Muscle-specific mRNA isoform encodes a protein composed mainly of the N-terminal 175 residues of type 2 Ins(1,4,5)P3 receptor.
Ontology highlight
ABSTRACT:
SUBMITTER: Futatsugi A
PROVIDER: S-EPMC1219723 | biostudies-other | 1998 Sep
REPOSITORIES: biostudies-other
Similar Datasets
Project description:Dewaste et al. [Dewaste, Moreau, De Smedt, Bex, De Smedt, Wuytaack, Missiaen and Erneux (2003) Biochem. J. 374, 41-49] showed that over-expressed EGFP (enhanced green fluorescent protein) fused to Ins(1,4,5)P3 3-kinase B (IP3K-B) co-localizes with the cytoskeleton, as well as with the endoplasmic reticulum and the plasma membrane. The domains responsible for these subcellular localizations are not yet identified. For the endogenous enzyme, we confirmed both actin and endoplasmic reticulum localization by employing a high affinity antibody against IP3K-B. F-actin targeting is exclusively dependent on the non-catalytic N-terminal region of IP3K-B. By expressing fragments of this N-terminal domain as EGFP-fusion proteins and inspecting transfected cells by confocal microscopy, we characterized a distinct 63-amino-acid domain comprising amino acids 108-170 of the enzyme which is responsible for F-actin targeting. A truncation of this fragment from both sides revealed that the full size of this segment is essential for this function. Deletion of this segment in a full-length over-expressed IP3K-B-EGFP-fusion protein completely abolished F-actin interaction. Direct interaction of this actin-binding segment with only F-actin, but not with G-actin, was observed in vitro using a bacterially expressed, affinity-purified GST (glutathione S-transferase)-Rattus norvegicus IP3K (aa 108-170) fusion protein. Helix-breaking mutations within this isolated segment abolished the F-actin binding properties both in vitro and when over-expressed in cells, indicating that an intact secondary structure is essential for actin targeting. The segment shows sequence similarities to the actin-binding region in IP3K-A, but no similarity to other actin-binding domains.
| S-EPMC1133948 | biostudies-literature
Project description:The role of intracellular Ca2+ signaling in starvation-induced autophagy remains unclear. Here, we examined Ca2+ dynamics during starvation-induced autophagy and the underlying molecular mechanisms. Tightly correlating with autophagy stimulation, we observed a remodeling of the Ca2+ signalosome. First, short periods of starvation (1 to 3 h) caused a prominent increase of the ER Ca2+-store content and enhanced agonist-induced Ca2+ release. The mechanism involved the upregulation of intralumenal ER Ca2+-binding proteins, calreticulin and Grp78/BiP, which increased the ER Ca2+-buffering capacity and reduced the ER Ca2+ leak. Second, starvation led to Ins(1,4,5)P3R sensitization. Immunoprecipitation experiments showed that during starvation Beclin 1, released from Bcl-2, first bound with increasing efficiency to Ins(1,4,5)P3Rs; after reaching a maximal binding after 3 h, binding, however, decreased again. The interaction site of Beclin 1 was determined to be present in the N-terminal Ins(1,4,5)P3-binding domain of the Ins(1,4,5)P3R. The starvation-induced Ins(1,4,5)P3R sensitization was abolished in cells treated with BECN1 siRNA, but not with ATG5 siRNA, pointing toward an essential role of Beclin 1 in this process. Moreover, recombinant Beclin 1 sensitized Ins(1,4,5)P3Rs in 45Ca2+-flux assays, indicating a direct regulation of Ins(1,4,5)P3R activity by Beclin 1. Finally, we found that Ins(1,4,5)P3R-mediated Ca2+ signaling was critical for starvation-induced autophagy stimulation, since the Ca2+ chelator BAPTA-AM as well as the Ins(1,4,5)P3R inhibitor xestospongin B abolished the increase in LC3 lipidation and GFP-LC3-puncta formation. Hence, our results indicate a tight and essential interrelation between intracellular Ca2+ signaling and autophagy stimulation as a proximal event in response to starvation.
| S-EPMC3327615 | biostudies-literature
Project description:We have characterized in detail the Ca(2+)-dependent inhibition of [(3)H]Ins(1,4,5)P(3) ([(3)H]InsP(3)) binding to sheep cerebellar microsomes, over a short duration (3 s), with the use of a perfusion protocol. This procedure prevented artifacts previously identified in studies of this Ca(2+) effect. In a cytosol-like medium at pH 7.1 and 20 degrees C, a maximal inhibition of approx. 50% was measured. Both inhibition and its reversal were complete within 3 s. Ca(2+) decreased the affinity of the receptor for InsP(3) by approx. 50% (K(d) 146+/-24 nM at pCa 9 and 321+/-56 nM at pCa 5.3), without changing the total number of binding sites. Conversely, increasing the [(3)H]InsP(3) concentration from 30 to 400 nM tripled the IC(50) for Ca(2+) and decreased the maximal inhibition by 63%. This is similar to a partial competitive inhibition between InsP(3) binding and inhibitory Ca(2+) binding and is consistent with InsP(3) and Ca(2+) converting InsP(3) receptor into two different states with different affinities for these ligands. Mn(2+) and Sr(2+) also inhibited [(3)H]InsP(3) binding but were respectively only 1/10 and 1/200 as effective as Ca(2+). No inhibition was observed with Ba(2+). This selectivity is the same as that previously reported for the inhibitory Ca(2+) site of InsP(3)-induced Ca(2+) flux, suggesting that the same site is used by Ca(2+) to convert cerebellar InsP(3) receptor to a low-affinity state and to inhibit its channel activity. Our results also suggest a mechanism by which InsP(3) counteracts this Ca(2+)-dependent inhibition.
| S-EPMC1220408 | biostudies-other
Project description:The pleckstrin homology domain of phospholipase Cdelta1 (PH(PLCdelta)) binds Ins(1,4,5)P(3) and PtdIns(4,5)P(2) specifically, and can be used to detect changes in Ins(1,4,5)P(3) in single cells. A fusion construct of PH(PLCdelta) and enhanced green fluorescent protein (EGFP-PH(PLCdelta)) associates with the plasma membrane due to its association with PtdIns(4,5)P(2). However, PH(PLCdelta) has greater affinity for Ins(1,4,5)P(3) than PtdIns(4,5)P(2), and translocates to the cytosol as Ins(1,4,5)P(3) levels rise. Prolonged activation of group I metabotropic glutamate receptor 1alpha expressed in Chinese-hamster ovary cells or endogenous M(3) muscarinic receptors in SH-SY5Y neuroblastoma cells gave an initial transient peak in translocation, followed by a sustained plateau phase. This closely followed changes in cell population Ins(1,4,5)P(3) mass, but not PtdIns(4,5)P(2) levels, which decreased monophasically, as determined by radioreceptor assay. Translocation thus provides a real-time method to follow increases in Ins(1,4,5)P(3). Graded changes in Ins(1,4,5)P(3) in Chinese-hamster ovary-lac-mGlu1alpha cells could be detected with increasing glutamate concentrations, and dual loading with fura 2 and EGFP-PH(PLCdelta) showed that changes in intracellular Ca(2+) concentration closely paralleled Ins(1,4,5)P(3) production. Moreover, Ins(1,4,5)P(3) accumulation and intracellular Ca(2+) mobilization within single cells is graded in nature and dependent on both agonist concentration and receptor density.
| S-EPMC1221821 | biostudies-literature
Project description:Inositol (1,4,5)-trisphosphate [Ins(1,4,5)P(3)] originating in the vascular smooth-muscle cells (VSMCs) has been shown to modulate the Ca(2+) stores in endothelial cells (ECs). However, the reverse is not found, suggesting that Ins(1,4,5)P(3) movement might be unidirectional across gap junctions at the myoendothelial junction (MEJ), or that distribution of the Ins(1,4,5)P(3) receptor [Ins(1,4,5)P(3)-R] is different between the two cell types. To study trans-junctional communication at the MEJ, we used a vascular-cell co-culture model system and selectively modified the connexin composition in gap junctions in the two cell types. We found no correlation between modification of connexin expression and Ins(1,4,5)P(3) signaling between ECs and VSMCs. We next explored the distribution of Ins(1,4,5)P(3)-R isoforms in the two cell types and found that Ins(1,4,5)P(3)-R1 was selectively localized to the EC side of the MEJ. Using siRNA, selective knockdown of Ins(1,4,5)P(3)-R1 in ECs eliminated the secondary Ins(1,4,5)P(3)-induced response in these cells. By contrast, siRNA knockdown of Ins(1,4,5)P(3)-R2 or Ins(1,4,5)P(3)-R3 in ECs did not alter the EC response to VSMC stimulation. The addition of 5-phosphatase inhibitor (5-PI) to ECs that were transfected with Ins(1,4,5)P(3)-R1 siRNA rescued the Ins(1,4,5)P(3) response, indicating that metabolic degradation of Ins(1,4,5)P(3) is an important part of EC-VSMC coupling. To test this concept, VSMCs were loaded with 5-PI and BAPTA-loaded ECs were stimulated, inducing an Ins(1,4,5)P(3)-mediated response in VSMCs; this indicated that Ins(1,4,5)P(3) is bidirectional across the gap junction at the MEJ. Therefore, localization of Ins(1,4,5)P(3)-R1 on the EC side of the MEJ allows the ECs to respond to Ins(1,4,5)P(3) from VSMCs, whereas Ins(1,4,5)P(3) moving from ECs to VSMCs is probably metabolized before binding to a receptor. This data implicates the MEJ as being a unique cell-signaling domain in the vasculature.
| S-EPMC2782698 | biostudies-literature
Project description:Most intracellular Ca(2+) signals result from opening of Ca(2+) channels in the plasma membrane or endoplasmic reticulum (ER), and they are reversed by active transport across these membranes or by shuttling Ca(2+) into mitochondria. Ca(2+) channels in lysosomes contribute to endo-lysosomal trafficking and Ca(2+) signalling, but the role of lysosomal Ca(2+) uptake in Ca(2+) signalling is unexplored. Inhibition of lysosomal Ca(2+) uptake by dissipating the H(+) gradient (using bafilomycin A1), perforating lysosomal membranes (using glycyl-L-phenylalanine 2-naphthylamide) or lysosome fusion (using vacuolin) increased the Ca(2+) signals evoked by receptors that stimulate inositol 1,4,5-trisphosphate [Ins(1,4,5)P(3)] formation. Bafilomycin A1 amplified the Ca(2+) signals evoked by photolysis of caged Ins(1,4,5)P(3) or by inhibition of ER Ca(2+) pumps, and it slowed recovery from them. Ca(2+) signals evoked by store-operated Ca(2+) entry were unaffected by bafilomycin A1. Video-imaging with total internal reflection fluorescence microscopy revealed that lysosomes were motile and remained intimately associated with the ER. Close association of lysosomes with the ER allows them selectively to accumulate Ca(2+) released by Ins(1,4,5)P(3) receptors.
| S-EPMC3603520 | biostudies-literature
Project description:The role of Ins(1,4,5)P3 in receptor-induced Ca2+ mobilization in pituitary cells was studied at the single-cell level. Experimental strategies were developed which allowed a comparative analysis of the effects of Ins(1,4,5)P3 with those of receptor activation under identical conditions. These include microfluorimetry as well as a novel technique which permits the controlled and rapid application of intracellular messenger molecules to individual cells. This latter approach is based on the tight-seal whole-cell recording (WCR) technique, and utilizes two patch-clamp micropipettes, one for electrical recording and the second for the controlled pressure injection. Ins(1,4,5)P3, when applied with this dual-WCR (DWCR) technique, leads rapidly to a marked rise in cytosolic free Ca2+ [( Ca2+]i) and a concomitant stimulation of Ca2(+)-activated K+ current; Ins(1,4,5)P3 can thus mimic the effects of thyrotropin-releasing hormone (TRH) in the same cells under identical conditions. In cells dialysed intracellularly with heparin, a potent antagonist of Ins(1,4,5)P3 action, the rapid response to extracellular stimulation with TRH was abolished, as were the effects of intracellular application of Ins(1,4,5)P3. Heparin, which abolished Ins(1,4,5)P3 action completely, blocked responses to TRH in some cells only partially, revealing that Ca2+ mobilization response to TRH is in part slower in onset than the response to Ins(1,4,5)P3. It is concluded (1) that Ins(1,4,5)P3 is an essential element for the action of TRH, providing a rapid mechanism for Ca2+ mobilization induced by the releasing hormone and (2) that TRH action in mobilizing intracellular Ca2+ is sustained by a slower mechanism which is independent of Ins(1,4,5)P3.
| S-EPMC1131438 | biostudies-other
Project description:Inositol (1,4,5)-trisphosphate [Ins(1,4,5)P3] is one of the key intracellular second messengers in cells and mobilizes Ca2+ stores in the ER (endoplasmic reticulum). Ins(1,4,5)P3 has a short half-life within the cell, and is rapidly metabolized through one of two pathways, one of which involves further phosphorylation of the inositol ring: Ins(1,4,5)P3 3-kinase (IP3-3K) phosphorylates Ins(1,4,5)P3, resulting in the formation of inositol (1,3,4,5)-tetrakisphosphate [Ins(1,3,4,5)P4]. There are three known isoforms of IP3-3K, designated IP3-3KA, IP3-3KB and IP3-3KC. These have differing N-termini, but highly conserved C-termini harbouring the catalytic domain. The three IP3-3K isoforms have different subcellular locations and the B-kinase is uniquely present in both cytosolic and membrane-bound pools. As it is the N-terminus of the B-kinase that differs most from the A- and C-kinases, we have hypothesized that this portion of the protein may be responsible for membrane localization. Although there are no known membrane-targeting protein motifs within the sequence of IP3-3KB, it is found to be tightly associated with the ER membrane. Here, we show that specific regions of the N-terminus of IP3-3KB are necessary and sufficient for efficient membrane localization of the protein. We also report that, in the presence of Ca2+, the kinase domain of IP3-3KB is cleaved from the membrane-anchoring region by calpain.
| S-EPMC1223724 | biostudies-other
Project description:Mass spectrometry identification of Trypanosoma brucei bloodstream form proteins that interacts with Ins(1,4,5)P3 and Ins(1,3,4,5)P4.
2018-10-26 | PXD005907 | Pride
Project description:The phosphorylation of Ins(1,4,5)P3 (InsP3) to Ins(1,3,4,5)P4 (InsP4) is catalysed by InsP3 3-kinase. Molecular-biological data have shown the presence of two human isoenzymes of InsP3 3-kinase, namely InsP3 3-kinases A and B. We have isolated from a rat thymus cDNA library a 2235 bp cDNA (clone B15) encoding rat InsP3 3-kinase B. Northern-blot analysis of mRNA isolated from rat tissues (thymus, testis, brain, spleen, liver, kidney, heart, lung and intestine) revealed that a rat InsP3 3-kinase B probe hybridized to a 6 kb mRNA in lung, thymus, testis, brain and heart. In contrast, Northern-blot analysis of the same tissues probed under stringent conditions with a rat InsP3 3-kinase A probe hybridized to a 2 kb mRNA only in brain and a 1.8-2.0 kb mRNA species in testis. Northern-blot analysis of three human cell lines (HL-60, SH-SY5Y and HTB-138) probed with a human InsP3 3-kinase B probe showed the presence of a 6 kb mRNA in all cell lines, except in the human neuroblastoma cell line (SH-SY5Y), where two mRNA species of 5.7 and 6 kb were detected. Using the same blot, no hybridization signal could be seen with a human InsP3 3-kinase A probe. Altogether, our data are consistent with the notion that the two InsP3 3-kinase isoenzymes, A and B, are specifically expressed in different tissues and cells.
| S-EPMC1136537 | biostudies-other