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PubMed1999

Modulation of inositol 1,4,5-trisphosphate binding to the recombinant ligand-binding site of the type-1 inositol 1,4, 5-trisphosphate receptor by Ca2+ and calmodulin.

A recombinant protein (Lbs-1) containing the N-terminal 581 amino acids of the mouse type 1 inositol 1,4,5-trisphosphate receptor (IP3R-1), including the complete IP3-binding site, was expressed in the soluble fraction of E. coli. The characteristics of IP3 binding to this protein were similar as observed previously for the intact IP3R-1. Ca2+ dose-dependently inhibited IP3 binding to Lbs-1 with an IC50 of about 200 nM. This effect represented a decrease in the affinity of Lbs-1 for IP3, because the Kd increased from 115 +/- 15 nM in the absence to 196 +/- 18 nM in the presence of 5 microM Ca2+. The maximal effect of Ca2+ on Lbs-1 (5 microM Ca2+, 42.0 +/- 6.4% inhibition) was similar to the maximal inhibition observed for microsomes of insect Sf9 cells expressing full-length IP3R-1 (33.8 +/- 10.2%). Conceivably, the two contiguous Ca2+-binding sites (residues 304-450 of mouse IP3R-1) previously found by us (Sienaert, I., Missiaen, L., De Smedt, H., Parys, J.B., Sipma, H., and Casteels, R. (1997) J. Biol. Chem. 272, 25899-25906) mediate the effect of Ca2+ on IP3 binding to IP3R-1. Calmodulin also dose-dependently inhibited IP3 binding to Lbs-1 with an IC50 of about 3 microM. Maximal inhibition (10 microM calmodulin, 43.1 +/- 5.9%) was similar as observed for Sf9-IP3R-1 microsomes (35.8 +/- 8.7%). Inhibition by calmodulin occurred independently of Ca2+ and was additive to the inhibitory effect of 5 microM Ca2+ (together 74.5 +/- 5.1%). These results suggest that the N-terminal ligand-binding region of IP3R-1 contains a calmodulin-binding domain that binds calmodulin independently of Ca2+ and that mediates the inhibition of IP3 binding to IP3R-1.

Animals↗

PubMed1999

Inositol 1,4,5-trisphosphate receptor down-regulation is activated directly by inositol 1,4,5-trisphosphate binding. Studies with binding-defective mutant receptors.

Activation of certain phosphoinositidase C-linked cell surface receptors is known to cause an acceleration of the proteolysis of inositol 1,4,5-trisphosphate (InsP3) receptors and, thus, lead to InsP3 receptor down-regulation. To gain insight into this process, we examined whether or not InsP3 receptor degradation is a direct consequence of InsP3 binding by analyzing the down-regulation of exogenous wild-type and binding-defective mutant InsP3 receptors expressed in SH-SY5Y human neuroblastoma cells. Stimulation of these cells with carbachol showed that wild-type exogenous receptors could be down-regulated but that the binding-defective mutant exogenous receptors were not. Thus, InsP3 binding appears to mediate down-regulation. To validate this conclusion, a comprehensive analysis of the effects of the exogenous receptors was undertaken. This showed that exogenous receptors (i) are localized appropriately within the cell, (ii) enhance InsP3-induced Ca2+ release in permeabilized cells, presumably by increasing the number of InsP3-sensitive Ca2+ channels, (iii) have minimal effects on Ca2+ mobilization and InsP3 formation in intact cells, (iv) form heteromers with endogenous receptors, and (v) do not alter the down-regulation of endogenous receptors. In total, these data show that the introduction of exogenous receptors into SH-SY5Y cells does not compromise intracellular signaling or the down-regulatory process. We can thus conclude that InsP3 binding directly activates InsP3 receptor degradation. Because InsP3 binding induces a conformational change in the InsP3 receptor, these data suggest that this change provides the signal for accelerated proteolysis.

Amino Acid Sequence↗

PubMed2004

Bcl-2 functionally interacts with inositol 1,4,5-trisphosphate receptors to regulate calcium release from the ER in response to inositol 1,4,5-trisphosphate.

Inositol 1,4,5-trisphosphate (InsP3) receptors (InsP3Rs) are channels responsible for calcium release from the endoplasmic reticulum (ER). We show that the anti-apoptotic protein Bcl-2 (either wild type or selectively localized to the ER) significantly inhibited InsP3-mediated calcium release and elevation of cytosolic calcium in WEHI7.2 T cells. This inhibition was due to an effect of Bcl-2 at the level of InsP3Rs because responses to both anti-CD3 antibody and a cell-permeant InsP3 ester were decreased. Bcl-2 inhibited the extent of calcium release from the ER of permeabilized WEHI7.2 cells, even at saturating concentrations of InsP3, without decreasing luminal calcium concentration. Furthermore, Bcl-2 reduced the open probability of purified InsP3Rs reconstituted into lipid bilayers. Bcl-2 and InsP3Rs were detected together in macromolecular complexes by coimmunoprecipitation and blue native gel electrophoresis. We suggest that this functional interaction of Bcl-2 with InsP3Rs inhibits InsP3R activation and thereby regulates InsP3-induced calcium release from the ER.

CD3 Complex↗

PubMed2002

Caenorhabditis elegans inositol 5-phosphatase homolog negatively regulates inositol 1,4,5-triphosphate signaling in ovulation.

Ovulation in Caenorhabditis elegans requires inositol 1,4,5-triphosphate (IP(3)) signaling activated by the epidermal growth factor (EGF)-receptor homolog LET-23. We generated a deletion mutant of a type I 5-phosphatase, ipp-5, and found a novel ovulation phenotype whereby the spermatheca hyperextends to engulf two oocytes per ovulation cycle. The temporal and spatial expression of IPP-5 is consistent with its proposed inhibition of IP(3) signaling in the adult spermatheca. ipp-5 acts downstream of let-23, and interacts with let-23-mediated IP(3) signaling pathway genes. We infer that IPP-5 negatively regulates IP(3) signaling to ensure proper spermathecal contraction.

Amino Acid Sequence↗

PubMed2005

Fertilization and inositol 1,4,5-trisphosphate (IP3)-induced calcium release in type-1 inositol 1,4,5-trisphosphate receptor down-regulated bovine eggs.

It is widely believed that stimulation of the phosphoinositide pathway and production of 1,4,5-inositol trisphosphate (IP(3)) underlies the oscillatory changes in the concentration of intracellular free calcium ions ([Ca(2+)](i)) seen during mammalian fertilization. IP(3) promotes Ca(2+) release in eggs by binding to its receptor, the type-1 IP(3) receptor (IP(3)R-1, also known as ITPR1), a ligand-gated Ca(2+) channel located in the membrane of the endoplasmic reticulum, the main Ca(2+) store of the cell. While IP(3)R-1 has been shown to mediate all Ca(2+) release during mouse fertilization, whether or not it plays such an essential role in fertilization-induced Ca(2+) release in large domestic species such as bovine and porcine is presently not known. Accordingly, we have generated metaphase II bovine eggs with a approximately 70%-80% reduction in the number of intact IP(3)R-1 by inducing receptor down-regulation during oocyte maturation. We did so by injecting the nonhydrolyzable IP(3) analogue, adenophostin A. Functional Ca(2+) release analysis revealed that IP(3)R-1 is the predominant Ca(2+) release channel in bovine eggs, requiring as little as 20% of total intact receptor to mount persistent [Ca(2+)](i) oscillations in response to fertilization, expression of PLCzeta (also known as PLCZ1), and adenophostin A. However, lower concentrations of IP(3) and near-physiological concentrations of porcine sperm extract were unable to trigger [Ca(2+)](i) oscillations in this reduced IP(3)R-1 model. Furthermore, we present evidence that the sensitivity of bovine IP(3)R-1 is impaired at the first embryonic interphase. Together, these results demonstrate the essential role of IP(3)R-1-mediated Ca(2+) release during fertilization in bovine eggs, and identify cell cycle regulatory mechanisms of [Ca(2+)](i) oscillations at the level of IP(3)R-1.

Adenosine↗

PubMed1997

Structural analogues of D-myo-inositol-1,4,5-trisphosphate and adenophostin A: recognition by cerebellar and platelet inositol-1,4,5-trisphosphate receptors.

Adenophostins A and B, which are metabolic products of the fungus Penicillium brevicompactum, are potent agonists at the D-myo-inositol-1,4,5-trisphosphate [Ins(1,4,5)P3] receptor. In the current study, adenophostin A was approximately 50-fold more potent than Ins(1,4,5)P3 at both releasing Ca2+ from the intracellular stores of permeabilized platelets and displacing [3H]Ins(1,4,5)P3 from its receptor on rat cerebellar membranes. Various analogues bearing structural features found in the adenophostins and/or Ins(1, 4,5)P3 were examined to elucidate the molecular basis for the observed enhanced potency. 2-AMP did not induce Ca2+ release from permeabilized platelets or have any effect on Ins(1,4,5)P3-induced Ca2+ release. Two carbohydrate-based analogues, (2-hydroxyethyl)-alpha-D-glucopyranoside-2',3,4-trisphosphate and alpha,alpha'-trehalose-3,4,3',4'-tetrakisphosphate, could induce release of Ca2+ and displace [3H]Ins(1,4,5)P3 from its binding site on rat cerebellar membranes, although both were less potent than Ins(1,4,5)P3. In common with adenophostin A, release of Ca2+ from the intracellular stores could be inhibited by heparin, and both analogues were metabolically resistant. This study is the first to demonstrate the activity of a synthetic disaccharide at the Ins(1,4, 5)P3 receptor and that the Ins(1,4,5)P3 receptor is capable of accommodating an increased steric bulk. The minimal importance of the 2-hydroxyl group of Ins(1,4,5)P3 (occupied by the pyranoside oxygen in adenophostin) was confirmed by comparing the activity of DL-scyllo-Ins(1,2,4)P3 [which differs from Ins(1,4,5)P3 solely by the orientation of this hydroxyl group] with that of Ins(1,4,5)P3. An analogue of this compound, namely, DL-6-CH2OH-scyllo-Ins(1,2,4)P3, which possesses an equatorial hydroxymethyl group analogous to the 5'-hydroxymethyl group of adenophostin, was found to be equipotent to Ins(1,4,5)P3, demonstrating the tolerance of the Ins(1,4,5)P3 receptor to additional steric bulk at this position.

Adenosine↗

PubMed1998

Differences among type I, II, and III inositol-1,4,5-trisphosphate receptors in ligand-binding affinity influence the sensitivity of calcium stores to inositol-1,4,5-trisphosphate.

Type I, II, and III inositol-1,4,5-trisphosphate (InsP3) receptors are expressed selectively in different cell lines and tissues. We examined whether type I, II, and III InsP3 receptors differ in ligand-binding affinity and whether such differences influence the sensitivity of Ca2+ stores to InsP3. Initially, SH-SY5Y human neuroblastoma cells, AR4-2J rat pancreatoma cells, and RINm5F rat insulinoma cells were studied because these cells express predominantly (>85%) type I, II, and III receptors, respectively. Immunopurification of receptors from these cell lines and measurement of InsP3 binding revealed that the rank order of affinity for InsP3 was type I > type II > type III (binding sites were half-maximally saturated at 1.5, 2.5, and 22.4 nM InsP3, respectively). Examination of Ca2+ store mobilization in permeabilized cells showed that InsP3 was equipotent in SH-SY5Y and AR4-2J cells but was approximately 5-fold less potent in RINm5F cells. In contrast, Ca2+ uptake and InsP3-independent Ca2+ release were very similar in the three cell types. The binding affinity of InsP3 in permeabilized SH-SY5Y, AR4-2J, and RINm5F cells correlated well with its potency as a Ca2+-mobilizing agent and with binding affinity to immunopurified type I, II, and III receptors. Thus, InsP3 receptor binding affinity seems to influence the potency of InsP3 as a Ca2+-mobilizing agent. Finally, immunopurification of type I, II, and III receptors from rat tissues revealed that the affinity differences seen in receptors purified from cultured cells are paralleled in vivo. In combination, the data from cell lines and rat tissues reveal that type I, II, and III receptors bind InsP3 with Kd values of approximately 1, approximately 2, and approximately 40 nM, respectively, and that the selective expression of a particular receptor type will influence the sensitivity of cellular Ca2+ stores to InsP3.

Animals↗

PubMed2000

The effect of inositol 1,3,4,5-tetrakisphosphate on inositol trisphosphate-induced Ca2+ mobilization in freshly isolated and cultured mouse lacrimal acinar cells.

Earlier reports have shown a remarkable synergism between InsP(4) and InsP(3) [either Ins(1,4,5)P(3) or Ins(2,4,5)P(3)] in activating Ca(2+)-dependent K(+) and Cl(-) currents in mouse lacrimal cells [Changya, Gallacher, Irvine, Potter and Petersen (1989) J. Membr. Biol. 109, 85-93; Smith (1992) Biochem. J. 283, 27-30]. However, Bird, Rossier, Hughes, Shears, Armstrong and Putney [(1991) Nature (London) 352, 162-165] reported that they could see no such synergism in the same cell type. A major experimental difference between the two laboratories lies in whether or not the cells were maintained in primary culture before use. Here we have compared directly the responses to inositol polyphosphates in freshly isolated cells versus cells cultured for 6-72 h. In the cultured cells, Ins(2,4,5)P(3) at 100 microM produced a robust stimulation of K(+) and Cl(-) currents, as much as an order of magnitude greater than that observed in the freshly isolated cells. However, the freshly isolated cells could be restored to a sensitivity similar to cultured cells by the addition of InsP(4) at a concentration two orders of magnitude lower than that of Ins(2,4,5)P(3). We discuss the implications of this with respect to the actions of InsP(4), including the possibility that disruption of the cellular structure during the isolation of the cells exposes an extreme manifestation of a possible physiological role for InsP(4) in controlling calcium-store integrity.

Acetylcholine↗

PubMed1986

The role of cytosolic free calcium in the generation of inositol 1,4,5-trisphosphate and inositol 1,3,4-trisphosphate in HL-60 cells. Differential effects of chemotactic peptide receptor stimulation at distinct Ca2+ levels.

The generation of the two inositol trisphosphate (IP3) isomers, 1,4,5-IP3 and 1,3,4-IP3, and its relation to changes in the cytosolic free calcium concentration, [Ca2+]i, in response to the chemotactic peptide fMet-Leu-Phe was studied in the human promyelocytic cell line HL-60, induced to differentiate with dimethyl sulfoxide. Stimulation by fMet-Leu-Phe within seconds transiently elevates 1,4,5-IP3 to peak values averaging 8-fold basal levels, and leads to a concomitant rise in [Ca2+]i and to degranulation. These responses are followed by a slower and more sustained rise in 1,3,4-IP3. Alterations in [Ca2+]i modulate differentially the generation of the two IP3 isomers. At [Ca2+]i lower than 30 nM, no IP3 is generated upon fMet-Leu-Phe stimulation. Working at normal resting [Ca2+]i, but preventing the fMet-Leu-Phe induced transient rise in [Ca2+]i (by prior depletion of intracellular Ca2+ stores and working in calcium-free medium) the fMet-Leu-Phe stimulation of 1,3,4-IP3 levels is attenuated, whereas the response of 1,4,5-IP3 is not significantly altered. Maintained elevation of [Ca2+]i to micromolar levels with the Ca2+ ionophore ionomycin generates enhanced 1,3,4-IP3 levels in the absence of fMet-Leu-Phe, whereas the fMet-Leu-Phe stimulation of 1,4,5-IP3 generation is markedly inhibited. Pertussis toxin selectively abolishes the fMet-Leu-Phe-induced IP3 production, whereas ionomycin stimulation of 1,3,4-IP3 generation is unaffected. These findings indicate that in intact cells: receptor-triggered phosphatidylinositol bisphosphate phosphodiesterase activation has a minimal Ca2+ requirement, but does not depend on a previous or concomitant rise in [Ca2+]i; Ca2+ elevations above micromolar levels decrease the fMet-Leu-Phe-induced generation of 1,4,5-IP3; and 1,3,4-IP3 generation is not directly linked to receptor activation and appears to result both from increased [Ca2+]i and 1,4,5-IP3 levels.

Calcium↗

PubMedDate not supplied

The effects of two inositol (1,4,5)-trisphosphate 5-phosphatase inhibitors on thyrotropin releasing hormone stimulated inositol (1,4,5)-trisphosphate production in GH3 cells.

The effects of FLA99 and EWP840, two disulfiram analogs which potently inhibit Ins(1,4,5)P3 5-phosphatase, upon basal and thyrotropin-releasing hormone (TRH)-stimulated inositol (1,4,5)-trisphosphate (Ins(1,4,5)P3) levels were investigated. Neither test compound affected the characteristics of the [3H]Ins(1,4,5)P3 binding site used in the competitive protein binding assay of Ins(1,4,5)P3 levels. In rat GH3 pituitary cell suspensions, TRH (100 nM) produced a large and time-dependent increase in Ins(1,4,5)P3 concentration, the maximum response being obtained within 5 seconds of stimulation in these cells. Neither FLA99 (100, 300 and 1000 microM) nor EWP840 (100 microM) produced obvious effects on the Ins(1,4,5)P3 response to TRH stimulation. Higher concentrations of EWP840 (300 and 1000 microM) abolished the Ins(1,4,5)P3 response to TRH stimulation. The lack of effect of the 5-phosphatase inhibition in the cells may indicate that 5-phosphatase is not the major metabolic pathway of this second messenger in this cell line under the assay conditions used.

Adenoma↗

PubMed1992

Synthetic D- and L-enantiomers of 2,2-difluoro-2-deoxy-myo-inositol 1,4,5-trisphosphate interact differently with myo-inositol 1,4,5-trisphosphate binding proteins: identification of a potent small molecule 3-kinase inhibitor.

The ability of two enantiomeric fluoro-analogues of D-myo-inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] to mobilize intracellular Ca2+ stores in SH-SY5Y neuroblastoma cells has been investigated. (-)-D-2,2-difluoro-2-deoxy-myo-Ins(1,4,5)P3 [D-2,2-F2-Ins(1,4,5)P3] was a full agonist [EC50 0.21 microM] and slightly less potent than D-Ins(1,4,5)P3 [EC50 0.13 microM]. (+)-L-2,2-F2Ins(1,4,5)P3 was a very poor agonist, confirming the stereospecificity of the Ins(1,4,5)P3 receptor. D-2,2-F2-Ins(1,4,5)P3 mobilized Ca2+ with broadly similar kinetics to Ins(1,4,5)P3 and was a substrate for Ins(1,4,5)P3 3-kinase inhibiting Ins(1,4,5)P3 phosphorylation (apparent Ki = 10.2 microM) but was recognised less well than Ins(1,4,5)P3. L-2,2-F2-Ins(1,4,5)P3 was a potent competitive inhibitor of 3-kinase (Ki = 11.9 microM). Whereas D-2,2-F2-Ins(1,4,5)P3 was a good substrate for Ins(1,4,5)P3 5-phosphatase, L-2,2-F2Ins(1,4,5)P3 was a relatively potent inhibitor (Ki = 19.0 microM).

Calcium↗

PubMed2000

Sperm-induced Ca(2+) oscillations in mouse oocytes and eggs can be mimicked by photolysis of caged inositol 1,4,5-trisphosphate: evidence to support a continuous low level production of inositol 1, 4,5-trisphosphate during mammalian fertilization.

During mouse fertilization the spermatozoon induces a series of low-frequency long-lasting Ca(2+) oscillations. It is generally accepted that these oscillations are due to Ca(2+) release through the inositol 1,4,5-trisphosphate (InsP(3)) receptor. However, InsP(3) microinjection does not mimic sperm-induced Ca(2+) oscillations, leading to the suggestion that the spermatozoon causes Ca(2+) release by sensitizing the InsP(3) receptor to basal levels of InsP(3). This contradicts recent evidence that the spermatozoon triggers Ca(2+) oscillations by introducing a phospholipase C or else an activator of phospholipase C. Here we show for the first time that sperm-induced Ca(2+) oscillations may be mimicked by the photolysis of caged InsP(3) in both mouse metaphase II eggs and germinal vesicle stage oocytes. Eggs, and also oocytes that had displayed spontaneous Ca(2+) oscillations, gave long-lasting Ca(2+) oscillations when fertilized or when caged InsP(3) was photolyzed. In contrast, oocytes that had shown no spontaneous Ca(2+) oscillations did not generate many oscillations when fertilized or following photolysis of caged InsP(3). Fertilization in eggs was most closely mimicked when InsP(3) was uncaged at relatively low amounts for extended periods. Here we observed an initial Ca(2+) transient with superimposed spikes, followed by a series of single transients with a low frequency; all characteristics of the Ca(2+) changes at fertilization. We therefore show that InsP(3) can mimic the distinctive pattern of Ca(2+) release in mammalian eggs at fertilization. It is proposed that a sperm Ca(2+)-releasing factor operates by generating a continuous small amount of InsP(3) over an extended period of time, consistent with the evidence for the involvement of a phospholipase C.

Animals↗

PubMed1978

The effect of myo-inositol deficiency on lipid metabolism in rats. III. The mechanism of an enhancement in lipolysis due to myo-inositol deficiency in rats.

myo-Inositol deficiency in rats produced an overaccumulation of triacylglycerols in the liver due to stimulated lipolysis in the adipose tissue (Hayashi, E., Maeda, T. and Tomita, T. (1974) Biochim. Biophys. Acta 360, 134--155). The mechanism of the enhancement in lipolysis has now been investigated. The lipolytic response to adrenalin, corticotropin and insulin of the epididymal adipose tissue did not change due to the deficiency, but hormone-sensitive lipase activity, plasma adrenalin level and blood pressure were higher in the deficient rats. Adrenalectomy had no influence, but administration of sympathetic nervous blockers (reserpine, hexamethonium and bupranolol) inhibited the liver lipid deposition and an increase of serum free fatty acids in the deficient rats. These results indicate that the enhancement in lipolysis is mediated by an excitation of sympathetic nerve terminals innervating in the adipose tissues.

Adipose Tissue↗

PubMed1991

Complexation studies on inositol-phosphates. II. Alkali-metal complexes of D-myo-inositol 1,2,6 trisphosphate.

The complexation properties of the D-myo-inositol 1,2,6 trisphosphate (Ins(1,2,6)P3) towards Li+, Na+, K+, Rb+, and Cs+ cations were studied at 25 degrees C in a 0.1 M tetra-n-butylammonium bromide medium. For all cations, mononuclear and protonated species were found. For smaller cations (Li+, Na+, and K+) a dinuclear complex was also put into evidence. The main characteristic of the complexes is its high stability; and of the ligand, its nonselectivity. The Ins(1,2,6)P3-K system was ascertained using Sammartano's method which additionally enabled the influence of various K+ concentrations on the protonations constants to be considered.

Cations, Monovalent↗

PubMed1992

Complexation studies on inositol-phosphates: IV. Ca(II) complexes of myo-inositol 1,4,5-trisphosphate.

The stability constants of the complexes formed between Ca2+ and the myo-inositol 1,4,5-triphosphate (Ins(1,4,5)P3) were determined by potentiometric titration in two different media and temperature conditions (medium 1: I = 0.1 M But4NBr, 25 degrees C; medium 2: I = 0.2 M KCl, 37 degrees C). Mainly because of the presence of potassium the results obtained in these media show large differences in both the nature and the stability of the complexes. In medium 1, MH2L and M2L species are formed along with the ML and MHL species which also exist in medium 2. In addition, the stability of the latter species decreases by more than one log unit in going from medium 1 to medium 2. In an attempt to assess the biological significance of the metal binding to Ins(1,4,5)P3, the results were compared to the Ca2+-ATP complexes that form in the same media conditions. Taking into account the relative stability of the complexes of both systems, it is likely that the action or metabolism of Ins(1,4,5)P3 may be influenced by coordination of either alkali or alkali-earth cations.

Calcium↗

PubMed2005

Fcepsilon RI control of Ras via inositol (1,4,5) trisphosphate 3-kinase and inositol tetrakisphosphate.

The inositol (1,4,5) trisphosphate 3-kinase (ITP3K) phosphorylates Ins (1,4,5) P3 to produce Ins (1,3,4,5) P4. The ITP3K substrate, InsP3, and its product, InsP4, both have the potential to regulate mast cell function. Here, we explore the effects of dominant inhibition of ITP3K upon secretory responses and Ras GTPase activation following antigenic cross-linking of the mast cell immunoreceptor, FcvarepsilonRI. Inhibition of ITP3K potentiates both calcium release from intracellular stores and calcium-dependent secretory responses in mast cells. Moreover, mast cells with dominantly inhibited ITP3K display constitutive activation of Ras and certain Ras effector pathways. We propose three mechanisms by which ITP3K inhibition could influence Ras activation. The protection of InsP3 that results from ITP3K inhibition may lead to enhanced activation of calcium-sensitive Ras-GAPs or -GRFs. Similarly, the deficit in InsP4 may change the behavior of the InsP4 receptor, the GAP1(IP4BP). Our data are inconsistent with calcium-sensitive Ras-GAP activation being the primary consequence of ITP3K inhibition in mast cells. Rather, we observe potentiation of Ras responses in mast cells transfected with dominant negative GAP1(IP4BP). Moreover, shRNA-mediated knockdown of GAP1(IP4BP) potentiates FcvarepsilonRI-mediated Ras activation, indicating that this InsP4-binding GAP protein may be used by the FcvarepsilonRI immunoreceptor to regulate Ras.

Animals↗

PubMed1994

The inositol trisphosphate calcium channel is inactivated by inositol trisphosphate.

Activation of intracellular Ca2+ channels by inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) represents the initial Ca2+ mobilization step in response to many extracellular signals. Here we report that Ins(1,4,5)P3-induced channel activation in permeabilized hepatocytes is followed by a time-dependent inactivation, which is a direct consequence of ligand binding. The inactivation by Ins(1,4,5)P3 parallels the quantal character of channel opening, giving rise to a unique process of incremental inactivation whereby discrete channel populations are inhibited at each Ins(1,4,5)P3 dose. Ins(1,4,5)P3 can induce inactivation in the absence of stored Ca2+, but the inactivation rate is enhanced by increases of cytosolic Ca2+. The inhibitory effect of Ins(1,4,5)P3 can be reversed by Ins(1,4,5)P3 washout, or by chelation of cytosolic Ca2+. Thus, Ins(1,4,5)P3 and Ca2+ act as coinhibitors of the Ins(1,4,5)P3-sensitive Ca2+ channel. Inactivation is an inherent consequence of Ins(1,4,5)P3-induced channel opening which can terminate increases of cytosolic Ca2+.

Calcium↗

PubMed1990

Identification in extracts from AR4-2J cells of inositol 1,4,5-trisphosphate by its susceptibility to inositol 1,4,5-trisphosphate 3-kinase and 5-phosphatase.

The identity of 3H-labelled material ascribed to Ins(1,4,5)P3 in resting or bombesin-stimulated myo-[3H]inositol-labelled AR4-2J cells was investigated by determining its ability to serve as substrate for partially purified Ins(1,4,5)P3/Ins(1,3,4,5)-P4 5-phosphatase and Ins(1,4,5)P3 3-kinase. This 3H-labelled material was metabolized by these two enzymes at rates which were indistinguishable from those for an internal [32P]Ins(1,4,5)P3 standard, establishing its identity as authentic Ins(1,4,5)P3. In addition, and in contrast with findings in earlier studies utilizing substance P as an agonist, prolonged stimulation with bombesin resulted in an increase in an InsP4 which was degraded by Ins(1,4,5)P3/Ins(1,3,4,5)P4 5-phosphatase. These findings serve to confirm the previous estimate of Horstman, Takemura & Putney [(1988) J. Biol. Chem. 263, 15297-15303] for the intracellular concentrations of Ins(1,4,5)P3 in resting (2 microM) and agonist-stimulated (25 microM) AR4-2J cells. The implications of these findings for the physiological regulation of intracellular Ca2+ through this intracellular messenger are discussed.

Bombesin↗