. These results strongly suggest that IRBIT associates with IP 3 R1 in basal states. Moreover, the physiological concentration of IP 3 , but not of other inositol polyphosphates, dissociated IRBIT from IP 3 R1. IRBIT bound to the IP 3 binding region of IP 3 R1, and Lys-508 of IP 3 R1 was essential for the interactions with both IP 3 and IRBIT. These results suggest that IRBIT is released from IP 3 R1 with IP 3 produced in response to extracellular stimuli. Although many IP 3 R-binding proteins have been reported (18 -39), IRBIT is the first molecule for which the interaction with IP 3 R was shown to be regulated by IP 3 .
IRBIT is composed of two regions, the N-terminal region (residues 1-104) essential for the interaction with IP 3 R1, and the C-terminal region (residues 105-530) homologous to Sadenosylhomocysteine hydrolase (48). Crystallographic studies (55,56) and site-directed mutagenesis studies (57-60) have determined amino acid residues of S-adenosylhomocysteine hydrolase involved in substrate binding or NAD ϩ binding (Fig.  1C). Although most of these residues were well conserved in IRBIT, we did not detect enzyme activity of recombinant IRBIT expressed in bacteria. We concluded that the IRBIT does not have S-adenosylhomocysteine hydrolase activity, probably due to substitution of amino acids important for substrate binding, such as Leu-54, Phe-302, and His-353 of S-adenosylhomocysteine hydrolase (Fig. 1C), as discussed by another group (49). Domains that are homologous to certain enzymes, but are catalytically inactive, such as the esterase domain of the neu-  , upper). B, the intensity of the immunoreactive bands of IRBIT was quantified by infrared imaging system, and relative intensity was plotted against concentration. Results are shown as the mean Ϯ S.D. from at least three independent experiments.

FIG. 8. IRBIT interacted with the IP 3 -binding region of IP 3 R1
and Lys-508 was critical for this interaction. A, schematic representation of the structure of mouse IP 3 R1 and the recombinant GST fusion proteins used in this study. The IP 3 binding core region is indicated with a gray box. Putative membrane-spanning regions are indicated by solid vertical bars. Roman numbers below IP 3 R1 indicate the domain structure determined by the limited trypsin digestion (54). Numbers above the lines represent corresponding amino acid numbers. B, determination of the IRBIT binding region of IP 3 R1. The high salt extract of crude mouse cerebellar microsomes was incubated with GST fusion proteins described in A. Bound proteins were pulled down with glutathione-Sepharose, eluted with glutathione, and analyzed by Western blotting using anti-IRBIT antibody. C, site-directed mutagenesis analysis. The high salt extract was processed for pull-down assay with GST-IbIIa, R441Q, and K508A as described in B. Bound proteins were analyzed by Western blotting using anti-IRBIT antibody (upper panel). GST fusion proteins pulled down with glutathione-Sepharose were visualized by staining with Coomassie Brilliant Blue (lower panel). roligin family (61) and the carbonic anhydrase domain of receptor tyrosine phosphatase ␤ (62), are reportedly involved in protein-protein interactions. The C-terminal region of IRBIT may be one such domain. However, the possibility that IRBIT has enzyme activity with a different substrate specificity cannot be excluded.
In vitro binding experiments and immunostaining studies showed the N-terminal region of IRBIT to be essential, though not sufficient, for the interaction with IP 3 R1. The IRBIT-binding region of IP 3 R1 was shown to be its IP 3 binding region, and Lys-508 of IP 3 R1, the critical amino acid for IP 3 binding, was required for this interaction. Based on mutagenesis analysis, Yoshikawa et al. (12) proposed that basic amino acid residues, including Lys-508, contribute to form a positively charged pocket for binding to the three negatively charged phosphate groups of IP 3 . This model leads us to speculate that acidic or phosphorylated amino acid residues in the N-terminal region of IRBIT may be involved in interaction with the positively charged IP 3 -binding pocket of IP 3 R1. This hypothesis is supported by the following findings: 1) although IRBIT is a neutral protein (calculated pI of 6.48), its N-terminal region is relatively acidic (calculated pI of 4.98), 2) seven potential phosphorylation sites are concentrated in the N-terminal region of IRBIT, and phosphorylation was supposed to be required for the interaction, 3) Lys-508 of IP 3 R1 was essential for the interaction with IRBIT, 4) IP 3 disrupted the interaction, and 5) a high salt buffer disrupted the interaction between IRBIT and GST-EL 2 and extracted IRBIT from crude microsomes, indicating that the interaction is dependent on an electrostatic bond. Deletion mutagenesis results also indicate that residues 105-277 of IRBIT, which contain a coiled-coil region, contribute to the interaction. The crystal structure of the IP 3 binding region of mouse IP 3 R1 in the complex with IP 3 was recently resolved (63). IP 3 bound to the positively charged cleft of the IP 3 binding region, and the side chain of Lys-508 formed the hydrogen bond with the phosphate group at the 5-position of IP 3 (63). Remarkably, the C-terminal region of the IP 3 binding domain containing Lys-508 (residues 437-604) formed an armadillo repeatlike fold (63), which generally acts as a protein-protein interaction motif (64). IRBIT may interact with IP 3 R1 via this motif. However, the armadillo repeat-like fold is not sufficient for interaction, since GST-IIab (residues 341-923 of IP 3 R1) did not interact with IRBIT.
IRBIT was dissociated from IP 3 R1 selectively with IP 3 at an EC 50 of ϳ0.5 M. This EC 50 value is higher than the K d of purified IP 3 R1 for IP 3 (K d ϭ 83-100 nM) determined by conventional IP 3 binding assays (46,65). This difference may be attributable to different buffer conditions because the IP 3 binding affinity of IP 3 R depends strongly on pH and ionic strength (66 -68). Conventional IP 3 binding assays were performed under optimal binding conditions, with a higher pH (8.0 -8.3) and a lower ionic strength (salt-free). Surface plasmon resonance biosensor studies using the N-terminal region of IP 3 R1 (residues 1-604) demonstrated the K d value determined under near physiological conditions (pH 7.4 and 150 mM NaCl) to be 336 nM (68), i.e. ϳ7.5-fold lower than the affinity determined by the conventional IP 3 binding assay (69), and close to the EC 50 (ϳ0.5 M) required for the dissociation of IRBIT from GST-EL determined at pH 7.4 and 100 mM NaCl. Therefore, taken together with the findings that IRBIT bound to the IP 3 binding region of IP 3 R1 and that both IRBIT and IP 3 were dependent on Lys-508 of IP 3 R1 for the interaction, these results indicate that IRBIT is released from IP 3 R1 upon IP 3 binding to IP 3 R1, probably via a competitive mechanism.
Phosphorylation, as well as IP 3 , is considered to regulate the interaction between IRBIT and IP 3 R. In vitro binding experiments showed IRBIT extracted from the membrane fraction with a high salt buffer to interact with IP 3 R1, whereas IRBIT in the cytosolic fraction did not. The difference in the phosphorylation state of IRBIT may account for this discrepancy, because alkaline phosphatase treatment of the high salt extract disrupted the interaction between IRBIT and IP 3 R1. IRBIT has 17 potential phosphorylation sites, and seven of these sites are concentrated in the N-terminal region, which is necessary for the interaction with IP 3 R1. These findings raise the possibility that the dephosphorylated form of IRBIT is free in the cytosol, whereas the phosphorylated form is membrane-bound via the interaction with IP 3 R1, although we could not rule out the possibility that phosphorylation of other proteins may regulate the interaction. We propose that the interaction between IRBIT and IP 3 R1 is dualistically regulated by IP 3 and, either directly or indirectly, by phosphorylation. Further studies are needed to determine whether or not the interaction is regulated by direct phosphorylation of IRBIT.
Using the detector cell/capillary electrophoresis system, Luzzi et al. (53) estimated intracellular IP 3 concentrations before and after stimulation to be tens of nanomolar and a few micromolar, respectively. Because the EC 50 of IP 3 (ϳ0.5 M) required for the dissociation of IRBIT from IP 3 R1 was between these concentrations, IRBIT was assumed to be released from IP 3 R1 after IP 3 production has been induced by extracellular stimuli. What is the physiological significance of the dissociation of IRBIT from IP 3 R1 after stimulation and what is the function of IRBIT? We propose four possible roles of IRBIT. First, IRBIT may modulate the channel activity of IP 3 R1. Recently, Yang et al. (35) showed that CaBP family members can act as direct ligands of IP 3 R. Interestingly, the CaBP-binding region of IP 3 R was within its 600 N-terminal residues (35), which also contain the IRBIT-binding region. Considering our preliminary data showing that IRBIT does not directly modulate the channel activity of IP 3 R, 2 IRBIT may block the binding of CaBP to IP 3 R1 and inhibit IP 3 -independent activation of IP 3 R1. Second, IRBIT may regulate the stability of IP 3 R. IP 3generating stimuli cause degradation of IP 3 R (13, 14, 50, 70 -72). Zhu et al. (13,14) proposed that the conformational change in IP 3 R induced by IP 3 binding unmasks the putative sites that facilitate ubiquitin conjugation, resulting in degradation of IP 3 R by the ubiquitin/proteasome pathway (71,72). Alternatively, dissociation of IRBIT induced by IP 3 binding may reveal the putative degradation signals or protease attack sites of IP 3 R. Third, IRBIT may play the role of a linker molecule coupling IP 3 R and other proteins to allow efficient signal propagation. Proteins possibly linked with IP 3 R include proteins whose activities are regulated by Ca 2ϩ released from IP 3 R, or plasma membrane receptors, analogous with mGluR (36) and B 2 R (37). Indeed, substantial amounts of IRBIT were present in the stripped microsome fraction (Fig. 2C), which might represent IRBIT tightly bound to membrane proteins other than IP 3 R. IP 3 may disrupt these complexes, resulting in desensitization of signals and/or translocation of linked proteins. To identify molecules possibly coupled with IP 3 R, we are now searching for IRBIT-interacting proteins. Fourth, IRBIT may be a direct downstream signal transducer of IP 3 R1. It has been thought that the only direct downstream molecule of IP 3 R1 is the calcium ion, which acts on a wide variety of target molecules. Besides a multifunctional and universal second messenger like Ca 2ϩ , IP 3 R1 may utilize IRBIT as a downstream signaling molecule with more restricted target molecules than Ca 2ϩ . In this model, IRBIT released from IP 3 -bound IP 3 R1 must be different (for example, in terms of phosphorylation state) from IRBIT originally present in the cytosol, because significant amounts of IRBIT already exist in the cytosol in the basal state. In this respect, the model in which only phosphorylated IRBIT binds to IP 3 R1 appears to be reasonable. Screening of IRBIT-binding proteins may reveal the target molecules of IRBIT.
Finally, the dissociation of IRBIT from IP 3 R in the presence of IP 3 is a feature which may be utilized for the development of a new IP 3 indicator based on fluorescence resonance energy transfer (FRET). FRET occurs when two fluorophores are in proximity and in the right orientation such that an excited donor fluorophore can transfer its energy to a second, acceptor fluorophore (73). Based on the cAMP-dependent dissociation of catalytic and regulatory subunits of cAMP-dependent protein kinase, Adams et al. (74) developed a fluorescent indicator for cAMP. Similarly, Miyawaki et al. (75) reported a genetically encoded Ca 2ϩ indicator based on the Ca 2ϩ -dependent interaction between calmodulin and calmodulin-binding peptide. Although IP 3 concentration changes could be detected by monitoring translocation of the GFP-tagged pleckstrin homology domain (76), a FRET-based IP 3 indicator has yet to be developed due to lack of suitable molecules. IP 3 -dependent dissociation of IRBIT and IP 3 R1 is a characteristic that can provide a new tool allowing real-time imaging of the spatiotemporal dynamics of IP 3 concentrations in living cells, although further studies focusing on the regulation of this interaction by phosphorylation are needed.
In summary, we identified IRBIT, a novel IP 3 R1-interacting protein, which was released from IP 3 R1 in the presence of IP 3 . Further studies aimed at elucidating the function of IRBIT, including the screening of IRBIT-interacting proteins, are anticipated to provide important insights into IP 3 /Ca 2ϩ signaling.