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PubMed1988

Inositol phosphate production and Ca2+ mobilization in human umbilical-vein endothelial cells stimulated by thrombin and histamine.

Human umbilical-vein endothelial cells (HUVECs) were cultured, and their inositol phosphate formation and Ca2+ mobilization in response to thrombin and histamine were studied. Evidence from measurement of intracellular Ca2+ in the absence of extracellular Ca2+ established that the two agonists were both acting on a single cell population, and suggested that a Ca2+-influx component was stimulated which was dependent on receptor-occupancy. After 30 s of stimulation in the presence of 10 mM-LiCl, the effects of 20 microM-histamine and 1 unit of thrombin/ml on formation of inositol phosphates were additive, but at 5 min they were not. HUVECs labelled with myo-[3H]inositol for 72 h synthesized radiolabelled inositol pentakis- and hexakis-phosphate. The predominant isomers of inositol mono-, bis- and tris-phosphates whose formation was stimulated were the 4-phosphate, the 1,4-bisphosphate and the 1,3,4-trisphosphate.

Calcium↗
PubMed1989

Bombesin and platelet-derived growth factor stimulate formation of inositol phosphates and Ca2+ mobilization in Swiss 3T3 cells by different mechanisms.

Highly purified platelet-derived growth factor (PDGF) or recombinant PDGF stimulate DNA synthesis in quiescent Swiss 3T3 cells. The dose-response curves for the natural and recombinant factors were similar, with half-maximal responses at 2-3 ng/ml and maximal responses at approx. 10 ng/ml. Over this dose range, both natural and recombinant PDGF stimulated a pronounced accumulation of [3H]inositol phosphates in cells labelled for 72 h with [3H]inositol. In addition, mitogenic concentrations of PDGF stimulated the release of 45Ca2+ from cells prelabelled with the radioisotope. However, in comparison with the response to the peptide mitogens bombesin and vasopressin, a pronounced lag was evident in both the generation of inositol phosphates and the stimulation of 45Ca2+ efflux in response to PDGF. Furthermore, although the bombesin-stimulated efflux of 45Ca2+ was independent of extracellular Ca2+, the PDGF-stimulated efflux was markedly inhibited by chelation of external Ca2+ by using EGTA. Neither the stimulation of formation of inositol phosphates nor the stimulation of 45Ca2+ efflux in response to PDGF were affected by tumour-promoting phorbol esters such as 12-O-tetradecanoylphorbol 13-acetate (TPA). In contrast, TPA inhibited phosphoinositide hydrolysis and 45Ca2+ efflux stimulated by either bombesin or vasopressin. Furthermore, whereas formation of inositol phosphates in response to both vasopressin and bombesin was increased in cells in which protein kinase C had been down-modulated by prolonged exposure to phorbol esters, the response to PDGF was decreased in these cells. These results suggest that, in Swiss 3T3 cells, PDGF receptors are coupled to phosphoinositidase activation by a mechanism that does not exhibit protein kinase C-mediated negative-feedback control and which appears to be fundamentally different from the coupling mechanism utilized by the receptors for bombesin and vasopressin.

Animals↗
PubMed1990

Activation of inositol phospholipid breakdown by prostaglandin F2 alpha without any stimulation of proliferation in quiescent NIH-3T3 fibroblasts.

Stimulation of NIH-3T3 cells with prostaglandin F2 alpha (PGF2 alpha) caused a dose- and time-dependent generation of inositol phosphates. The first detectable changes were in the levels of Ins(1,4,5)P3 and Ins(1,3,4,5)P4. Increases in Ins(1,3,4)P3, InsP2 and InsP were detected later, and only minor changes were observed in putative InsP5 or InsP6. The accumulation of inositol phosphates was synergistically increased by the addition of calf serum, whereas PGF2 alpha had no effects on cell proliferation in either the presence or the absence of calf serum. Stimulation of a different clone of NIH-3T3 cells (AmNIH-3T3) or Swiss 3T3 cells with PGF2 alpha resulted in both inositol phospholipid breakdown and cell proliferation. No differences were found in the characteristics of PGF2 alpha-stimulated inositol phosphate generation between the two clones of NIH-3T3 cells, nor was there any difference in receptor number of Kd. These results question the role of inositol phospholipid breakdown in mitogenesis and demonstrate significant differences in the biochemical properties of apparently the 'same' cells.

Animals↗
PubMed1990

Down's syndrome fibroblasts exhibit enhanced inositol uptake.

The inositol metabolism of Down's syndrome (DS, trisomy 21) skin fibroblasts was examined. We report that DS cells accumulated [3H]inositol 2-3-fold faster than did other aneuploid or diploid controls. In contrast, trisomy 21 did not affect the uptake of choline, serine or glucose. Kinetic analysis demonstrated an increased maximal velocity of high-affinity, Na(+)-dependent, inositol transport, consistent with the expression of higher numbers of transporters by DS cells. Enhanced uptake was accompanied by a proportional increase in the incorporation of radiolabelled inositol into phospholipid. We suggest that an imbalance of inositol metabolism may contribute to plasma membrane abnormalities characteristic of DS cells.

Alzheimer Disease↗
PubMed1990

Development of muscarinic-cholinergic stimulation of inositol phosphate production in cultured embryonic chick atrial cells. Evidence for a switch in guanine-nucleotide-binding protein coupling.

We have demonstrated that muscarinic stimulation of inositol phosphate production in cultured atrial cells from chicks at 14 days in ovo is partially sensitive to inhibition by pertussis toxin. In these cells, muscarinic agonist binding is coupled to phospholipase C activity via at least two guanine-nucleotide-binding proteins (G-proteins), one sensitive to pertussis toxin and the other (Gp) insensitive to pertussis toxin [Barnett, Shamah, Lassegue, Griendling & Galper (1990) Biochem. J. 271, 437-442]. In the current study we demonstrate that during embryonic development of the chick heart, muscarinic stimulation of inositol phosphate production decreases by 50% between days 5 and 14 in ovo in cells cultured from both atrium and ventricle. In atrial cells, however, pertussis toxin-sensitive muscarinic stimulation of inositol phosphate production increased from undetectable levels at day 5 in ovo to 40% of total stimulation at day 12 in ovo. Muscarinic stimulation of inositol phosphate production in the ventricle did not become sensitive to pertussis toxin at any age studied. In permeabilized atrial cells from embryonic chicks at 5 days in ovo, guanosine 5'-[gamma-thio]triphosphate (GTP[S]) stimulated InsP1 levels by 40 +/- 10% (mean +/- S.E.M., n = 3), InsP2 levels by 117 +/- 18% and InsP3 levels by 51 +/- 8%, suggesting that at day 5 in ovo all of the muscarinic-stimulated inositol phosphate production was coupled to phospholipase C via Gp. H.p.l.c. analysis demonstrated that, in spite of these changes in coupling of phospholipase C to different G-proteins, no changes could be demonstrated in the isomers of InsP3 produced in response to carbamylcholine at both days 5 and 14 in ovo. These data demonstrate that embryonic development of the chick atrium is associated with a switch in coupling of muscarinic receptors to phospholipase C from Gp to a pertussis toxin substrate. This developmental switch in coupling of G-proteins may be related to possible developmental switches in levels of muscarinic receptor isoforms or switches in the subtype of phospholipase C.

Animals↗
PubMed1991

Masses of inositol phosphates in resting and tetanically stimulated vertebrate skeletal muscles.

The masses of inositol phosphates have been determined in isolated skeletal muscles from Xenopus laevis (sartorius, tibialis anterior and iliofibularis) and rat (gastrocnemius and soleus) which were quick-frozen in the resting state and at different stages of an isometric (Xenopus) or isotonic (rat) tetanus. The isomeric spectrum of inositol phosphates detected was similar to that in other tissues and cell types. The total sarcoplasmic concentrations of the isomers Ins-(1,4,5,6)P4/Ins(3,4,5,6)P4 (0.2-0.9 microM), Ins(1,3,4,6)P4 (not detectable), Ins(1,3,4,5,6)P5 (about 1 microM) and InsP6 (3.2-4.6 microM) were lower than in other cell types. Variations in these concentrations were due to the muscle type rather than to the donor species. The putative second messenger Ins(1,4,5)P3, as well as its dephosphorylation product Ins(1,4)P2, were present at surprisingly high total myoplasmic resting concentrations, ranging from 1.2 to 2.5 microM and 3.5 to 6.9 microM respectively. Upon tetanic stimulation these two inositol phosphates in particular exhibited significantly increased total sarcoplasmic concentrations, up to 4.2 microM and 11.3 microM respectively, with a time scale of seconds. From the initial rate of increase in the total sarcoplasmic concentrations of Ins(1,4,5)P3 and its rapidly formed metabolic products, a minimal phosphoinositidase C (PIC) activity in tetanically activated Xenopus skeletal muscle of about 1.7-2.6 microM/s can be estimated. This PIC activity observed in vivo seems to be far too low to account for a functional role for Ins(1,4,5)P3 as a chemical transmitter in the fast excitation-contraction coupling (ECC) process in skeletal muscle. The presence of Ins(1,3,4,5)P4 in all muscle types is indicative of a Ca(2+)-activated Ins(1,4,5)P3 3-kinase activity. The rapid transient increases in Ins(1,3,4)P3 and Ins(1,3)P2 in isometrically contracting Xenopus muscles suggest that corresponding Ins(1,3,4,5)P4 phosphatases are operating in skeletal muscle as well. In all muscles investigated except rat soleus, the fructose 1,6-bisphosphate [Fru(1,6)P2] concentration increased substantially during a tetanus, up to about 2 mM. This increase is correlated with a simultaneous decrease in phosphocreatine, whereas the energy charge of the muscles was essentially unaffected by the applied tetani. The time course of the rise in Fru(1,6)P2 was used to model changes in the free concentrations of high-affinity aldolase-binding inositol phosphates during the course of a tetanus. These calculations demonstrate that the free concentration of Ins(1,4,5)P3 and other aldolase-bound inositol phosphates can increase much faster and to a larger extent than the corresponding total concentrations as a result of their competitive displacement from aldolase-binding sites by the rapidly rising concentration of Fru(1,6)P2.

Animals↗
PubMed1994

Antigen and thapsigargin promote influx of Ca2+ in rat basophilic RBL-2H3 cells by ostensibly similar mechanisms that allow filling of inositol 1,4,5-trisphosphate-sensitive and mitochondrial Ca2+ stores.

In single, Fura 2-loaded RBL-2H3 cells, antigen and thapsigargin depleted the same intracellular pool of Ca2+ in the absence of external Ca2+; provision of external Ca2+ induced immediate increases in levels of free Ca2+ ([Ca2+]i). These increases were dependent on the presence of external Ca2+ and, presumably, on influx of Ca2+ across the cell membrane. Both stimulants enhanced intracellular accumulation of 45Ca2+ through ostensibly similar mechanisms because accumulation was blocked to similar extents by various multivalent cations or by depolarization with K+. Because thapsigargin blocked reuptake of Ca2+ into inositol 1,4,5-trisphosphate sensitive stores, uptake occurred independently of the refilling of these stores. Uptake was dependent instead on sequestration of 45Ca2+ in a pool of high capacity that was insensitive to thapsigargin, caffeine, GTP and inositol 1,4,5-trisphosphate but sensitive to ionomycin and mitochondrial inhibitors. The existence of an inositol 1,4,5-trisphosphate-insensitive pool was also apparent in permeabilized cells; at 0.1 microM [Ca2+]i, uptake of 45Ca2+ was largely confined (> 80%) to the inositol 1,4,5-trisphosphate-sensitive pool, but at 2 microM [Ca2+]i uptake was largely (> 60%) into the inositol 1,4,5-trisphosphate-insensitive pool. Provision of mitochondrial inhibitors along with thapsigargin to block uptake into both pools, did not impair the thapsigargin-induced increase in [Ca2+]i or influx of Ca2+, as indicated by changes in Fura 2 fluorescence, but did block the intracellular accumulation of 45Ca2+. The studies illustrate the utility of simultaneous measurements of [Ca2+]i and 45Ca2+ uptake for a full accounting of Ca2+ homoeostasis as exemplified by the ability to distinguish between influx and mitochondrial uptake of Ca2+.

Animals↗
PubMed1996

Inositol phosphates in barley (Hordeum vulgare L.) aleurone tissue are stereochemically similar to the products of breakdown of InsP6 in vitro by wheat-bran phytase.

Partisphere SAX HPLC analysis of endogenous inositol phosphates in [3H]inositol-labelled barley aleurone tissue revealed a range of isomers, including D- and/or L-Ins3P, D- and/or L-Ins(1,4)P2, D- and/or L-Ins(1,2)P2, a third unidentified InsP2, Ins(1,2,3)P3, D- and/or L-Ins(1,2,6)P3, D-and/or L-Ins(1,2,3,4)P4, D- and/or L-Ins(1,2,5,6)P4, Ins(1,3,4,5,6)P5, D- and/or L-Ins(1,2,3,4,5)P5, Ins(1,2,3,4,6)P5, InsP6 and a molecule with the chromatographic properties of an inositol pyrophosphate. The striking match between the identities of the stereoisomers, and in some cases enantiomers, detected in vivo and those stereoisomers produced in vitro by the action of wheat-bran phytase on InsP6 [Cosgrove (1980) Inositol Phosphates: Their Chemistry, Bio-chemistry and Physiology. Elsevier, Amsterdam] strongly suggests that most of the inositol phosphates identified are products of the breakdown of InsP4 by endogenous phytase(s) with stereospecificity similar to that of the wheat-bran enzyme(s).

6-Phytase↗
PubMed2003

Arg82p is a bifunctional protein whose inositol polyphosphate kinase activity is essential for nitrogen and PHO gene expression but not for Mcm1p chaperoning in yeast.

In Saccharomyces cerevisiae, the synthesis of inositol pyrophosphates is essential for vacuole biogenesis and the cell's response to certain environmental stresses. The kinase activity of Arg82p and Kcs1p is required for the production of soluble inositol phosphates. To define physiologically relevant targets of the catalytic products of Arg82p and Kcs1p, we used DNA microarray technology. In arg82delta or kcs1delta cells, we observed a derepressed expression of genes regulated by phosphate (PHO) on high phosphate medium and a strong decrease in the expression of genes regulated by the quality of nitrogen source (NCR). Arg82p and Kcs1p are required for activation of NCR-regulated genes in response to nitrogen availability, mainly through Nil1p, and for repression of PHO genes by phosphate. Only the catalytic activity of both kinases was required for PHO gene repression by phosphate and for NCR gene activation in response to nitrogen availability, indicating a role for inositol pyrophosphates in these controls. Arg82p also controls expression of arginine-responsive genes by interacting with Arg80p and Mcm1p, and expression of Mcm1-dependent genes by interacting with Mcm1p. We show here that Mcm1p and Arg80p chaperoning by Arg82p does not involve the inositol polyphosphate kinase activity of Arg82p, but requires its polyaspartate domain. Our results indicate that Arg82p is a bifunctional protein whose inositol kinase activity plays a role in multiple signalling cascades, and whose acidic domain protects two MADS-box proteins against degradation.

Gene Expression Regulation, Fungal↗
PubMed1993

A plant gene with homology to D-myo-inositol-3-phosphate synthase is rapidly and spatially up-regulated during an abscisic-acid-induced morphogenic response in Spirodela polyrrhiza.

The induction of turions (dormant vegetative buds) by the plant growth regulator abscisic acid (ABA) in the aquatic angiosperm Spirodela polyrrhiza is one of the few examples of a plant hormone triggering a morphogenic response. In order to aid our understanding of the molecular basis of this phenomenon a cDNA library has been prepared from ABA-induced S. polyrrhiza and by differential screening a number of early ABA-up-regulated genes have been isolated. This paper reports the characterization of one such cDNA (tur1) from S. polyrrhiza which codes for a protein highly homologous to yeast D-myo-inositol-3-phosphate synthase (EC.5.5.1.4), the catalyst for the first committed step in inositol biosynthesis. This is the first reported cloning of a plant gene involved in inositol production. It is shown by Northern analysis and in situ hybridization that ABA induces a rapid preferential up-regulation of this gene and that this is localized to the stolon tissue. The stolon connects the developing turion to the node of the mother frond (the meristematic region involved in the production of turions which arise as a morphogenic response to this hormone). This increase in tur1 transcript level is accompanied by a rise in the activity of D-myo-inositol-3-phosphate synthase. The possible role of inositol in ABA-induced events during turion formation is discussed.

Abscisic Acid↗
PubMed2000

Coupling of canine serotonin 5-HT(1B) and 5-HT(1D) receptor subtypes to the formation of inositol phosphates by dual interactions with endogenous G(i/o) and recombinant G(alpha15) proteins.

Molecular cloning and expression of canine (ca) serotonin 5-HT(1B) and ca 5-HT(1D) receptor subtypes showed that besides the lower binding affinity of ketanserin for the ca 5-HT(1D) receptor, the ligand binding profiles were similar to their human homologues. Site-directed mutagenesis studies suggest that a Gln(189) residue in the second extracellular loop of the ca 5-HT(1D) receptor may partially account for the lower binding affinity of ketanserin. The coupling of ca 5-HT(1B) and ca 5-HT(1D) receptor subtypes to the phospholipase C pathway was analyzed by measuring stimulation of inositol phosphate formation in COS-7 cells. Zolmitriptan potently stimulated (EC(50) = 4.9 nM) the inositol phosphate formation at ca 5-HT(1D) receptors in a fully pertussis toxin (PTX)-dependent manner, whereas only a weak PTX-resistant inositol phosphate response (26-29% at 10 microM zolmitriptan) could be detected for the ca 5-HT(1B) receptor at a similar expression level. In contrast, both ca 5-HT(1B) and ca 5-HT(1D) receptor subtypes yielded a similar maximal magnitude of inositol phosphate formation (300-340% at 10 microM zolmitriptan) upon co-expression with a mouse (m) G(alpha15) protein. PTX treatment and co-expression with a beta-adrenergic receptor kinase C-terminal polypeptide partially (20-46%) abolished the m G(alpha15) protein-dependent ca 5-HT(1B) and ca 5-HT(1D) receptor-mediated stimulation of inositol phosphate formation. This study suggests both 5-HT receptor subtypes can activate betagamma subunits of endogenous G(i/o) proteins besides their coupling to recombinant m G(alpha15) protein.

Amino Acid Sequence↗
PubMed1985

Inositol cyclic phosphates are produced by cleavage of phosphatidylphosphoinositols (polyphosphoinositides) with purified sheep seminal vesicle phospholipase C enzymes.

Previous studies have shown that metabolism of phosphatidylinositol by phospholipase C produces a mixture of two water-soluble products: inositol 1-phosphate and inositol 1,2-(cyclic)phosphate. In the present study, we demonstrate that the water-soluble products of phosphatidylphosphoinositol (polyphosphoinositide) cleavage by purified ram seminal vesicle phospholipase C enzymes also contain cyclic phosphates. Inositol cyclic phosphates were detected by 18O labeling. In the presence of acid, cyclic phosphates are rapidly hydrolyzed to phosphomonoesters, and when the hydrolysis is carried out in H2 18O, the resultant phosphomonoesters will contain 18O. The 18O content of the phosphomonoesters was measured following alkaline phosphatase treatment and conversion of the inorganic phosphate to a volatile derivative for gas chromatography/mass spectrometry. Inositol cyclic phosphates were found in the phospholipase C cleavage products of all three phosphoinositides, but the ratio of cyclic to noncyclic product was found to decrease in the order phosphatidylinositol greater than phosphatidylinositol 4-phosphate greater than phosphatidylinositol 4,5-bisphosphate. The formation of myo-inositol 1,2(cyclic)-4-bisphosphate was further substantiated by anion-exchange HPLC of the water-soluble products of [32P]phosphatidylinositol 4-phosphate metabolism by phospholipase C. Two peaks were detected one of which, on acid treatment, incorporated 18O from H2 18O into phosphate groups, consistent with this peak containing the cyclic phosphate product. These results suggest that polyphosphoinositide breakdown in stimulated cells may occur via a cyclic phosphate intermediate, as has been described for phosphatidylinositol. These cyclic phosphates contain a reactive bond that may play a role in phosphoinositide-derived signal transduction.

Animals↗
PubMed1994

Insulin-induced activation of glycerol-3-phosphate acyltransferase by a chiro-inositol-containing insulin mediator is defective in adipocytes of insulin-resistant, type II diabetic, Goto-Kakizaki rats.

Type II diabetic Goto-Kakizaki (GK) rats were insulin-resistant in euglycemic-hyperinsulinemic clamp studies. We therefore examined insulin signaling systems in control Wistar and diabetic GK rats. Glycerol-3-phosphate acyltransferase (G3PAT), which is activated by headgroup mediators released from glycosyl-phosphatidylinositol (GPI), was activated by insulin in intact and cell-free adipocyte preparations of control, but not diabetic, rats. A specific chiro-inositol-containing inositol phosphoglycan (IPG) mediator, prepared from beef liver, bypassed this defect and comparably activated G3PAT in cell-free adipocyte preparations of both diabetic GK and control rats. A myo-inositol-containing IPG mediator did not activate G3PAT. Relative to control adipocytes, labeling of GPI by [3H]glucosamine was diminished by 50% and insulin failed to stimulate GPI hydrolysis in GK adipocytes. In contrast to GPI-dependent G3PAT activation, insulin-stimulated hexose transport was intact in adipocytes and soleus and gastrocnemius muscles of the GK rat, as was insulin-induced activation of mitogen-activated protein kinase and protein kinase C. We conclude that (i) chiro-inositol-containing IPG mediator activates G3PAT during insulin action, (ii) diabetic GK rats have a defect in synthesizing or releasing functional chiro-inositol-containing IPG, and (iii) defective IPG-regulated intracellular glucose metabolism contributes importantly to insulin resistance in diabetic GK rats.

Adipocytes↗
PubMed1994

The inositol high-polyphosphate series blocks synaptic transmission by preventing vesicular fusion: a squid giant synapse study.

Presynaptic injection of inositol 1,3,4,5-tetraphosphate, inositol 1,3,4,5,6-pentakisphosphate, or inositol 1,2,3,4,5,6-hexakisphosphate--which we denote here the inositol high-polyphosphate series (IHPS)--is shown to block synaptic transmission when injected into the preterminal of the squid giant synapse. This effect is not produced by injection of inositol 1,4,5-trisphosphate. The synaptic block is characterized by a time course in the order of 15-45 min, depending on the injection site in the preterminal fiber; the fastest block occurs when the injection is made at the terminal release site. Presynaptic voltage clamp during transmitter release demonstrates that IHPS block did not modify the presynaptic inward, calcium current. Analysis of synaptic noise at the postsynaptic axon shows that both the evoked and spontaneous transmitter release are blocked by the IHPS. Tetanic stimulation of the presynaptic fiber at frequencies of 100 Hz indicates that block is accompanied by gradual reduction of the postsynaptic response, demonstrating that the block interferes with vesicular fusion rather than with vesicular docking. These results, in combination with the recently demonstrated observation that the IHPS bind the C2B domain in synaptotagmin [Fukada, M., Aruga, J., Niinobe, M., Aimoto, S. & Mikoshiba, K. (1994) J. Biol. Chem. 269, 29206-29211], suggest that IHPS elements are involved in vesicle fusion and exocytosis. In addition, a scheme is proposed in which synaptotagmin triggers transmitter release directly by promoting the fusion of synaptic vesicles with the presynaptic plasmalemma, in agreement with the very rapid nature of transmitter release in chemical synapses.

Animals↗
PubMed1996

Purified inositol hexakisphosphate kinase is an ATP synthase: diphosphoinositol pentakisphosphate as a high-energy phosphate donor.

Diphosphoinositol pentakisphosphate (PP-IP5) and bis(diphospho)inositol tetrakisphosphate (bis-PP-IP4) are recently identified inositol phosphates that possess pyrophosphate bonds. We have purified an inositol hexakisphosphate (IP6) kinase from rat brain supernatants. The pure protein, a monomer of 54 kDa, displays high affinity (Km = 0.7 microM) and selectivity for inositol hexakisphosphate as substrate. It can be dissociated from bis(diphospho)inositol tetrakisphosphate synthetic activity. The purified enzyme transfers a phosphate from PP-IP5 to ADP to form ATP. This ATP synthase activity indicates the high phosphate group transfer potential of PP-IP5 and may represent a physiological role for PP-IP5.

Animals↗
PubMed1997

Aspartate 19 and glutamate 121 are critical for transport function of the myo-inositol/H+ symporter from Leishmania donovani.

The protozoan flagellate Leishmania donovani has an active myo-inositol/proton symporter (MIT), which is driven by a proton gradient across the parasite membrane. We have used site-directed mutagenesis in combination with functional expression of transporter mutants in Xenopus oocytes and overexpression in Leishmania transfectants to investigate the significance of acidic transmembrane residues for proton relay and inositol transport. MIT has only three charged amino acids within predicted transmembrane domains. Two of these residues, Asp19 (TM1) and Glu121 (TM4), appeared to be critical for transport function of MIT, with a reduction of inositol transport to about 2% of wild-type activity when mutated to the uncharged amides D19N or E121Q and 20% (D19E) or 4% (E121D) of wild-type activity for the conservative mutations that retained the charge. Immunofluorescence microscopy of oocyte cryosections showed that MIT mutants were expressed on the oocyte surface at a similar level as MIT wild type, confirming that these mutations affect transport function and do not prevent trafficking of the transporter to the plasma membrane. The proton uncouplers carbonylcyanide-4-(trifluoromethoxy)phenylhydrazone and dinitrophenol inhibited inositol transport by 50-70% in the wild-type as well as in E121Q, despite its reduced transport activity. The mutant D19N, however, was stimulated about 4-fold by either protonophore and 2-fold by cyanide or increase of pH 7.5 to 8.5 but inhibited at pH 6.5. The conservative mutant D19E, in contrast, showed an inhibition profile similar to MIT wild type. We conclude that Asp19 and Glu121 are critical for myo-inositol transport, while the negatively charged carboxylate at Asp19 may be important for proton coupling of MIT.

Animals↗
PubMed2000

Identification and characterization of a novel inositol polyphosphate 5-phosphatase.

We have identified a cDNA encoding a novel inositol polyphosphate 5-phosphatase. It contains two highly conserved catalytic motifs for 5-phosphatase, has a molecular mass of 51 kDa, and is ubiquitously expressed and especially abundant in skeletal muscle, heart, and kidney. We designated this 5-phosphatase as SKIP (Skeletal muscle and Kidney enriched Inositol Phosphatase). SKIP is a simple 5-phosphatase with no other motifs. Baculovirus-expressed recombinant SKIP protein exhibited 5-phosphatase activities toward inositol 1,4,5-trisphosphate, inositol 1,3,4,5-tetrakisphosphate, phosphatidylinositol (PtdIns) 4,5-bisphosphate, and PtdIns 3,4, 5-trisphosphate but has 6-fold more substrate specificity for PtdIns 4,5-bisphosphate (K(m) = 180 microM) than for inositol 1,4, 5-trisphosphate (K(m) = 1.15 mM). The ectopic expression of SKIP protein in COS-7 cells and immunostaining of neuroblastoma N1E-115 cells revealed that SKIP is expressed in cytosol and that loss of actin stress fibers occurs where the SKIP protein is concentrated. These results imply that SKIP plays a negative role in regulating the actin cytoskeleton through hydrolyzing PtdIns 4,5-bisphosphate.

Amino Acid Sequence↗
PubMed2001

Reassessment of the Ca2+ sensing property of a type I metabotropic glutamate receptor by simultaneous measurement of inositol 1,4,5-trisphosphate and Ca2+ in single cells.

Transient transfection of Chinese hamster ovary or baby hamster kidney cells expressing the Group I metabotropic glutamate receptor mGlu1alpha with green fluorescent protein-tagged pleckstrin homology domain of phospholipase Cdelta1 allows real-time detection of inositol 1,4,5-trisphosphate. Loading with Fura-2 enables simultaneous measurement of intracellular Ca(2+) within the same cell. Using this technique we have studied the extracellular calcium sensing property of the mGlu1alpha receptor. Quisqualate, in extracellular medium containing 1.3 mm Ca(2+), increased inositol 1,4,5-trisphosphate in all cells. This followed a typical peak and plateau pattern and was paralleled by concurrent increases in intracellular Ca(2+) concentration. Under nominally Ca(2+)-free conditions similar initial peaks in inositol 1,4,5-trisphosphate and Ca(2+) concentration occurred with little change in either agonist potency or efficacy. However, sustained inositol 1,4,5-trisphosphate production was substantially reduced and the plateau in Ca(2+) concentration absent. Depletion of intracellular Ca(2+) stores using thapsigargin abolished quisqualate-induced increases in intracellular Ca(2+) and markedly reduced inositol 1,4,5-trisphosphate production. These data suggest that the mGlu1alpha receptor is not a calcium-sensing receptor because the initial response to agonist is not sensitive to extracellular Ca(2+) concentration. However, prolonged activation of phospholipase C requires extracellular Ca(2+), while the initial burst of activity is highly dependent on Ca(2+) mobilization from intracellular stores.

Animals↗