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• W1976134600 abstract "The inositol 1,4,5-trisphosphate (IP3) receptors (IP3Rs) are IP3-gated intracellular Ca2+ channels. We previously identified an IP3R binding protein, IRBIT, which binds to the IP3 binding domain of IP3R and is dissociated from IP3R in the presence of IP3. In the present study, we showed that IRBIT suppresses the activation of IP3R by competing with IP3 by [3H]IP3 binding assays, in vitro Ca2+ release assays, and Ca2+ imaging of intact cells. Multiserine phosphorylation of IRBIT was essential for the binding, and 10 of the 12 key amino acids in IP3R for IP3 recognition participated in binding to IRBIT. We propose a unique mode of IP3R regulation in which IP3 sensitivity is regulated by IRBIT acting as an endogenous “pseudoligand” whose inhibitory activity can be modulated by its phosphorylation status. The inositol 1,4,5-trisphosphate (IP3) receptors (IP3Rs) are IP3-gated intracellular Ca2+ channels. We previously identified an IP3R binding protein, IRBIT, which binds to the IP3 binding domain of IP3R and is dissociated from IP3R in the presence of IP3. In the present study, we showed that IRBIT suppresses the activation of IP3R by competing with IP3 by [3H]IP3 binding assays, in vitro Ca2+ release assays, and Ca2+ imaging of intact cells. Multiserine phosphorylation of IRBIT was essential for the binding, and 10 of the 12 key amino acids in IP3R for IP3 recognition participated in binding to IRBIT. We propose a unique mode of IP3R regulation in which IP3 sensitivity is regulated by IRBIT acting as an endogenous “pseudoligand” whose inhibitory activity can be modulated by its phosphorylation status. Intracellular Ca2+ signaling regulates various aspects of cellular and physiological functions, and increases in cytoplasmic Ca2+ concentrations are mediated by either Ca2+ influx from the extracellular space or Ca2+ release from intracellular Ca2+ storage sites, such as the endoplasmic reticulum (ER) (Berridge et al., 2000Berridge M.J. Lipp P. Bootman M.D. The versatility and universality of calcium signalling.Nat. Rev. Mol. Cell Biol. 2000; 1: 11-21Crossref PubMed Scopus (4425) Google Scholar, Berridge et al., 2003Berridge M.J. Bootman M.D. Roderick H.L. Calcium signalling: dynamics, homeostasis and remodelling.Nat. Rev. Mol. Cell Biol. 2003; 4: 517-529Crossref PubMed Scopus (4192) Google Scholar). Inositol 1,4,5-trisphosphate (IP3) receptors (IP3Rs) are Ca2+ release channels on the ER that are activated by binding to their ligand IP3, a second messenger produced by hydrolysis of phosphatidylinositol 4,5-bisphosphate in response to activation of cell surface receptors. IP3Rs play pivotal roles in the regulation of numerous cell processes, including fertilization, development, secretion, gene expression, synaptic plasticity, and cell death (Berridge, 1993Berridge M.J. Inositol trisphosphate and calcium signalling.Nature. 1993; 361: 315-325Crossref PubMed Scopus (6165) Google Scholar, Furuichi and Mikoshiba, 1995Furuichi T. Mikoshiba K. Inositol 1,4,5-trisphosphate receptor-mediated Ca2+ signaling in the brain.J. Neurochem. 1995; 64: 953-960Crossref PubMed Scopus (180) Google Scholar, Patel et al., 1999Patel S. Joseph S.K. Thomas A.P. Molecular properties of inositol 1,4,5-trisphosphate receptors.Cell Calcium. 1999; 25: 247-264Crossref PubMed Scopus (371) Google Scholar). There are three distinct types of IP3Rs in mammals (Furuichi and Mikoshiba, 1995Furuichi T. Mikoshiba K. Inositol 1,4,5-trisphosphate receptor-mediated Ca2+ signaling in the brain.J. Neurochem. 1995; 64: 953-960Crossref PubMed Scopus (180) Google Scholar, Patel et al., 1999Patel S. Joseph S.K. Thomas A.P. Molecular properties of inositol 1,4,5-trisphosphate receptors.Cell Calcium. 1999; 25: 247-264Crossref PubMed Scopus (371) Google Scholar). Type 1 IP3R (IP3R1), the dominant subtype in the brain, consists of 2749 amino acids and contains an IP3 binding core domain near the N terminus, a channel-forming domain near the C terminus, and a modulatory domain separating the other two domains (Bosanac et al., 2004Bosanac I. Michikawa T. Mikoshiba K. Ikura M. Structural insights into the regulatory mechanism of IP3 receptor.Biochim. Biophys. Acta. 2004; 1742: 89-102Crossref PubMed Scopus (97) Google Scholar). Examination of the crystal structure of the IP3 binding domain of IP3R1 revealed that the IP3 binding domain consists of two subdomains, β domain and α domain, and that IP3 binds to a highly positively charged pocket at the interface between these two subdomains (Bosanac et al., 2002Bosanac I. Alattia J.R. Mal T.K. Chan J. Talarico S. Tong F.K. Tong K.I. Yoshikawa F. Furuichi T. Iwai M. et al.Structure of the inositol 1,4,5-trisphosphate receptor binding core in the complex with its ligand.Nature. 2002; 420: 696-700Crossref PubMed Scopus (276) Google Scholar). Extensive site-directed mutagenesis analysis has revealed that a number of highly conserved Arg and Lys residues are required for ligand binding (Yoshikawa et al., 1996Yoshikawa F. Morita M. Monkawa T. Michikawa T. Furuichi T. Mikoshiba K. Mutational analysis of the ligand binding site of the inositol 1,4,5-trisphosphate receptor.J. Biol. Chem. 1996; 271: 18277-18284Abstract Full Text Full Text PDF PubMed Scopus (197) Google Scholar). Most of these basic amino acids are located at the interface between the β domain and α domain and are involved in interaction with the phosphoryl groups of IP3 (Bosanac et al., 2002Bosanac I. Alattia J.R. Mal T.K. Chan J. Talarico S. Tong F.K. Tong K.I. Yoshikawa F. Furuichi T. Iwai M. et al.Structure of the inositol 1,4,5-trisphosphate receptor binding core in the complex with its ligand.Nature. 2002; 420: 696-700Crossref PubMed Scopus (276) Google Scholar). IP3 binding to the IP3 binding domain is assumed to induce a conformational change in IP3R, which may cause channel opening (Bosanac et al., 2004Bosanac I. Michikawa T. Mikoshiba K. Ikura M. Structural insights into the regulatory mechanism of IP3 receptor.Biochim. Biophys. Acta. 2004; 1742: 89-102Crossref PubMed Scopus (97) Google Scholar). We previously identified an IP3R binding protein termed IRBIT (IP3R binding protein released with inositol 1,4,5-trisphosphate) that interacts with the IP3 binding core domain of IP3R (Ando et al., 2003Ando H. Mizutani A. Matsu-ura T. Mikoshiba K. IRBIT, a novel inositol 1,4,5-trisphosphate (IP3) receptor-binding protein, is released from the IP3 receptor upon IP3 binding to the receptor.J. Biol. Chem. 2003; 278: 10602-10612Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar). IRBIT is divided into two regions: an N-terminal region (residues 1–104), which contains a serine-rich region and is essential for the interaction with IP3R, and a C-terminal region (residues 105–530), which has homology with the methylation pathway enzyme S-adenosylhomocysteine hydrolase (Ando et al., 2003Ando H. Mizutani A. Matsu-ura T. Mikoshiba K. IRBIT, a novel inositol 1,4,5-trisphosphate (IP3) receptor-binding protein, is released from the IP3 receptor upon IP3 binding to the receptor.J. Biol. Chem. 2003; 278: 10602-10612Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar). Interaction between IRBIT and IP3R is regulated by phosphorylation (Ando et al., 2003Ando H. Mizutani A. Matsu-ura T. Mikoshiba K. IRBIT, a novel inositol 1,4,5-trisphosphate (IP3) receptor-binding protein, is released from the IP3 receptor upon IP3 binding to the receptor.J. Biol. Chem. 2003; 278: 10602-10612Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar), but whether direct phosphorylation of IRBIT is required for the interaction with IP3R remains unclear. Strikingly, in vitro binding experiments revealed that physiological concentrations of IP3, but not of other related inositol polyphosphates, selectively dissociate IRBIT from IP3R (Ando et al., 2003Ando H. Mizutani A. Matsu-ura T. Mikoshiba K. IRBIT, a novel inositol 1,4,5-trisphosphate (IP3) receptor-binding protein, is released from the IP3 receptor upon IP3 binding to the receptor.J. Biol. Chem. 2003; 278: 10602-10612Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar), suggesting that IRBIT may interact with IP3R in the resting state and be released from IP3R when IP3 production is induced by extracellular stimuli. Although IRBIT is the only protein whose interaction with IP3R has been found to be regulated by IP3 thus far, the mechanism by which IP3 disrupts the interaction between IRBIT and IP3R is largely unknown. More importantly, the functions of IRBIT in either the IP3R-interacting form in the resting state or in the released form after IP3 production have not been clarified. In this study, we investigated the regulation of IP3R-mediated Ca2+ release by IRBIT. IP3 binding assays, in vitro Ca2+ release assays, and Ca2+ imaging of intact cells revealed that IRBIT suppresses the activation of IP3R by lowering its affinity for IP3. We also identified multiple phosphorylation sites within the serine-rich region of IRBIT that are essential for the interaction with IP3R. In addition, a Scatchard analysis and site-directed mutagenesis of basic amino acids at the interface of the β domain and α domain of the IP3 binding core indicated that IRBIT and IP3 compete with each other for their common binding site on IP3R, representing a unique mechanism of IP3R regulation. Our results indicate that IRBIT regulates the IP3 sensitivity of cells and suggest structural, but not functional, similarity between IRBIT and IP3. To confirm the direct interaction between IRBIT and IP3R, we prepared purified recombinant IRBIT expressed in E. coli or Sf9 insect cells and examined their interaction with GST-tagged IP3R1 with the channel domain deleted (GST-IP3R1ΔChn) by GST pull-down experiments. As shown in Figure 1A, purified IRBIT expressed in Sf9 cells bound to GST-IP3R1ΔChn efficiently. By contrast, little interaction was observed between IRBIT expressed in E. coli and GST-IP3R1ΔChn, probably due to lack of posttranslational modification, such as by phosphorylation, in E. coli. Exposure of the IRBIT from Sf9 cells to alkaline phosphatase markedly reduced its interaction with GST-IP3R1ΔChn (Figure 1B). These results indicate that phosphorylation of IRBIT is required for its interaction with IP3R. We also investigated whether IP3 disrupts the interaction between recombinant IRBIT from Sf9 cells and IP3R. As shown in Figure 1C, 1 μM IP3 efficiently dissociated IRBIT from GST-IP3R1ΔChn, as was reported with IRBIT in mouse cerebellar lysate (Ando et al., 2003Ando H. Mizutani A. Matsu-ura T. Mikoshiba K. IRBIT, a novel inositol 1,4,5-trisphosphate (IP3) receptor-binding protein, is released from the IP3 receptor upon IP3 binding to the receptor.J. Biol. Chem. 2003; 278: 10602-10612Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar). These results indicate that the interaction between IRBIT and IP3R is direct and is regulated by IP3 and phosphorylation of IRBIT. We examined the effect of PKA phosphorylation of IP3R on the interaction with IRBIT, and the results showed that in vitro phosphorylation of GST-IP3R1ΔChn by PKA did not affect the interaction with IRBIT (see Figure S1 in the Supplemental Data available with this article online). To explore regulation of the interaction between IRBIT and IP3R by phosphorylation, we attempted to identify the critical phosphorylation site(s) on IRBIT that are essential for its interaction with IP3R. IRBIT was predominantly phosphorylated at serine residues and, to a lesser extent, at threonine residues in Sf9 cells (Figure S2) and COS-7 cells (data not shown). Because IRBIT has a serine-rich region (residues 62–103) containing a cluster of 16 serines (Figure 2A), we examined the role of the serine-rich region in the interaction with IP3R by using N-terminally truncated mutants of IRBIT. IRBIT-(60–530), which contains the entire serine-rich region, bound to GST-IP3R1ΔChn, whereas IRBIT-(78–530), which lacks the first half of the serine-rich region, did not bind to GST-IP3R1ΔChn (Figure 2B), indicating that the serine-rich region is required for the interaction with IP3R. To identify phosphorylation site(s) essential for the interaction with IP3R, we carried out an intensive site-directed mutagenesis analysis of the serine-rich region of IRBIT. We mutated serine or threonine residues in the serine-rich region (Figure 2A, squares) into alanine or glycine and examined the interaction with IP3R. As shown in Figure 2C, the S68A, S71A, and S74G mutants did not bind to GST-IP3R1ΔChn, and the S70A, T72A, and S77A mutants showed reduced interaction with GST-IP3R1ΔChn. These findings are consistent with the results shown in Figure 2B, suggesting the importance of residues 60–77 to the interaction with IP3R. Interestingly, all six residues are well conserved among various species (Figure 2D). We also noticed that the S68A, S71A, S74G, and S77A mutants migrated slightly faster in SDS-PAGE gel than the wild-type (Figure 2E), suggesting that these mutants were less phosphorylated than the wild-type. To determine their phosphorylation level, we metabolically labeled COS-7 cells overexpressing site-directed mutants with [32P]orthophosphate. As shown in Figure 2F, the phosphorylation level of the S68A and S71A mutants was markedly reduced compared to the wild-type (31% and 37%, respectively), suggesting that Ser68 and Ser71 are the predominant phosphorylation sites that are essential for the interaction with IP3R. [32P]phosphate incorporation into S70A, S74G, and S77A was very slightly decreased, and incorporation into T72A was rather increased, although all of these mutants showed weak binding to IP3R. Since their phosphorylation levels reflect only overall phosphate incorporation into each mutant, we could not conclude that the four Ser and Thr residues were actually phosphorylated. Nevertheless, the reproducible decrease in phosphorylation levels of S70A, S74G, and S77A strongly suggested that these three Ser residues were also phosphorylated and involved in the interaction with IP3R. The most important specificity determinant of casein kinase I (CKI) is a phosphoserine located three amino acids upstream of target Ser/Thr [-S(P)-X-X-S/T-] (Flotow et al., 1990Flotow H. Graves P.R. Wang A. Fiol C.J. Roeske R.W. Roach P.J. Phosphate groups as substrate determinants for casein kinase I action.J. Biol. Chem. 1990; 265: 14264-14269Abstract Full Text PDF PubMed Google Scholar), and the cluster of four serines (Ser68, Ser71, Ser74, and Ser77), each separated by two intervening residues, led us to speculate that CKI is involved in the phosphorylation of IRBIT. Treatment of HeLa cells with the specific CKI inhibitor D4476 (Rena et al., 2004Rena G. Bain J. Elliott M. Cohen P. D4476, a cell-permeant inhibitor of CKI, suppresses the site-specific phosphorylation and nuclear exclusion of FOXO1a.EMBO Rep. 2004; 5: 60-65Crossref PubMed Scopus (215) Google Scholar) decreased the phosphorylation of IRBIT (Figure S3A), and an in vitro kinase assay revealed that CKI phosphorylated IRBIT only when Ser68 was phosphorylated by other kinases (Figure S3B). These results suggest that CKI is indeed involved in the phosphorylation of IRBIT. The kinases that phosphorylate Ser68 of IRBIT in vivo are unknown; however, we found that wild-type IRBIT, but not the S68A mutant, was phosphorylated in Xenopus oocytes during progesterone-induced maturation (Figure S4), suggesting that the kinases responsible for the phosphorylation of Ser68 are activated during oocyte maturation. Because IRBIT binds to the IP3 binding domain of IP3R (Ando et al., 2003Ando H. Mizutani A. Matsu-ura T. Mikoshiba K. IRBIT, a novel inositol 1,4,5-trisphosphate (IP3) receptor-binding protein, is released from the IP3 receptor upon IP3 binding to the receptor.J. Biol. Chem. 2003; 278: 10602-10612Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar), we examined the influence of IRBIT on the IP3 binding of IP3R by a [3H]IP3 binding assay with purified recombinant IRBIT. As shown in Figure 3A, IRBIT from Sf9 cells, but not from E. coli, efficiently suppressed IP3 binding of GST-IP3R1ΔChn in a dose-dependent manner. The concentration of IRBIT required for 50% inhibition of IP3 binding of GST-IP3R1ΔChn was about 0.1 μM. IRBIT itself had no IP3 binding activity (data not shown). When IRBIT expressed in Sf9 cells had been exposed to alkaline phosphatase, it no longer suppressed IP3 binding of GST-IP3R1ΔChn (Figure 3B). These results indicate that IRBIT suppresses IP3 binding of IP3R in a phosphorylation-dependent manner. We then examined the effect of S68A mutation of IRBIT on its suppression activity using recombinant IRBIT-S68A expressed in Sf9 cells (Figure S5A). The ability of IRBIT to suppress the IP3 binding was markedly attenuated by the S68A mutation (Figure 3C and Figure S5B), confirming the phosphorylation dependency of its inhibitory activity. Wild-type IRBIT, but not IRBIT-S68A, also suppressed the IP3 binding of cerebellar microsomes (Figure 3D), indicating that IRBIT inhibited the IP3 binding of full-length IP3R. We next performed a Scatchard analysis in the absence and presence of 170 nM IRBIT. IRBIT increased the Kd values of GST-IP3R1ΔChn for IP3 from 6.1 ± 0.1 nM to 87.8 ± 20.4 nM (Figures 3E and 3F), but the Bmax values were not significantly different (Figures 3E and 3G). Thus, IRBIT decreased the affinity of IP3R for IP3 about 14-fold without affecting the maximal number of IP3 binding sites, thereby suggesting competitive inhibition. To clarify the mechanism of the inhibitory effect of IRBIT on IP3 binding of IP3R, we analyzed the interaction between IRBIT and various deletion or site-directed mutants of the IP3 binding domain (residues 224–604) of IP3R1, which consists of β domain (residues 224–436) and α domain (residues 437–604) (Bosanac et al., 2002Bosanac I. Alattia J.R. Mal T.K. Chan J. Talarico S. Tong F.K. Tong K.I. Yoshikawa F. Furuichi T. Iwai M. et al.Structure of the inositol 1,4,5-trisphosphate receptor binding core in the complex with its ligand.Nature. 2002; 420: 696-700Crossref PubMed Scopus (276) Google Scholar). Pull-down assay using deletion mutants of the IP3 binding domain (Figure 4A) showed that the β domain alone did not bind IRBIT and that the α domain alone showed decreased IRBIT binding activity (Figure 4B). Thus, both the β domain and the α domain were required for the interaction with IRBIT, indicating that the entire IP3 binding domain is necessary and sufficient for the interaction with IRBIT, the same as for interaction with IP3 (Yoshikawa et al., 1996Yoshikawa F. Morita M. Monkawa T. Michikawa T. Furuichi T. Mikoshiba K. Mutational analysis of the ligand binding site of the inositol 1,4,5-trisphosphate receptor.J. Biol. Chem. 1996; 271: 18277-18284Abstract Full Text Full Text PDF PubMed Scopus (197) Google Scholar). Site-directed mutagenesis studies have revealed that 12 amino acids (R241, K249, R265, T267, R269, R504, R506, K508, R511, Y567, R568, and K569) in the IP3 binding domain of IP3R1 (Figure 4A) are critical for IP3 binding (Yoshikawa et al., 1996Yoshikawa F. Morita M. Monkawa T. Michikawa T. Furuichi T. Mikoshiba K. Mutational analysis of the ligand binding site of the inositol 1,4,5-trisphosphate receptor.J. Biol. Chem. 1996; 271: 18277-18284Abstract Full Text Full Text PDF PubMed Scopus (197) Google Scholar, Bosanac et al., 2002Bosanac I. Alattia J.R. Mal T.K. Chan J. Talarico S. Tong F.K. Tong K.I. Yoshikawa F. Furuichi T. Iwai M. et al.Structure of the inositol 1,4,5-trisphosphate receptor binding core in the complex with its ligand.Nature. 2002; 420: 696-700Crossref PubMed Scopus (276) Google Scholar). We examined the influence of the same mutations on the interaction with IRBIT by using GST fusion proteins of these 12 mutants of the IP3 binding domain of IP3R1. As shown in Figure 4C, 10 of the 12 mutants lost their IRBIT binding activity, indicating that these 10 amino acids are involved in both IP3 binding and IRBIT binding. The exceptions were R265 and T267, both of which are in the β domain. Surprisingly, all seven residues in the α domain (R504, R506, K508, R511, Y567, R568, and K569) were required for IRBIT binding. These findings are in agreement with the results of a deletion mutagenesis analysis that suggested greater importance of the α domain for the interaction with IRBIT (Figure 4B). The inability of site-directed mutants to bind to IRBIT was unlikely to be due to a general disruption of the overall structure of the mutant proteins, because the mutants retained a decreased but substantial amount of IP3 binding activity (data not shown). These findings indicate that IRBIT and IP3 recognize common amino acids in the IP3 binding domain of IP3R for the interaction, raising the possibility that a portion of IRBIT may interact with IP3R in a manner similar to IP3. These findings, together with the observations that IP3 dissociated IRBIT from IP3R and IRBIT inhibited IP3 binding of IP3R, led us to conclude that IRBIT and IP3 bind an overlapping region of IP3R in a mutually exclusive manner. The results showing that IRBIT suppressed IP3 binding of IP3R suggested that IRBIT inhibits the activation of IP3R. Alternatively, since IRBIT and IP3 bound IP3R in a similar manner by recognizing common amino acids in the IP3 binding domain, IRBIT may mimic the function of IP3; that is, IRBIT may directly activate IP3R in the absence of IP3, as was reported with neuronal Ca2+ binding proteins (CaBPs) (Yang et al., 2002Yang J. McBride S. Mak D.D. Vardi N. Palczewski K. Haeseleer F. Foskett J.K. Identification of a family of calcium sensors as protein ligands of inositol trisphosphate receptor Ca2+ release channels.Proc. Natl. Acad. Sci. USA. 2002; 99: 7711-7716Crossref PubMed Scopus (168) Google Scholar). To test these possibilities, we first investigated whether IRBIT suppresses the channel activity of IP3R by an in vitro Ca2+ release assay using mouse cerebellar microsomes. Microsomes were preincubated without or with 330 nM recombinant IRBIT expressed in Sf9 cells, and Ca2+ release was then induced by addition of various concentration of IP3. As shown in Figure 5A, wild-type IRBIT reduced Ca2+ release in response to low concentrations (10–200 nM) of IP3, and the S68A mutation notably attenuated the ability to suppress Ca2+ release. By contrast, IRBIT did not suppress Ca2+ release at the maximal concentration of IP3 (10 μM), a finding consistent with the model in which IRBIT and IP3 compete with each other for the IP3 binding domain of IP3R. We next tested the second possibility, that IRBIT functions as a protein ligand of IP3R, by single-channel current recording in planar lipid bilayers fused with mouse cerebellar microsome. As shown in Figure 5B, addition of 1 μM recombinant IRBIT did not induce activation of IP3R. These results indicate that IRBIT suppresses the channel activity of IP3R but does not activate IP3R. To determine whether IRBIT suppresses IP3R-mediated Ca2+ release in intact cells, we utilized RNA interference to suppress the expression of IRBIT in HeLa cells. Because small interfering RNA (siRNA) can have nonspecific effects (Scherer and Rossi, 2003Scherer L.J. Rossi J.J. Approaches for the sequence-specific knockdown of mRNA.Nat. Biotechnol. 2003; 21: 1457-1465Crossref PubMed Scopus (402) Google Scholar), we used two siRNA duplexes, IRBIT-1 and IRBIT-2, which are specific to different sequences in IRBIT. Control-1 and control-2 are control siRNAs, each of which was introduced three base mutations in IRBIT-1 and IRBIT-2 sequences, respectively. Both IRBIT-1 and IRBIT-2 suppressed the expression of IRBIT in HeLa cells but had no effect on expression of IP3Rs (Figure 6A and Figure S6). We analyzed the effect of IRBIT depletion on IP3R function by Ca2+ imaging in HeLa cells. Responses to the IP3-generating agonist ATP in the absence of extracellular Ca2+ were examined in cells treated with siRNAs. As shown in Figure 6B, depletion of IRBIT by siRNA IRBIT-1 or IRBIT-2 resulted in an increase in the number of cells that responded to the threshold dose, 0.25 or 0.5 μM, of ATP stimulation. Figure 6C shows representative traces of the [Ca2+]i of single cells that responded to 0.25 or 0.5 μM ATP. Most responsive HeLa cells not treated with siRNA or treated with control siRNAs showed only one or two Ca2+ transients, whereas some IRBIT-depleted cells released Ca2+ more frequently (Figures 6C–6E). On the other hand, knockdown of IRBIT had little effect on the number of cells that responded to the maximal dose of agonist stimulation (10 μM ATP) (Figure 6F). These results indicate that IP3R was more susceptible to activation in response to weak agonist stimulation in IRBIT-depleted cells. Next, we examined the effect of overexpression of IRBIT on IP3-induced Ca2+ release. Overexpression of IRBIT in HeLa cells had little effect on the response to 0.25 μM ATP stimulation (Figure 7A). Similar results were obtained when we overexpressed IRBIT in HEK293 cells (Figure S7). Possible explanations for the failure of exogenous IRBIT to suppress the IP3-induced Ca2+ release are that the endogenous level of phosphorylated IRBIT may be high enough to saturate the interaction with IP3R (Figure S8). By contrast, overexpression of the IRBIT-S68A mutant significantly increased the number of responsive cells without affecting the maximal peak amplitude of Ca2+ transients (Figure 7A and Figure S7). We speculated that IRBIT-S68A binds to endogenous IRBIT and suppresses its function, because the C-terminal region of IRBIT possesses homology with S-adenosylhomocysteine hydrolase, which forms a tetramer (Turner et al., 1998Turner M.A. Yuan C.S. Borchardt R.T. Hershfield M.S. Smith G.D. Howell P.L. Structure determination of selenomethionyl S-adenosylhomocysteine hydrolase using data at a single wavelength.Nat. Struct. Biol. 1998; 5: 369-376Crossref PubMed Scopus (140) Google Scholar). We investigated whether IRBIT-S68A interacts with wild-type IRBIT by coimmunoprecipitation assay using COS-7 cells overexpressing the S68A mutant of HA-IRBIT or FLAG-IRBIT. As shown in Figure 7B, HA-IRBIT interacted with FLAG-IRBIT, indicating that IRBIT formed a multimer. IRBIT-S68A retained the ability to form complexes with wild-type IRBIT. We confirmed that IRBIT formed a multimer through its C-terminal region (residues 105–530) by immunoprecipitation with deletion mutants of IRBIT (Figure 7C). In addition, a pull-down assay showed that the heteromultimer formed by wild-type IRBIT and IRBIT-S68A had weaker affinity for IP3R than a homomultimer of wild-type IRBIT (Figure 7D). These results suggest that overexpression of IRBIT-S68A reduces the interaction between wild-type IRBIT and IP3R and results in enhanced IP3-induced Ca2+ release. Together with the results of the knockdown experiments, these findings indicate that IRBIT lowered the sensitivity of IP3R to threshold concentrations of IP3-generating agonist stimulation. IRBIT is unique in that it is the only protein that has been reported to bind to the IP3 binding core domain of IP3R and to be dissociated from IP3R in the presence of IP3. In this study, we investigated the functional relationship between IRBIT and IP3R in the resting state and found that IRBIT efficiently inhibited IP3 binding to IP3R and that it suppressed IP3-induced Ca2+ release in vitro and in intact cells. We also identified multiple phosphorylation sites critical to the interaction with IP3R. Moreover, we demonstrated by intensive site-directed mutagenesis that IRBIT bound to IP3R in a similar manner to IP3. These observations suggest a unique mechanism of IP3R regulation. The mechanism by which IRBIT suppresses IP3R activity is assumed to be direct competition with IP3 for binding to the same region of IP3R. This model is supported by the following findings: (1) the IRBIT binding site to IP3R was mapped to the IP3 binding domain; (2) the interaction of IRBIT with IP3R was dependent on amino acids involved in IP3 binding of IP3R; (3) IP3 disrupted the interaction between IRBIT and IP3R; (4) IRBIT suppressed IP3 binding of IP3R; (5) Scatchard analysis indicated competitive inhibition; (6) IRBIT reduced Ca2+ release from microsomes in response to low concentrations of IP3, but not to a saturating concentration of IP3; and (7) knockdown of IRBIT enhanced the occurrence of IP3-induced Ca2+ release in response to the very low concentration of agonist in intact cells but did not affect the response to high concentration of agonist. Thus, IRBIT functions as a cover of the IP3 binding domain that prevents the activation of IP3R by inhibiting access of IP3 to IP3R when the IP3 concentration is low. When agonist stimulation results in high concentrations of IP3, IP3 displaces IRBIT and activates IP3R. Thus, the function of IRBIT is presumable to set the threshold IP3 concentration required for activation of IP3R. The expression level and/or phosphorylation status of IRBIT are assumed to regulate the IP3 sensitivity of IP3R. IRBIT may also be involved in the tuning of the spatial patterning of Ca2+ signaling by regulating local Ca2+ rises, such as Ca2+ puffs (Berridge et al., 2000Berridge M.J. Lipp P. Bootman M.D. The versatility and universality of calcium signalling.Nat. Rev. Mol. Cell Biol. 2000; 1: 11-21Crossref PubMed Scopus (4425) Google Scholar, Bootman et al., 2001Bootman M.D. Lipp P. Berridge M.J. The organization and functions of local Ca2+ signals.J. Cell Sci. 2001; 114: 2213-2222PubMed Google Scholar). Boulware and Marchant have demonstrated that the relative threshold of IP3 concentration needed to evoke Ca2+ puffs within the vegetal hemisphere of Xenopus oocytes increases during maturation (Boulware and Marchant, 2005Boulware M.J. Marchant J.S. IP3 receptor activity is differentially regulated in endoplasmic reticulum subdomains during oocyte maturation.Curr." @default.
• W1976134600 created "2016-06-24" @default.
• W1976134600 creator A5007846122 @default.
• W1976134600 creator A5011302857 @default.
• W1976134600 creator A5022833180 @default.
• W1976134600 creator A5035555934 @default.
• W1976134600 creator A5080938100 @default.
• W1976134600 creator A5087759769 @default.
• W1976134600 date "2006-06-01" @default.
• W1976134600 modified "2023-10-14" @default.
• W1976134600 title "IRBIT Suppresses IP3 Receptor Activity by Competing with IP3 for the Common Binding Site on the IP3 Receptor" @default.
• W1976134600 cites W1485297825 @default.
• W1976134600 cites W1599277155 @default.
• W1976134600 cites W1866820856 @default.
• W1976134600 cites W1964103059 @default.
• W1976134600 cites W1966035156 @default.
• W1976134600 cites W1980166477 @default.
• W1976134600 cites W2001907971 @default.
• W1976134600 cites W2014985826 @default.
• W1976134600 cites W2018696172 @default.
• W1976134600 cites W2020828897 @default.
• W1976134600 cites W2045399399 @default.
• W1976134600 cites W2051404222 @default.
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