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.     

Binding of IRBIT to the IP3 receptor: determinants and functional effects

IRBIT has previously been shown to interact with the inositol 1,4,5-trisphosphate (IP3) receptor (IP3R) in an IP3-sensitive way. So far it remained to be elucidated whether this interaction was direct or indirect, and whether it was functionally relevant. We now show that IRBIT can directly interact with the IP3R, and that both the suppressor domain and the IP3-binding core of the IP3R are essential for a strong interaction. Moreover, we identified a PEST motif and a PDZ-ligand on IRBIT which were critical for the interaction with the IP3R. Furthermore, we identified Asp-73 as a critical residue for this interaction. Finally, we demonstrated that this interaction functionally affects the IP3R: IRBIT inhibits both IP3 binding and IP3-induced Ca2+ release.     

Protein phosphatase-1 is a novel regulator of the interaction between IRBIT and the inositol 1,4,5-trisphosphate receptor

IRBIT is an IP3R [IP3 (inositol 1,4,5-trisphosphate) receptor]-binding protein that competes with IP3 for binding to the IP3R. Phosphorylation of IRBIT is essential for the interaction with the IP3R. The unique N-terminal region of IRBIT, residues 1-104 for mouse IRBIT, contains a PEST (Pro-Glu-Ser-Thr) domain with many putative phosphorylation sites. In the present study, we have identified a well-conserved PP1 (protein phosphatase-1)-binding site preceeding this PEST domain which enabled the binding of PP1 to IRBIT both in vitro and in vivo. IRBIT emerged as a mediator of its own dephosphorylation by associated PP1 and, hence, as a novel substrate specifier for PP1. Moreover, IRBIT-associated PP1 specifically dephosphorylated Ser68 of IRBIT. Phosphorylation of Ser68 was required for subsequent phosphorylation of Ser71 and Ser74, but the latter two sites were not targeted by PP1. We found that phosphorylation of Ser71 and Ser74 were sufficient to enable inhibition of IP3 binding to the IP3R by IRBIT. Finally, we have shown that mutational inactivation of the docking site for PP1 on IRBIT increased the affinity of IRBIT for the IP3R. This pinpoints PP1 as a key player in the regulation of IP3R-controlled Ca2+ signals.     

The IP3 receptor/Ca2+ channel and its cellular function

The IP3R [IP3 (inositol 1,4,5-trisphosphate) receptor] is responsible for Ca2+ release from the ER (endoplasmic reticulum). We have been working extensively on the P400 protein, which is deficient in Purkinje-neuron-degenerating mutant mice. We have discovered that P400 is an IP3R and we have determined the primary sequence. Purified IP3R, when incorporated into a lipid bilayer, works as a Ca2+ release channel and overexpression of IP3R shows enhanced IP3 binding and channel activity. Addition of an antibody blocks Ca2+ oscillations indicating that IP3R1 works as a Ca2+ oscillator. Studies on the role of IP3R during development show that IP3R is involved in fertilization and is essential for determination of dorso-ventral axis formation. We found that IP3R is involved in neuronal plasticity. A double homozygous mutant of IP3R2 (IP3R type 2) and IP3R3 (IP3R type 3) shows a deficit of saliva secretion and gastric juice secretion suggesting that IP3Rs are essential for exocrine secretion. IP3R has various unique properties: cryo-EM (electron microscopy) studies show that IP3R contains multiple cavities; IP3R allosterically and dynamically changes its form reversibly (square form-windmill form); IP3R is functional even though it is fragmented by proteases into several pieces; the ER forms a meshwork but also forms vesicular ER and moves along microtubules using a kinesin motor; X ray analysis of the crystal structure of the IP3 binding core consists of an N-terminal beta-trefoil domain and a C-terminal alpha-helical domain. We have discovered ERp44 as a redox sensor in the ER which binds to the luminal part of IP3R1 and regulates its activity. We have also found the role of IP3 is not only to release Ca2+ but also to release IRBIT which binds to the IP3 binding core of IP3R.     

IP3 receptor/Ca2+ channel: from discovery to new signaling concepts

Inositol 1,4,5-trisphosphate (IP(3)) is a second messenger that induces the release of Ca(2+) from the endoplasmic reticulum (ER). The IP(3) receptor (IP(3)R) was discovered as a developmentally regulated glyco-phosphoprotein, P400, that was missing in strains of mutant mice. IP(3)R can allosterically and dynamically change its form in a reversible manner. The crystal structures of the IP(3)-binding core and N-terminal suppressor sequence of IP(3)R have been identified. An IP(3) indicator (known as IP(3)R-based IP(3) sensor) was developed from the IP(3)-binding core. The IP(3)-binding core's affinity to IP(3) is very similar among the three isoforms of IP(3)R; instead, the N-terminal IP(3) binding suppressor region is responsible for isoform-specific IP(3)-binding affinity tuning. Various pathways for the trafficking of IP(3)R have been identified; for example, the ER forms a meshwork upon which IP(3)R moves by lateral diffusion, and vesicular ER subcompartments containing IP(3)R move rapidly along microtubles using a kinesin motor. Furthermore, IP(3)R mRNA within mRNA granules also moves along microtubules. IP(3)Rs are involved in exocrine secretion. ERp44 works as a redox sensor in the ER and regulates IP(3)R1 activity. IP(3) has been found to release Ca(2+), but it also releases IRBIT (IP(3)R-binding protein released with IP(3)). IRBIT is a pseudo-ligand for IP(3) that regulates the frequency and amplitude of Ca(2+) oscillations through IP(3)R. IRBIT binds to pancreas-type Na, bicarbonate co-transporter 1, which is important for acid-base balance. The presence of many kinds of binding partners, like homer, protein 4.1N, huntingtin-associated protein-1A, protein phosphatases (PPI and PP2A), RACK1, ankyrin, chromogranin, carbonic anhydrase-related protein, IRBIT, Na,K-ATPase, and ERp44, suggest that IP(3)Rs form a macro signal complex and function as a center for signaling cascades. The structure of IP(3)R1, as revealed by cryoelectron microscopy, fits closely with these molecules.     

IRBIT, inositol 1,4,5-triphosphate (IP3) receptor-binding protein released with IP3, binds Na+/H+ exchanger NHE3 and activates NHE3 activity in response to calcium

Calcium (Ca2+) is a highly versatile second messenger that regulates various cellular processes. Previous studies showed that elevation of intracellular Ca2+ regulates the activity of Na+/H+ exchanger 3 (NHE3). However, the effect of Ca2+-dependent signaling on NHE3 activity varies depending on cell types. In this study, we report the identification of IP3 receptor-binding protein released with IP3 (IRBIT) as a NHE3 interacting protein and its role in regulation of NHE3 activity. IRBIT bound to the carboxyl-terminal domain of NHE3, which is necessary for acute regulation of NHE3. Ectopic expression of IRBIT resulted in Ca2+-dependent activation of NHE3 activity, whereas silencing of endogenous IRBIT resulted in inhibition of NHE3 activity. Ca2+-dependent stimulation of NHE3 activity was dependent on the binding of IRBIT to NHE3. Previously Ca2+-dependent inhibition of NHE3 was demonstrated in the presence of NHERF2. Co-expression of IRBIT was able to reverse the NHERF2-dependent inhibition of NHE3. We also showed that IRBIT-dependent activation of NHE3 involves exocytic trafficking of NHE3 to the plasma membrane and this activation was blocked by inhibition of calmodulin (CaM) or CaM-dependent kinase II. These results suggest that the overall effect of Ca2+ on NHE3 activity is balanced by IRBIT-dependent activation and NHERF2-dependent inhibition.     

Inositol 1,4,5-trisphosphate IP(3) receptors and their role in neuronal cell function

Inositol 1,4,5-trisphosphate (IP(3)) receptor is a Ca(2+) release channel localized on the endoplasmic reticulum (ER) and plays an important role in neuronal function. IP(3) receptor was discovered as a developmentally regulated protein missing in the cerebellar mutant mice. Recent studies indicate that IP(3)Rs are involved in early development and neuronal plasticity. IP(3) works to release IRBIT from the IP(3) binding core in addition to release Ca(2+). IRBIT binds to and activates Na, Bicarbonate cotransporter. Electron microscopic study show the IP(3) receptor has allosteric property to change its form from square to windmill in the presence of Ca(2+). IP(3)R associates with ERp44, a redox sensor, Homer, other proteins and is transported as vesicular ER on microtubules. All these data suggests IP(3) receptor/CA(2+) channel works as a signaling center inside cells.     

Rapid functional assays of recombinant IP3 receptors

Functional assays of inositol 1,4,5-trisphosphate receptors (IP3R) currently use 45Ca2+ release methods, fluorescent Ca2+ indicators within either the ER or cytosol, or electrophysiological analyses of IP3R in the nuclear envelope or artificial bilayers. None of the methods is presently amenable to the rapid, high-throughput quantitative analyses of IP3R function needed to address the structural determinants of IP3R behavior. We use a low-affinity Ca2+ indicator (Mag-fluo-4) to measure free [Ca2+] within the ER of permeabilized DT40 cells expressing only rat type 1 IP(3)R, and establish that the indicator is capable of reliably reporting the Ca(2+) release evoked by IP3. A 96-well fluorescence plate reader equipped for automated fluid additions (FlexStation, Molecular Devices) is used to monitor IP3-evoked Ca2+ release. The method allows quick and economical functional assays of recombinant IP3R in small volumes (< or = 100 microl).     

Regulation of the type 1 inositol 1,4,5-trisphosphate receptor by phosphorylation at tyrosine 353

The inositol 1,4,5-trisphosphate receptor (IP3R) plays an essential role in Ca2+ signaling during lymphocyte activation. Engagement of the T cell or B cell receptor by antigen initiates a signal transduction cascade that leads to tyrosine phosphorylation of IP3R by Src family nonreceptor protein tyrosine kinases, including Fyn. However, the effect of tyrosine phosphorylation on the IP3R and subsequent Ca2+ release is poorly understood. We have identified tyrosine 353 (Tyr353) in the IP3-binding domain of type 1 IP3R (IP3R1) as a phosphorylation site for Fyn both in vitro and in vivo. We have developed a phosphoepitope-specific antibody and shown that IP3R1-Y353 becomes phosphorylated during T cell and B cell activation. Furthermore, tyrosine phosphorylation of IP3R1 increased IP3 binding at low IP3 concentrations (<10 nm). Using wild-type IP3R1 or an IP3R1-Y353F mutant that cannot be tyrosine phosphorylated at Tyr353 or expressed in IP3R-deficient DT40 B cells, we demonstrated that tyrosine phosphorylation of Tyr353 permits prolonged intracellular Ca2+ release during B cell activation. Taken together, these data suggest that one function of tyrosine phosphorylation of IP3R1-Y353 is to enhance Ca2+ signaling in lymphocytes by increasing the sensitivity of IP3R1 to activation by low levels of IP3.     

Unbiased proteomic profiling reveals the IP3R modulator AHCYL1/IRBIT as a novel interactor of microtubule-associated protein tau

Microtubule-associated protein tau is a naturally unfolded protein that can modulate a vast array of physiological processes through direct or indirect binding with molecular partners. Aberrant tau homeostasis has been implicated in the pathogenesis of several neurodegenerative disorders, including Alzheimer’s disease. In this study, we performed an unbiased high-content protein profiling assay by incubating recombinant human tau on microarrays containing thousands of human polypeptides. Among the putative tau-binding partners, we identify SAH hydrolase–like protein 1/inositol 1,4,5-trisphosphate receptor (IP3R)–binding protein (AHCYL1/IRBIT), a member of the SAH hydrolase family and a previously described modulator of IP3R activity. Using coimmunoprecipitation assays, we show that endogenous as well as overexpressed tau can physically interact with AHCYL1/IRBIT in brain tissues and cultured cells. Proximity ligation assay experiments demonstrate that tau overexpression may modify the close localization of AHCYL1/IRBIT to IP3R at the endoplasmic reticulum. Together, our experimental evidence indicates that tau interacts with AHCYL1/IRBIT and potentially modulates AHCYL1/IRBIT function.     

A novel recombinant hyperaffinity inositol 1,4,5-trisphosphate (IP(3)) absorbent traps IP(3), resulting in specific inhibition of IP(3)-mediated calcium signaling.

We have developed a novel recombinant hyperaffinity inositol 1,4,5-trisphosphate (IP(3)) absorbent, called the "IP(3) sponge," which we constructed on the basis of the ligand-binding site of the mouse type 1 IP(3) receptor (IP(3)R1). The IP(3) sponge exhibited approximately 1000-fold higher affinity for IP(3) than the parental IP(3)R1 and specifically competed with the endogenous IP(3)R for binding to IP(3). Trapping IP(3) with the IP(3) sponge inhibited IP(3)-induced Ca(2+) release (IICR) from cerebellar microsomes in a dose-dependent manner. The IP(3) sponge expressed in HEK293 cells also inhibited IICR in response to stimulation with carbachol or ATP. Its inhibitory effects were dependent upon the level of its expression over the increased IP(3) contents. Moreover, the IP(3) sponge significantly reduced the carbachol-induced phosphorylation of cAMP-response element-binding protein in HEK293 cells, indicating that the activation of cAMP-response element-binding protein by Ca(2+)-dependent phosphorylation may be partly attributable to IICR.     

Minimal requirements for calcium oscillations driven by the IP3 receptor.

Hormones and neurotransmitters that act through inositol 1,4,5-trisphosphate (IP3) can induce oscillations of cytosolic Ca2+ ([Ca2+]c), which render dynamic regulation of intracellular targets. Imaging of fluorescent Ca2+ indicators located within intracellular Ca2+ stores was used to monitor IP3 receptor channel (IP3R) function and to demonstrate that IP3-dependent oscillations of Ca2+ release and re-uptake can be reproduced in single permeabilized hepatocytes. This system was used to define the minimum essential components of the oscillation mechanism. With IP3 clamped at a submaximal concentration, coordinated cycles of IP3R activation and subsequent inactivation were observed in each cell. Cycling between these states was dependent on feedback effects of released Ca2+ and the ensuing [Ca2+]c increase, but did not require Ca2+ re-accumulation. [Ca2+]c can act at distinct stimulatory and inhibitory sites on the IP3R, but whereas the Ca2+ release phase was driven by a Ca2+-induced increase in IP3 sensitivity, Ca2+ release could be terminated by intrinsic inactivation after IP3 bound to the Ca2+-sensitized IP3R without occupation of the inhibitory Ca2+-binding site. These findings were confirmed using Sr2+, which only interacts with the stimulatory site. Moreover, vasopressin induced Sr2+ oscillations in intact cells in which intracellular Ca2+ was completely replaced with Sr2+. Thus, [Ca2+]c oscillations can be driven by a coupled process of Ca2+-induced activation and obligatory intrinsic inactivation of the Ca2+-sensitized state of the IP3R, without a requirement for occupation of the inhibitory Ca2+-binding site.     

Encoding of Ca2+ signals by differential expression of IP3 receptor subtypes

Inositol 1,4,5-trisphosphate (IP3) plays a key role in Ca2+ signalling, which exhibits a variety of spatio-temporal patterns that control important cell functions. Multiple subtypes of IP3 receptors (IP3R-1, -2 and -3) are expressed in a tissue- and development-specific manner and form heterotetrameric channels through which stored Ca2+ is released, but the physiological significance of the differential expression of IP3R subtypes is not known. We have studied the Ca2+-signalling mechanism in genetically engineered B cells that express either a single or a combination of IP3R subtypes, and show that Ca2+-signalling patterns depend on the IP3R subtypes, which differ significantly in their response to agonists, i.e. IP3, Ca2+ and ATP. IP3R-2 is the most sensitive to IP3 and is required for the long lasting, regular Ca2+ oscillations that occur upon activation of B-cell receptors. IP3R-1 is highly sensitive to ATP and mediates less regular Ca2+ oscillations. IP3R-3 is the least sensitive to IP3 and Ca2+, and tends to generate monophasic Ca2+ transients. Furthermore, we show for the first time functional interactions between coexpressed subtypes. Our results demonstrate that differential expression of IP3R subtypes helps to encode IP3-mediated Ca2+ signalling.     

Na/K-ATPase tethers phospholipase C and IP3 receptor into a calcium-regulatory complex

We have shown that the caveolar Na/K-ATPase transmits ouabain signals via multiple signalplexes. To obtain the information on the composition of such complexes, we separated the Na/K-ATPase from the outer medulla of rat kidney into two different fractions by detergent treatment and density gradient centrifugation. Analysis of the light fraction indicated that both PLC-gamma1 and IP3 receptors (isoforms 2 and 3, IP3R2 and IP3R3) were coenriched with the Na/K-ATPase, caveolin-1 and Src. GST pulldown assays revealed that the central loop of the Na/K-ATPase alpha1 subunit interacts with PLC-gamma1, whereas the N-terminus binds IP3R2 and IP3R3, suggesting that the signaling Na/K-ATPase may tether PLC-gamma1 and IP3 receptors together to form a Ca(2+)-regulatory complex. This notion is supported by the following findings. First, both PLC-gamma1 and IP3R2 coimmunoprecipitated with the Na/K-ATPase and ouabain increased this interaction in a dose- and time-dependent manner in LLC-PK1 cells. Depletion of cholesterol abolished the effects of ouabain on this interaction. Second, ouabain induced phosphorylation of PLC-gamma1 at Tyr(783) and activated PLC-gamma1 in a Src-dependent manner, resulting in increased hydrolysis of PIP2. It also stimulated Src-dependent tyrosine phosphorylation of the IP3R2. Finally, ouabain induced Ca(2+) release from the intracellular stores via the activation of IP3 receptors in LLC-PK1 cells. This effect required the ouabain-induced activation of PLC-gamma1. Inhibition of Src or depletion of cholesterol also abolished the effect of ouabain on intracellular Ca(2+).     

Dynamic clustering of IP3 receptors by IP3

The versatility of Ca2+ as an intracellular messenger stems largely from the impressive, but complex, spatiotemporal organization of the Ca2+ signals. For example, the latter when initiated by IP3 (inositol 1,4,5-trisphosphate) in many cells manifest hierarchical recruitment of elementary Ca2+ release events ('blips' and then 'puffs') en route to global regenerative Ca2+ waves as the cellular IP3 concentration rises. The spacing of IP3Rs (IP3 receptors) and their regulation by Ca2+ are key determinants of these spatially organized Ca2+ signals, but neither is adequately understood. IP3Rs have been proposed to be pre-assembled into clusters, but their composition, geometry and whether clustering affects IP3R behaviour are unknown. Using patch-clamp recording from the outer nuclear envelope of DT40 cells expressing rat IP3R1 or IP3R3, we have recently shown that low concentrations of IP3 cause IP3Rs to aggregate rapidly and reversibly into small clusters of approximately four IP3Rs. At resting cytosolic Ca2+ concentrations, clustered IP3Rs open independently, but with lower open probability, shorter open duration and lesser IP3-sensitivity than lone IP3Rs. This inhibitory influence of clustering on IP3R is reversed when the [Ca2+]i (cytosolic free Ca2+ concentration) increases. The gating of clustered IP3Rs exposed to increased [Ca2+]i is coupled: they are more likely to open and close together, and their simultaneous openings are prolonged. Dynamic clustering of IP3Rs by IP3 thus exposes them to local Ca2+ rises and increases their propensity for a CICR (Ca2+-induced Ca2+ rise), thereby facilitating hierarchical recruitment of the elementary events that underlie all IP3-evoked Ca2+ signals.     

80K-H interacts with inositol 1,4,5-trisphosphate (IP3) receptors and regulates IP3-induced calcium release activity

Inositol 1,4,5-trisphosphate receptors (IP3Rs) are intracellular channel proteins that mediate calcium (Ca2+) release from the endoplasmic reticulum, and they are involved in many biological processes (e.g. fertilization, secretion, and synaptic plasticity). Recent reports show that IP3R activity is strictly regulated by several interacting molecules (e.g. IP3R binding protein released with inositol 1,4,5-trisphosphate, huntingtin, presenilin, DANGER, and cytochrome c), and perturbation of this regulation causes intracellular Ca2+ elevation leading to several diseases (e.g. Huntington disease and Alzheimer disease). In this study, we identified protein kinase C substrate 80K-H (80K-H) to be a novel molecule interacting with the COOH-terminal tail of IP3Rs by yeast two-hybrid screening. 80K-H directly interacted with IP3R type 1 (IP3R1) in vitro and co-immunoprecipitated with IP3R1 in cell lysates. Immunocytochemical and immunohistochemical staining revealed that 80K-H colocalized with IP3R1 in COS-7 cells and in hippocampal neurons. We also showed that the purified recombinant 80K-H protein directly enhanced IP3-induced Ca2+ release activity by a Ca2+ release assay using mouse cerebellar microsomes. Furthermore 80K-H was found to regulate ATP-induced Ca2+ release in living cells. Thus, our findings suggest that 80K-H is a novel regulator of IP3R activity, and it may contribute to neuronal functions.     

The IRBIT domain adds new functions to the AHCY family

During the past few years, the IRBIT domain has emerged as an important add-on of S-adenosyl-L-homocystein hydrolase (AHCY), thereby creating the new family of AHCY-like proteins. In this review, we discuss the currently available data on this new family of proteins. We describe the IRBIT domain as a unique part of these proteins and give an overview of its regulation via (de)phosphorylation and proteolysis. The second part of this review is focused on the potential functions of the AHCY-like proteins. We propose that the IRBIT domain serves as an anchor for targeting AHCY-like proteins towards cytoplasmic targets. This leads to regulation of (i) intracellular Ca2+ via the inositol 1,4,5-trisphosphate receptor (IP3R), (ii) intracellular pH via the Na+/HCO3 - cotransporters (NBCs); whereas inactivation of the IRBIT domain induces (iii) nuclear translocation and regulation of AHCY activity. Dysfunction of AHCY-like proteins will disturb these three important functions, with various biological implications.     

A kinetic model of single and clustered IP3 receptors in the absence of Ca2+ feedback

Ca2+ liberation through inositol 1,4,5-trisphosphate receptor (IP3R) channels generates complex patterns of spatiotemporal cellular Ca2+ signals owing to the biphasic modulation of channel gating by Ca2+ itself. These processes have been extensively studied in Xenopus oocytes, where imaging studies have revealed local Ca2+ signals ("puffs") arising from clusters of IP3R, and patch-clamp studies on isolated oocyte nuclei have yielded extensive data on IP3R gating kinetics. To bridge these two levels of experimental data, we developed an IP3R model and applied stochastic simulation and transition matrix theory to predict the behavior of individual and clustered IP3R channels. The channel model consists of four identical, independent subunits, each of which has an IP3-binding site together with one activating and one inactivating Ca2+-binding site. The channel opens when at least three subunits undergo a conformational change to an "active" state after binding IP3 and Ca2+. The model successfully reproduces patch-clamp data; including the dependence of open probability, mean open duration, and mean closed duration on [IP3] and [Ca2+]. Notably, the biexponential distribution of open-time duration and the dependence of mean open time on [Ca2+] are explained by populations of openings involving either three or four active subunits. As a first step toward applying the single IP3R model to describe cellular responses, we then simulated measurements of puff latency after step increases of [IP3]. Assuming that stochastic opening of a single IP3R at basal cytosolic [Ca2+] and any given [IP3] has a high probability of rapidly triggering neighboring channels by calcium-induced calcium release to evoke a puff, optimal correspondence with experimental data of puff latencies after photorelease of IP3 was obtained when the cluster contained a total of 40-70 IP3Rs.     

Cdc2/cyclin B1 interacts with and modulates inositol 1,4,5-trisphosphate receptor (type 1) functions

The resistance of inositol 1,4,5-trisphosphate receptor (IP3R)-deficient cells to multiple forms of apoptosis demonstrates the importance of IP3-gated calcium (Ca2+) release to cellular apoptosis. However, the specific upstream biochemical events leading to IP3-gated Ca2+ release during apoptosis induction are not known. We have shown previously that the cyclin-dependent kinase 1/cyclin B (cdk1/CyB or cdc2/CyB) complex phosphorylates IP3R1 in vitro and in vivo at Ser421 and Thr799. In this study, we show that: 1) the cdc2/CyB complex directly interacts with IP3R1 through Arg391, Arg441, and Arg871; 2) IP3R1 phosphorylation at Thr799 by the cdc2/CyB complex increases IP3 binding; and 3) cdc2/CyB phosphorylation increases IP3-gated Ca2+ release. Taken together, these results demonstrate that cdc2/CyB phosphorylation positively regulates IP3-gated Ca2+ signaling. In addition, identification of a CyB docking site(s) on IP3R1 demonstrates, for the first time, a direct interaction between a cell cycle component and an intracellular calcium release channel. Blocking this phosphorylation event with a specific peptide inhibitor(s) may constitute a new therapy for the treatment of several human immune disorders.     

Three-dimensional rearrangements within inositol 1,4,5-trisphosphate receptor by calcium

Allosteric binding of calcium ion (Ca2+) to inositol 1,4,5-trisphosphate (IP3) receptor (IP3R) controls channel gating within IP3R. Here, we present biochemical and electron microscopic evidence of Ca2+-sensitive structural changes in the three-dimensional structure of type 1 IP3R (IP3R1). Low concentrations of Ca2+ and high concentrations of Sr2+ and Ba2+ were shown to be effective for the limited proteolysis of IP3R1, but Mg2+ had no effect on the proteolysis. The electron microscopy and the limited proteolysis consistently demonstrated that the effective concentration of Ca2+ for conformational changes in IP3R1 was <10(-7) m and that the IP3 scarcely affected the conformational states. The structure of IP3R1 without Ca2+, as reconstructed by three-dimensional electron microscopy, had a "mushroom-like" appearance consisting of a large square-shaped head and a small channel domain linked by four thin bridges. The projection image of the "head-to-head" assembly comprising two particles confirmed the mushroom-like side view. The "windmill-like" form of IP3R1 with Ca2+ also contains the four bridges connecting from the IP3-binding domain toward the channel domain. These data suggest that the Ca2+-specific conformational change structurally regulates the IP3-triggered channel opening within IP3R1.