Expression of green fluorescent protein and inositol 1,4,5-triphosphate receptor in Xenopus laevis oocytes.
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Animals↗
Biomedical subjects
T Furuichi
Publications and source records attributed to T Furuichi.
Calmodulin mediates calcium-dependent inactivation of the cerebellar type 1 inositol 1,4,5-trisphosphate receptor.
The dependency of purified mouse cerebellar type 1 inositol 1,4,5-trisphosphate receptor (IP3R1)/Ca2+ channel function on cytoplasmic Ca2+ was examined. In contrast to the channels in crude systems, the purified IP3R1 reconstituted into planar lipid bilayers did not show the bell-shaped dependence on Ca2+. It was activated with increasing Ca2+ sublinearly without inhibition even up to 200 microM. The addition of calmodulin to the cytoplasmic side inhibited the channel at high Ca2+ concentrations. Calmodulin antagonists reversed the Ca2+-dependent inactivation of the native channels in cerebellar microsomes. These results indicate that the bell-shaped dependence on cytoplasmic Ca2+ is not an intrinsic property of the IP3R1, and the Ca2+-dependent inactivation is directly mediated by calmodulin.
Animals↗
Transcriptional regulation of mouse type 1 inositol 1,4,5-trisphosphate receptor gene by NeuroD-related factor.
The type 1 inositol 1,4,5-trisphosphate receptor (IP3R1) is a Ca2+ channel protein that is expressed abundantly in the CNS, such as in the cerebellar Purkinje cells and hippocampus. We previously demonstrated that the box-I element, which is located -334 relative to the transcription initiation site of the mouse IP3R1 gene and includes an E-box consensus sequence, is involved in the up-regulation of such IP3R1 gene expression. Furthermore, the previous study also indicated that some CNS-related basic helix-loop-helix (bHLH) factors bind to the box-I and activate IP3R1 gene expression. In this study, we demonstrated that one of the CNS-related bHLH factors, neuronal differentiation factor (NeuroD)-related factor (NDRF), specifically bound to the box-I sequence with a ubiquitously expressed bHLH protein, E47, and activated IP3R1 gene expression. In situ hybridization of adult mouse brain revealed that IP3R1 and NDRF mRNA were co-expressed in many subsets of neurons, highly in Purkinje cells and hippocampus and moderately in cerebral cortex, olfactory bulb, and caudate putamen. Furthermore, the spatiotemporal expression patterns of these two genes resembled one another throughout postnatal development of the mouse CNS. From these results, we suggest that NDRF is involved in the tissue-specific regulation of IP3R1 gene expression in the CNS.
Animals↗
Effects of long-term use of benzodiazepines on gait and standing balance in the elderly.
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Accidental Falls↗
Cloning and characterization of two genes encoding dihydroxyacetone kinase from Schizosaccharomyces pombe IFO 0354.
We report the cloning and characterization of two genes encoding dihydroxyacetone kinase (EC 2.7.1.29), SpDAK1 and SpDAK2, from Schizosaccharomyces pombe IFO 0354. The open reading frames of both genes encode 591 amino acids and have Mrs of 62158 and 62170, respectively. Both predicted amino acid sequences exhibited a high identity to each other (99.8%) and relatively high identities (30% to 76%) to other putative dihydroxyacetone kinase gene products. A Western blot analysis showed that these enzymes are induced by glycerol and repressed by glucose. A genomic Southern blot analysis indicated the presence of SpDAK1 and the absence of SpDAK2 in a standard laboratory strain, S. pombe 972h-.
Amino Acid Sequence↗
T-cell-receptor signalling in inositol 1,4,5-trisphosphate receptor (IP3R) type-1-deficient mice: is IP3R type 1 essential for T-cell-receptor signalling?
Stimulation of T-cells via the T-cell receptor (TCR) complex is accompanied by an increase in intracellular Ca2+ concentration ([Ca2+]i). Recently, it was reported that a stable transformant of the human T-cell line, Jurkat, expressing an antisense cDNA construct of inositol 1,4,5-trisphosphate receptor (IP3R) type 1 (IP3R1), failed to demonstrate increased [Ca2+]i or interleukin-2 production after TCR stimulation and was also resistant to apoptotic stimuli. This cell line lacked IP3R1 expression, but expressed the type-2 and -3 receptors, IP3R2 and IP3R3 respectively [Jayaraman, Ondriasova, Ondrias, Harnick and Marks (1995) Proc. Natl. Acad. Sci. U.S.A. 92, 6007-6011, and Jayaraman and Marks (1997) Mol. Cell. Biol. 17, 3005-3012]. The authors concluded that IP3R1 is essential for TCR signalling and suggested that Ca2+ release via IP3R1 is a critical mediator of apoptosis. To establish whether a loss of IP3R1 function in T-cells occurred in vivo and in vitro, we investigated Ca2+ signalling after TCR stimulation and the properties of T-cells using IP3R1-deficient (IP3R1-/-) mice. As IP3R1-/- mice die at weaning, we transplanted bone marrow cells of IP3R1-/- mice into irradiated wild-type mice. Western blot analysis showed that the recipient IP3R1-containing (IP3R1+/+) lymphocytes were replaced by the donor IP3R1-/- lymphocytes after transplantation and that expression of IP3R2 and IP3R3 was unaltered. In contrast with the previous reports, T-cells lacking IP3R1 were able to mobilize Ca2+ from intracellular Ca2+ stores after stimulation via the TCR. We observed no significant differences between IP3R1+/+ and IP3R1-/- T-cells in terms of the number of thymocytes and splenocytes, the proportion of the T-cell phenotype, proliferative response to anti-CD3 monoclonal antibody (mAb) stimulation and cell viability. Therefore IP3R1 is not essential for T-cell development and function.
Animals↗
Apical vesicles bearing inositol 1,4,5-trisphosphate receptors in the Ca2+ initiation site of ductal epithelium of submandibular gland.
In polarized epithelial cells, agonists trigger Ca2+ waves and oscillations. These patterns may be caused by the compartmentalization of inositol 1,4,5-trisphosphate (IP3)-sensitive Ca2+ pools into specific regions. We have investigated the relationship between the distribution of IP3 receptors (IP3Rs) and the spatiotemporal pattern of Ca2+ signaling in the duct cells of the rat submandibular gland (SMG). Using immunofluorescence, although labeling was somewhat heterogeneous, the IP3Rs were colocalized to the apical pole of the duct cells. Immunoelectron microscopy identified small apical vesicles bearing IP3R2 in some types of duct cells. Real-time confocal imaging of intact ducts demonstrated that, after carbachol stimulation, an initial Ca2+ spike occurred in the apical region. Subsequently, repetitive Ca2+ spikes spread from the apical to the middle cytoplasm. These apical Ca2+ initiation sites were found only in some "pioneer cells," rather than in all duct cells. We performed both Ca2+ imaging and immunofluorescence on the same ducts and detected the strongest immunosignals of IP3R2 in the Ca2+ initiation sites of the pioneer cells. The subcellular localization and expression level of IP3Rs correlated strongly with the spatiotemporal nature of the intracellular Ca2+ signal and distinct Ca2+ responses among the rat SMG duct cells.
Animals↗
Differential time course of induction of 1alpha,25-dihydroxyvitamin D3-24-hydroxylase mRNA expression in rats by 1alpha,25-dihydroxyvitamin D3 and its analogs.
In order to investigate the in vivo mechanisms of target gene activation by vitamin D3 analogs, we compared the effects of two vitamin D3 analogs, 22-oxa-1alpha,25-(OH)2D3 (OCT) and 2beta (3-hydroxypropoxy) -1alpha,25-(OH)2D3 (ED-71) with that of 1alpha,25-(OH)2D3 on 1alpha,25-(OH)2D3 -24-hydroxylase[24(OH)ase] mRNA expression in the kidney and intestine of normal rats. In these experiments, all three compounds induced 24(OH)ase mRNA, but the time course of induction for each respective treatment was clearly different. OCT caused the most rapid onset of increased 24(OH)ase mRNA expression and its subsequent return to pre-injection levels. In marked contrast, ED-71 was the slowest to increase expression which was prolonged over that observed with the other compounds tested. These differences probably relate to the pharmacokinetic properties of these analogs, which are mainly generated by the affinity of analogs for the vitamin D-binding protein(DBP).
Animals↗
Localization of inositol 1,4,5-trisphosphate receptors in the rat kidney.
Inositol 1,4,5-trisphosphate (IP3) receptors (IP3Rs) serve as intracellular calcium release channels involved in signal transduction of various hormones in the kidney. Molecular cloning studies have shown that there are three types of IP3R, designated type 1, type 2, and type 3. To characterize their localizations in the rat kidney, we employed immunohistochemical studies using type-specific monoclonal antibodies that were raised against the 15 C-terminal amino acids of each type of IP3R. Type 1 was detected in glomerular mesangial cells and vascular smooth muscle cells. Type 2 was expressed exclusively in intercalated cells of collecting ducts from the cortex to the inner medulla. Type 3 was expressed in vascular smooth muscle cells, glomerular mesangial cells, and some cells of cortical collecting ducts, probably principal cells. As to the subcellular distribution, type 1 and type 2 showed a homogenous distribution in the cytoplasm, whereas type 3 was present mainly in the basolateral portion of the cytoplasm. These results indicate that IP3R isoforms were expressed in a cell-specific manner. The heterogeneous subcellular localizations among the IP3R types suggests compartmentalization of distinct IP3-sensitive Ca2+ pools.
Animals↗
[IP3 receptor].
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Adenosine Triphosphate↗
Safety and kinetic properties of a humanized antibody to human interleukin-6 receptor in healthy non-human primates.
A monoclonal antibody, hPM-1, was constructed by grafting the complementarity determining regions to human interleukin-6 (IL-6) receptor, raised in mouse, onto a human antibody backbone (humanized antibody). It is expected to be useful as a therapeutic agent for IL-6-related diseases such as multiple myeloma. To investigate the toxicological and kinetic properties of hPM-1 preliminarily, normal cynomolgus monkeys, which showed cross-reactivity with hPM-1, were intravenously administered with hPM-1 at doses of 0 (vehicle), 4 or 40 mg/kg once a week for 13 weeks. Upon toxicological examination, there were no changes in clinical signs, food consumption, body weights, urinalyses, body temperatures, electrocardiograms, hematological and biochemical parameters including blood platelet counts, serum levels of immunoglobulin G and C-reactive protein, and pathological findings. In a kinetic study, serum concentrations of hPM-1 showed a linearity between doses of 4 and 40 mg/kg. The serum concentrations, even at a dose of 4 mg/kg, were maintained at a high enough level to inhibit the IL-6 functions throughout the period of the study. Concentrations of hPM-1 in bone marrow were almost equal to those in serum. The antibodies against hPM-1 were detected only in one of four monkeys receiving hPM-1. This study suggests that blockage of the IL-6 receptor by hPM-1 does not induce any influence on a healthy living body, and hPM-1 is not toxic under the conditions of this investigation.
Animals↗
Transcription initiation sites and promoter structure of the mouse type 2 inositol 1,4,5-trisphosphate receptor gene.
Transcription initiation sites and the promoter sequence of the ubiquitously expressed mouse type 2 inositol 1,4,5-trisphosphate receptor (IP3R2) gene were determined. In contrast to the nervous system-enriched IP3R1, the IP3R2 gene had multiple (seven major) transcription initiation sites located 334 to 269 bp upstream from the first ATG codon. Transient luciferase assay revealed promoter activity of the IP3R2 sequence upstream from the transcription initiation sites. The IP3R2 promoter was GC-rich and had no conventional TATA box, but had a GC box in the proximal promoter. Multiple transcription start sites were flanked by CpG islands, and various cis elements were located in the promoter. These structural features are considered to be responsible for a profile of IP3R2 gene expression.
Animals↗
Intracellular targeting and homotetramer formation of a truncated inositol 1,4,5-trisphosphate receptor-green fluorescent protein chimera in Xenopus laevis oocytes: evidence for the involvement of the transmembrane spanning domain in endoplasmic reticulum targeting and homotetramer complex formation.
In an attempt to define structural regions of the type I inositol 1, 4,5-trisphosphate [Ins(1,4,5)P3] receptor [Ins(1,4,5)P3R] involved in its intracellular targeting to the endoplasmic reticulum (ER), we have employed the use of green fluorescent protein (GFP) to monitor the localization of a truncated Ins(1,4,5)P3R mutant containing just the putative transmembrane spanning domain and the C-terminal cytoplasmic domain [amino acids 2216-2749; termed inositol trisphosphate receptor(ES)]. We expressed a chimeric GFP-Ins(1,4, 5)P3R(ES) fusion protein in Xenopus laevis oocytes, and used fluorescence confocal microscopy to monitor its intracellular localization. Fluorescence confocal microscopy data showed an intense fluorescence in the perinuclear region and in a reticular-network under the animal pole of the oocyte, consistent with the targeting of expressed GFP-Ins(1,4,5)P3R(ES) to perinuclear ER and ER under the animal pole. These findings are consistent with the intracellular localization of the endogenous Xenopus Ins(1,4, 5)P3R shown previously. Furthermore, electron microscopy data indicate that expressed GFP-Ins(1,4,5)P3R(ES) is in fact targeted to the ER. Sodium carbonate extraction of microsomal membranes and cross-linking experiments indicate that the expressed chimeric protein is in fact membrane anchored and able to form a homotetrameric complex. Our data provides evidence that Ins(1,4, 5)P3R(ES) constitutes the membrane spanning domain of the Ins(1,4, 5)P3R and is able to mediate homotetramer formation, without the need for the large N-terminal cytoplasmic domain. Furthermore, the localization of GFP-Ins(1,4,5)P3R(ES) on the ER indicates that an ER retention/targeting signal is contained within the transmembrane spanning domain of the inositol trisphosphate receptor.
Animals↗
Ca2+ differentially regulates the ligand-affinity states of type 1 and type 3 inositol 1,4,5-trisphosphate receptors.
To elucidate the functional difference between type 1 and type 3 Ins(1,4,5)P3 receptors [Ins(1,4,5)P3R1 and Ins(1,4,5)P3R3 respectively] we studied the effect of Ca2+ on the ligand-binding properties of both Ins(1,4,5)P3R types. We expressed full-length human Ins(1,4,5)P3R1 and Ins(1,4,5)P3R3 from cDNA species in insect ovary Sf9 cells, and the membrane fractions were used for Ins(1,4,5)P3-binding assays. The binding of Ins(1,4,5)P3 to Ins(1,4,5)P3R1 and Ins(1,4,5)P3R3 was differentially regulated by Ca2+. With increasing concentrations of free Ca2+ ([Ca2+]), Ins(1,4,5)P3 binding to Ins(1,4,5)P2R1 decreased, whereas that to Ins(1,4,5)P3R3 increased. Alteration of Ins(1,4,5)P3 binding to Ins(1,4,5)P3R1 was observed at [Ca2+] ranging from less than 1 nM to more than 10 microM. The EC50 of Ins(1,4,5)P3 binding was 100 nM Ca2+ for Ins(1,4,5)P3R1. In contrast, Ins(1,4,5)P3 binding to Ins(1,4,5)P3R3 was changed at high [Ca2+] with an EC50 value of 872 nM, and steeply between 100 nM and 10 microM. These Ca2+-dependent alterations of Ins(1,4,5)P3 binding to both Ins(1,4,5)P3R types were reversible. Scatchard analyses revealed that Ca2+ changed the affinity of both Ins(1,4,5)P3R types but not the total number of Ins(1,4,5)P3-binding sites. The Kd values of Ins(1,4,5)P3R1 for Ins(1,4,5)P3 were 78.5 nM with 3 nM free Ca2+, and 312 nM with 1.4 microM free Ca2+. In contrast, Ins(1,4,5)P3R3 exhibited an affinity for Ins(1,4,5)P3 with Kd values of 116 nM with 3 nM free Ca2+, and 62.2 nM with 1.4 microM free Ca2+. These results indicate that (1) both Ins(1,4,5)P3R1 and Ins(1,4,5)P3R3 have at least two affinity states, (2) Ca2+ regulates interconversions between these states, and (3) Ca2+ regulates the binding of Ins(1,4,5)P3 to Ins(1,4,5)P3R1 and Ins(1,4,5)P3R3 in opposite manners.
Calcium↗
Demonstration of an E-box and its CNS-related binding factors for transcriptional regulation of the mouse type 1 inositol 1,4,5-trisphosphate receptor gene.
The type 1 inositol 1,4,5-trisphosphate receptor (IP3R1) is expressed abundantly in the CNS, such as in cerebellar Purkinje cells and the hippocampus. We established a tissue-specific cell-free transcription system and studied regulatory properties of the 5' upstream region of the IP3R1 gene by use of this system. Deletion analyses of the promoter revealed several cis elements that function significantly in brain nuclear extracts. Among those elements, sequences from -398 to -295 showed the most predominant cerebellum-specific positive function. Footprint analyses demonstrated a factor-binding region from -334 to -318, termed box-I, that contained an E-box consensus sequence. Electrophoretic mobility shift assay revealed CNS-related basic helix-loop-helix proteins for the box-I. Mutational studies using the function assay and competitive electrophoretic mobility shift assays demonstrated a good correlation between the box-I-binding factors and the activated transcription. Box-I-binding factors were present abundantly in adult mouse CNS, whereas their existence was restricted in embryonic and nonneural tissues. Transient chloramphenicol acetyltransferase assay for the IP3R1 promoter revealed the requirement of box-I in Neuro2a neuroblastoma cells. In the postnatal CNS, multiple basic helix-loop-helix factors are expressed abundantly, some of which are suggested to activate IP3R1 gene expression in the mammalian CNS.
Animals↗
Mutational analysis of the ligand binding site of the inositol 1,4,5-trisphosphate receptor.
To define the structural determinants for inositol 1,4, 5-trisphosphate (IP3) binding of the type 1 inositol 1,4, 5-trisphosphate receptor (IP3R1), we developed a means of expressing the N-terminal 734 amino acids of IP3R1 (T734), which contain the IP3 binding region, in Escherichia coli. The T734 protein expressed in E. coli exhibited a similar binding specificity and affinity for IP3 as the native IP3R from mouse cerebellum. Deletion mutagenesis, in which T734 was serially deleted from the N terminus up to residue 215, markedly reduced IP3 binding activity. However, when deleted a little more toward the C terminus (to residues 220, 223, and 225), the binding activity was retrieved. Further N-terminal deletions over the first 228 amino acids completely abolished it again. C-terminal deletions up to residue 579 did not affect the binding activity, whereas those up to residue 568 completely abolished it. In addition, the expressed 356-amino acid polypeptide (residues 224-579) exhibited specific binding activity. Taken together, residues 226-578 were sufficient and close enough to the minimum region for the specific IP3 binding, and thus formed an IP3 binding "core." Site-directed mutagenesis was performed on 41 basic Arg and Lys residues within the N-terminal 650 amino acids of T734. We showed that single amino acid substitutions for 10 residues, which were widely distributed within the binding core and conserved among all members of the IP3R family, significantly reduced the binding activity. Among them, three (Arg-265, Lys-508, and Arg-511) were critical for the specific binding, and Arg-568 was implicated in the binding specificity for various inositol phosphates. We suggest that some of these 10 residues form a basic pocket that interacts with the negatively charged phosphate groups of IP3.
Amino Acid Sequence↗
Ataxia and epileptic seizures in mice lacking type 1 inositol 1,4,5-trisphosphate receptor.
The inositol 1,4,5-trisphosphate (InsP3) receptor acts as an InsP3-gated Ca2+ release channel in a variety of cell types. Type 1 InsP3 receptor (IP3R1) is the major neuronal member of the IP3R family in the central nervous system, predominantly enriched in cerebellar Purkinje cells but also concentrated in neurons in the hippocampal CA1 region, caudate-putamen, and cerebral cortex. Here we report that most IP3R1-deficient mice generated by gene targeting die in utero, and born animals have severe ataxia and tonic or tonic-clonic seizures and die by the weaning period. An electroencephalogram showed that they suffer from epilepsy, indicating that IP3R1 is essential for proper brain function. However, observation by light microscope of the haematoxylin-eosin staining of the brain and peripheral tissues of IP3R1-deficient mice showed no abnormality, and the unique electrophysiological properties of the cerebellar Purkinje cells of IP3R1-deficient mice were not severely impaired.
Animals↗
Functional expression of the type 1 inositol 1,4,5-trisphosphate receptor promoter-lacZ fusion genes in transgenic mice.
Inositol 1,4,5-trisphosphate receptor (IP3R) is an inositol 1,4,5-trisphosphate (InsP3)-gated Ca2+ release channel. Type 1 IP3R (IP3R1) is the neuronal member of the IP3R family in the CNS and is predominantly expressed in cerebellar Purkinje cells. To elucidate the molecular mechanisms responsible for coupling gene expression to neuronal InsP3/Ca2+ signaling, we have studied the structure and function of the 5'-flanking region of the mouse IP3R1 gene. The cloned 5'-flanking region has several sequences sharing identity with motifs for known transcriptional regulation. We have fused 5'-flanking regions 1N from -528 to +169 and 4N from -4,187 to +169 to a beta-galactosidase gene (lacZ) as a reporter marker and have characterized their in vivo gene expression. Both 1N and 4N fusion genes functioned as a strong promoter in a neuroblastoma-glioma hybrid cell line NG108-15. Moreover, both 1N and 4N transgenic mouse lines carrying these 1N and 4N fusion genes showed characteristic patterns of beta-galactosidase activity in the CNS that are almost consistent with that of the endogenous IP3R1 protein, thereby suggesting that the 1N region from -528 to +169 contains sequence elements responsible for regulating gene expression in neurons and for specifying predominant expression in cerebellar Purkinje cells.
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