Miwako Iwai

Structure of the inositol 1,4,5-trisphosphate receptor binding core in complex with its ligand

In a variety of cells, the Ca2+ signalling process is mediated by the endoplasmic-reticulum-membrane-associated Ca2+ release channel, inositol 1,4,5-trisphosphate (InsP3) receptor (InsP3R). Being ubiquitous and present in organisms ranging from humans to Caenorhabditis elegans, InsP3R has a vital role in the control of cellular and physiological processes as diverse as cell division, cell proliferation, apoptosis, fertilization, development, behaviour, memory and learning. Mouse type I InsP3R (InsP3R1), found in high abundance in cerebellar Purkinje cells, is a polypeptide with three major functionally distinct regions: the amino-terminal InsP3-binding region, the central modulatory region and the carboxy-terminal channel region. Here we present a 2.2-Å crystal structure of the InsP3-binding core of mouse InsP3R1 in complex with InsP3. The asymmetric, boomerang-like structure consists of an N-terminal β-trefoil domain and a C-terminal α-helical domain containing an ‘armadillo repeat’-like fold. The cleft formed by the two domains exposes a cluster of arginine and lysine residues that coordinate the three phosphoryl groups of InsP3. Putative Ca2+-binding sites are identified in two separate locations within the InsP3-binding core.

Molecular Cloning of Mouse Type 2 and Type 3 Inositol 1,4,5-Trisphosphate Receptors and Identification of a Novel Type 2 Receptor Splice Variant

We isolated cDNAs encoding type 2 and type 3 inositol 1,4,5-trisphosphate (IP3) receptors (IP3R2 and IP3R3, respectively) from mouse lung and found a novel alternative splicing segment, SIm2, at 176–208 of IP3R2. The long form (IP3R2 SIm2+) was dominant, but the short form (IP3R2 SIm2–) was detected in all tissues examined. IP3R2 SIm2– has neither IP3 binding activity nor Ca2+ releasing activity. In addition to its reticular distribution, IP3R2 SIm2+ is present in the form of clusters in the endoplasmic reticulum of resting COS-7 cells, and after ATP or Ca2+ ionophore stimulation, most of the IP3R2 SIm2+ is in clusters. IP3R3 is localized uniformly on the endoplasmic reticulum of resting cells and forms clusters after ATP or Ca2+ ionophore stimulation. IP3R2 SIm2– does not form clusters in either resting or stimulated cells. IP3 binding-deficient site-directed mutants of IP3R2 SIm2+ and IP3R3 fail to form clusters, indicating that IP3 binding is involved in the cluster formation by these isoforms. Coexpression of IP3R2 SIm2– prevents stimulus-induced IP3R clustering, suggesting that IP3R2 SIm2– functions as a negative coordinator of stimulus-induced IP3R clustering. Expression of IP3R2 SIm2– in CHO-K1 cells significantly reduced ATP-induced Ca2+ entry, but not Ca2+ release, suggesting that the novel splice variant of IP3R2 specifically influences the dynamics of the sustained phase of Ca2+ signals.

Molecular Basis of the Isoform-specific Ligand-binding Affinity of Inositol 1,4,5-Trisphosphate Receptors

Three isoforms of the inositol 1,4,5-trisphosphate (IP3) receptor (IP3R), IP3R1, IP3R2, and IP3R3, have different IP3-binding affinities and cooperativities. Here we report that the amino-terminal 604 residues of three mouse IP3R types exhibited Kd values of 49.5 ± 10.5, 14.0 ± 3.5, and 163.0 ± 44.4 nm, which are close to the intrinsic IP3-binding affinity previously estimated from the analysis of full-length IP3Rs. In contrast, residues 224–604 of IP3R1 and IP3R2 and residues 225–604 of IP3R3, which contain the IP3-binding core domain but not the suppressor domain, displayed an almost identical IP3-binding affinity with a Kd value of ∼2 nm. Addition of 100-fold excess of the suppressor domain did not alter the IP3-binding affinity of the IP3-binding core domain. Artificial chimeric proteins in which the suppressor domain was fused to the IP3-binding core domain from different isoforms exhibited IP3-binding affinity significantly different from those of the proteins composed of the native combination of the suppressor domain and the IP3-binding core domain. Systematic mutagenesis analyses showed that amino acid residues critical for type-3 receptor-specific IP3-binding affinity are involved in Glu-39, Ala-41, Asp-46, Met-127, Ala-154, Thr-155, Leu-162, Trp-168, Asn-173, Asn-176, and Val-179. These results indicate that the IP3-binding affinity of IP3Rs is specifically tuned through the intramolecular attenuation of IP3-binding affinity of the IP3-binding core domain by the amino-terminal suppressor domain. Moreover, the functional diversity in ligand sensitivity among IP3R isoforms originates from at least the structural difference identified on the suppressor domain.

Cluster Formation of Inositol 1,4,5-Trisphosphate Receptor Requires Its Transition to Open State

The inositol 1,4,5-trisphosphate (IP3) receptor (IP3R) Ca2+ channel plays pivotal roles in many aspects of physiological and pathological events. It was previously reported that IP3R forms clusters on the endoplasmic reticulum when cytosolic Ca2+ concentration ([Ca2+]C) is elevated. However, the molecular mechanism of IP3R clustering remains largely unknown, and thus its physiological significance is far from clear. In this study we found that the time course of clustering of green fluorescent protein-tagged IP3R type 1 (GFP-IP3R1), evoked by IP3-generating agonists, did not correlate with [Ca2+]C but seemed compatible with cytoplasmic IP3 concentration. IP3 production alone induced GFP-IP3R1 clustering in the absence of a significant increase in [Ca2+]C but elevated [Ca2+]C without IP3 production did not. Moreover IP3R1 mutants that do not undergo an IP3-induced conformational change failed to form clusters. Thus, IP3R clustering is induced by its IP3-induced conformational change to the open state. We also found that GFP-IP3R1 clusters colocalized with ERp44, a luminal protein of endoplasmic reticulum that inhibits its channel activity. This is the first example of ligand-induced clustering of a ligand-gated channel protein.

ATP Modulation of Ca2+ Release by Type-2 and Type-3 Inositol (1, 4, 5)-Triphosphate Receptors DIFFERING ATP SENSITIVITIES AND MOLECULAR DETERMINANTS OF ACTION

ATP enhances Ca2+ release from inositol (1,4,5)-trisphosphate receptors (InsP3R). However, the three isoforms of InsP3R are reported to respond to ATP with differing sensitivities. Ca2+ release through InsP3R1 is positively regulated at lower ATP concentrations than InsP3R3, and InsP3R2 has been reported to be insensitive to ATP modulation. We have reexamined these differences by studying the effects of ATP on InsP3R2 and InsP3R3 expressed in isolation on a null background in DT40 InsP3R knockout cells. We report that the Ca2+-releasing activity as well as the single channel open probability of InsP3R2 was enhanced by ATP, but only at submaximal InsP3 levels. Further, InsP3R2 was more sensitive to ATP modulation than InsP3R3 under similar experimental conditions. Mutations in the ATPB sites of InsP3R2 and InsP3R3 were generated, and the functional consequences of these mutations were tested. Surprisingly, mutation of the ATPB site in InsP3R3 had no effect on ATP modulation, suggesting an additional locus for the effects of ATP on this isoform. In contrast, ablation of the ATPB site of InsP3R2 eliminated the enhancing effects of ATP. Furthermore, this mutation had profound effects on the patterns of intracellular calcium signals, providing evidence for the physiological significance of ATP binding to InsP3R2.

Induction of brain-derived neurotrophic factor by convulsant drugs in the rat brain: involvement of region-specific voltage-dependent calcium channels

A high level of hippocampal brain‐derived neurotrophic factor (BDNF) in normally aged as compared with young rats suggests that it is important to maintain a considerable level of hippocampal BDNF during aging in order to keep normal hippocampal functions. To elucidate possible mechanisms of endogenous BDNF increase, changes in levels of BDNF were studied in the rat brain following systemic administration of various convulsant agents; excitotoxic glutamate agonists, NMDA, kainic acid and (+/–)‐α‐amino‐3‐hydroxy‐5‐methylisoxazole‐4‐propionic acid (AMPA); GABA receptor antagonists, picrotoxin, pentylenetetrazole (PTZ) and lindane (γ‐hexachlorocyclohexane); and l‐type voltage‐dependent calcium channel agonist, BAY‐K 8644. Kainic acid and AMPA, but not NMDA, caused remarkable increases in BDNF protein in the rat hippocampus and entorhinal cortex. Picrotoxin, PTZ and lindane stimulated BDNF production in the entorhinal cortex and also in the hippocampus of rats showing very severe convulsions. On the other hand, BAY‐K 8644 treatment increased BDNF levels in the neocortex and entorhinal cortex. Maximal levels of BDNF protein were observed at 12–24 h, 8–16 h and 6 h following administration of kainic acid, PTZ and BAY‐K 8644, respectively. Kainic acid stimulated BDNF synthesis in presynaptic hippocampal granule neurons, but not in postsynaptic neurons with its receptors, while PTZ and BAY‐K 8644 produced the same effects in postsynaptic neurons in the entorhinal cortex (in granule neurons in the hippocampus) and in the whole cortex, respectively. Nifedipine inhibited almost completely BAY‐K 8644, but not PTZ, effects. ω‐Conotoxin GVIA and DCG‐IV partially blocked kainic acid‐induced enhancement of BDNF, indicating involvement of l‐type and N‐type voltage‐dependent calcium channels, respectively. In addition, BDNF levels in the hippocampus of mice deficient in d‐myo‐inositol‐1,4,5‐triphosphate receptor gene were scarcely different from those in the same region of controls, suggesting little involvement of intracellular calcium increase through this receptor. BAY‐K 8644, but not kainic acid or PTZ, stimulated the phosphorylation of cyclic AMP responsive element binding protein. Our results indicate convulsant‐dependent stimulation of BDNF production and involvement of region‐specific voltage‐dependent calcium channels.

Mechanistic basis of bell-shaped dependence of inositol 1,4,5-trisphosphate receptor gating on cytosolic calcium

The inositol 1,4,5-trisphosphate (IP3) receptor (IP3R) is an intracellular Ca2+ release channel, and its opening is controlled by IP3 and Ca2+. A single IP3 binding site and multiple Ca2+ binding sites exist on single subunits, but the precise nature of the interplay between these two ligands in regulating biphasic dependence of channel activity on cytosolic Ca2+ is unknown. In this study, we visualized conformational changes in IP3R evoked by various concentrations of ligands by using the FRET between two fluorescent proteins fused to the N terminus of individual subunits. IP3 and Ca2+ have opposite effects on the FRET signal change, but the combined effect of these ligands is not a simple summative response. The bell-shaped Ca2+ dependence of FRET efficiency was observed after the subtraction of the component corresponding to the FRET change evoked by Ca2+ alone from the FRET changes evoked by both ligands together. A mutant IP3R containing a single amino acid substitution at K508, which is critical for IP3 binding, did not exhibit this bell-shaped Ca2+ dependence of the subtracted FRET efficiency. Mutation at E2100, which is known as a Ca2+ sensor, resulted in ∼10-fold reduction in the Ca2+ dependence of the subtracted signal. These results suggest that the subtracted FRET signal reflects IP3R activity. We propose a five-state model, which implements a dual-ligand competition response without complex allosteric regulation of Ca2+ binding affinity, as the mechanism underlying the IP3-dependent regulation of the bell-shaped relationship between the IP3R activity and cytosolic Ca2+.

Expression of a splicing variant of the CADM1 specific to small cell lung cancer.

CADM1, a member of the immunoglobulin superfamily cell adhesion molecule, acts as a tumor suppressor in a variety of human cancers. CADM1 is also ectopically expressed in adult T‐cell leukemia (ATL), conferring an invasive phenotype characteristic to ATL. Therefore, CADM1 plays dual roles in human oncogenesis. Here, we investigate the roles of CADM1 in small cell lung cancer (SCLC). Immunohistochemistry demonstrates that 10 of 35 (29%) primary SCLC tumors express CADM1 protein. Western blotting and RT‐PCR analyses reveal that CADM1 is significantly expressed in 11 of 14 SCLC cells growing in suspension cultures but in neither of 2 SCLC cells showing attached growth to plastic dishes, suggesting that CADM1 is involved in anchorage‐independent growth in SCLC. In the present study, we demonstrate that SCLC expresses a unique splicing variant of CADM1 (variant 8/9) containing additional extracellular fragments corresponding to exon 9 in addition to variant 8, a common isoform in epithelia. Variant 8/9 of CADM1 is almost exclusively observed in SCLC and testis, although this variant protein localizes along the membrane and shows similar cell aggregation activity to variant 8. Interestingly, both variant 8/9 and variant 8 of CADM1 show enhanced tumorigenicity in nude mice when transfected into SBC5, a SCLC cell lacking CADM1. Inversely, suppression of CADM1 expression by shRNA reduced spheroid‐like cell aggregation of NCI‐H69, an SCLC cell expressing a high amount of CADM1. These findings suggest that CADM1 enhances the malignant features of SCLC, as is observed in ATL, and could provide a molecular marker specific to SCLC. (Cancer Sci 2012; 103: 1051–1057)

Transcriptional regulation of the CADM1 gene by retinoic acid during the neural differentiation of murine embryonal carcinoma P19 cells

CADM1 is a multifunctional cell adhesion molecule expressed predominantly in the nerve system, testis and lung. The expression of the Cadm1 gene is induced during the neural differentiation of murine embryonal carcinoma P19 cells by treatment with retinoic acid (RA). Here, we show that the suppression of CADM1 expression using RNAi interfered with P19 cell aggregation and reduced cell populations expressing MAP2 after RA treatment. Nonaggregated P19 cells were not differentiated into neurons, suggesting that CADM1 participates in the aggregate formation and neuronal differentiation of P19 in vitro. A luciferase assay of a series of deletion mutants of the CADM1 promoter localized an RA‐responsive cis‐acting element to an approximately 90‐bp fragment upstream of the translational start site. This element contains a putative binding site for transcription factor Sp1, named Sp1‐binding site‐1 (Sp1BS‐1). Sp1BS‐1 and adjacent Sp1‐binding sites (Sp1BS‐2 and Sp1BS‐3) showed enhanced transcriptional activity by RA. Moreover, a chromatin immunoprecipitation showed that RA receptor (RAR)α was associated with a DNA fragment containing Sp1BS‐1, whereas suppression of RARα expression using siRNA reduced the responsiveness of the CADM1 promoter to RA. These results suggest that Sp1 plays a critical role in RA‐induced CADM1 expression through possible interaction with RARα in the neural differentiation of P19.

664. Therapeutic Effect of Oncolytic Herpes Simplex Virus Type 1 (G47Δ) in Combination with Chemotherapy on Colorectal Cancer

One of the reasons for the difficulty of curing unresectable advanced stage colorectal cancer is that cancer cells acquire resistance to anticancer drugs. Oncolytic virus therapy is a potent candidate for developing a new therapeutic approach that counteracts drug resistance, in which an oncolytic virus kills cancer cells in the course of tumor cell specific viral replication. G47Δ is a third-generation oncolytic herpes simplex virus type 1 with triple mutations in the γ34.5, ICP6, and α47 genes. G47Δ not only destroys cancer cells directly, but also induces systemic antitumor immunity efficiently. In the present study, we investigate the efficacy of G47Δ in combination with 5-fluorouracil (5-FU) or oxaliplatin for colorectal cancer. A human colorectal cancer cell line HCT116 and a murine colorectal cancer cell line CT26 were used in vitro to examine cytotopathic effects and replication capabilities of G47Δ. Combination index analyses demonstrated additive or synergistic effects of G47Δ when used in combination with chemotherapeutic drugs. Importantly, cytotoxic anticancer drugs did not affect the replication capability of G47Δ in vitro. In vivo experiments were performed using BALB/c mice bearing syngeneic subcutaneous CT26 tumors or athymic mice bearing subcutaneous HCT116 tumors. Each established tumor was treated with 2-time intratumoral injections with G47Δ (1×106 pfu) or mock, with concomitant intraperitoneal injections with chemotherapeutic drugs (3mg/kg 5-FU three times or 5mg/kg oxaliplatin four times) or vehicle. Combination therapy inhibited tumor growth significantly better than each therapy alone. Next, we established a 5-FU-resistant CT26 cell line (CT26FuR) by continuously exposing the naive CT26 cells to increasing concentrations of 5-FU. The tolerability of the CT26FuR against 5-FU was confirmed in vitro and in vivo. Cytotopathic effect and replication capability of G47Δ in CT26FuR cells were comparable to those in naive CT26 cells in vitro. Also in vivo, intratumoral administration of G47Δ was as efficacious in subcutaneous CT26FuR tumors as in CT26 tumors. In conclusion, G47Δ was shown efficacious for colorectal cancer both in vitro and in vivo. G47Δ may be especially useful for the treatment of multi-drug resistant colorectal cancer.