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1.
Triphosphoinositide breakdown and dense body release as the earliest events in thrombin-induced activation of human platelets 总被引:2,自引:0,他引:2
F Rendu P Marche J Maclouf A Girard S Levy-Toledano 《Biochemical and biophysical research communications》1983,116(2):513-519
The activation by thrombin of human platelets prelabelled with 32P induced a 30-40% decrease in 32P-triphosphoinositides (TPI) in the first 10 sec; the decrease in the other 32P-labelled phosphoinositides occurred by 20-30 sec. At 10 sec., the intensity of these effects was maximum with 0.2-0.4 U/ml thrombin. Under these conditions, 53, 20 and 15% of the dense granule, alpha-granule and lysosome constituents, respectively were released and thromboxane B2 synthesis reached only 10% of its maximum. Together with experiments carried out with chlorpromazine - or PGE1 - treated platelets, our results suggest the existence of a close relationship between TPI-breakdown and dense body release which appear to be the earliest events resulting from the activation of human platelets by thrombin. 相似文献
2.
Myotubularin lipid phosphatase binds the hVPS15/hVPS34 lipid kinase complex on endosomes 总被引:3,自引:1,他引:2
Cao C Laporte J Backer JM Wandinger-Ness A Stein MP 《Traffic (Copenhagen, Denmark)》2007,8(8):1052-1067
Myotubularins constitute a ubiquitous family of phosphatidylinositol (PI) 3-phosphatases implicated in several neuromuscular disorders. Myotubularin [myotubular myopathy 1 (MTM1)] PI 3-phosphatase is shown associated with early and late endosomes. Loss of endosomal phosphatidylinositol 3-phosphate [PI(3)P] upon overexpression of wild-type MTM1, but not a phosphatase-dead MTM1C375S mutant, resulted in altered early and late endosomal PI(3)P levels and rapid depletion of early endosome antigen-1. Membrane-bound MTM1 was directly complexed to the hVPS15/hVPS34 [vacuolar protein sorting (VPS)] PI 3-kinase complex with binding mediated by the WD40 domain of the hVPS15 (p150) adapter protein and independent of a GRAM-domain point mutation that blocks PI(3,5)P(2) binding. The WD40 domain of hVPS15 also constitutes the binding site for Rab7 and, as shown previously, contributes to Rab5 binding. In vivo, the hVPS15/hVPS34 PI 3-kinase complex forms mutually exclusive complexes with the Rab GTPases (Rab5 or Rab7) or with MTM1, suggesting a competitive binding mechanism. Thus, the Rab GTPases together with MTM1 likely serve as molecular switches for controlling the sequential synthesis and degradation of endosomal PI(3)P. Normal levels of endosomal PI(3)P and PI(3,5)P(2) are crucial for both endosomal morphology and function, suggesting that disruption of endosomal sorting and trafficking in skeletal muscle when MTM1 is mutated may be a key factor in precipitating X-linked MTM. 相似文献
3.
4.
Sheryl T. Homa Dolores M. Conroy Anthony D. Smith 《Biochemical and biophysical research communications》1980,95(3):1321-1327
Free fatty acids added in ethanol to human platelets prelabelled with []arachidonate induce an accumulation of radioactive diacylglycerol. Unsaturated fatty acids are ten times more potent than palmitate. Ethanol alone does not alter the distribution of radioactivity. Increasing the concentration of arachidonate leads to increased diacylglycerol formation. The fatty acid effect is independent of thrombin, which itself causes a relatively small change in diacylglycerol levels. Neither the labelled triacylglycerol nor the labelled free fatty acid appears to be the source of the diacylglycerol formed which may arise from the activation of phosphatidylinositol phosphodiesterase. 相似文献
5.
Overexpression of phosphatidylinositol phosphate 5-kinase (PIP5KI) isoforms alpha, beta, or gamma in CV-1 cells increased phosphatidylinositol 4,5-bisphosphate (PIP2) levels by 35, 180, and 0%, respectively. Endocytosis of transferrin receptors, association of AP-2 proteins with membranes, and the number of clathrin-coated pits at the plasma membrane increased when PIP2 increased. When expression of PIP5KIbeta was inhibited with small interference RNA in HeLa cells, expression of PIP5KIalpha was also reduced slightly, but PIP5KIgamma expression was increased. PIP2 levels and internalization of transferrin receptors dropped 50% in these cells; thus, PIP5KIgamma could not compensate for loss of PIP5KIbeta. When expression of PIP5KIalpha was reduced, expression of both PIP5KIbeta and PIP5KIgamma increased and PIP2 levels did not change. A similar increase of PIP5KIalpha and PIP5KIbeta occurred when PIP5KIgamma was inhibited. These results indicate that constitutive endocytosis in CV-1 and HeLa cells requires (and may be regulated by) PIP2 produced primarily by PIP5KIbeta. 相似文献
6.
Phosphatidylinositol 4,5-bisphosphate may represent the site of release of plasma membrane-bound calcium upon stimulation of human platelets 总被引:6,自引:0,他引:6
M J Broekman 《Biochemical and biophysical research communications》1984,120(1):226-231
Thrombin stimulation of human blood platelets caused an extensive (up to 45%) and rapid (5-10 s) decline in endogenous phosphatidylinositol 4,5-bisphosphate (PI-P2). Thrombin initiated an equally rapid loss of membrane-bound Ca, as indicated by the decrease in fluorescence of chlortetracycline (CTC)-loaded platelets. PI-P2 breakdown also correlated with decreased CTC fluorescence upon use of other platelet stimuli: Arachidonate caused moderate and slow decreases in both PI-P2 and CTC fluorescence, while ionophore only induced minimal changes. Thrombin-induced decreases in PI-P2 content could account for release of sufficient membrane-bound Ca to raise cytoplasmic free [Ca2+] to 1-2 microM, supporting the hypothesis that PI-P2 represents the Ca-binding site involved in the stimulus-dependent increase in cytoplasmic Ca2+ evoked by receptor-ligand interactions. 相似文献
7.
《Autophagy》2013,9(5):933-935
Phosphatidylinositol 3-phosphate (PtdIns3P) is a phospholipid essential for autophagy, but the detailed distribution of PtdIns3P in the membrane of autophagosomes, autophagic bodies, and other organelles is unclear due to technical difficulties. In the present study, we examined PtdIns3P distribution in autophagic membranes with an electron microscopy method called the quick-freeze freeze-fracture replica labeling method (QF-FRL), which can define the distribution of membrane lipids at the nanometer scale. In this method, membranes are split into 2 leaflets so that membrane asymmetry, i.e., differences between the 2 leaflets, can be defined unambiguously. As a result, PtdIns3P in the yeast autophagosome was found to exist much more abundantly in the lumenal leaflet (i.e., the leaflet facing the space between the outer and inner autophagosomal membranes) than in the cytoplasmic leaflet. In contrast, PtdIns3P in the mammalian autophagosome was confined to the cytoplasmic leaflet, showing an opposite asymmetry from that found in yeast. In yeast deleted for 2 cytoplasmic PtdIns3P phosphatases, Ymr1 and Sjl3, PtdIns3P distributed in an equivalent density in the 2 leaflets of the autophagosome membrane, suggesting that the asymmetry in wild-type yeast is generated as a result of unilateral PtdIns3P hydrolysis. The contrasting PtdIns3P distribution revealed in the present study suggested that formation of autophagic membranes may proceed in different ways in yeast and mammals. 相似文献
8.
Phosphatidylinositol 3-phosphate (PtdIns3P) is a phospholipid essential for autophagy, but the detailed distribution of PtdIns3P in the membrane of autophagosomes, autophagic bodies, and other organelles is unclear due to technical difficulties. In the present study, we examined PtdIns3P distribution in autophagic membranes with an electron microscopy method called the quick-freeze freeze-fracture replica labeling method (QF-FRL), which can define the distribution of membrane lipids at the nanometer scale. In this method, membranes are split into 2 leaflets so that membrane asymmetry, i.e., differences between the 2 leaflets, can be defined unambiguously. As a result, PtdIns3P in the yeast autophagosome was found to exist much more abundantly in the lumenal leaflet (i.e., the leaflet facing the space between the outer and inner autophagosomal membranes) than in the cytoplasmic leaflet. In contrast, PtdIns3P in the mammalian autophagosome was confined to the cytoplasmic leaflet, showing an opposite asymmetry from that found in yeast. In yeast deleted for 2 cytoplasmic PtdIns3P phosphatases, Ymr1 and Sjl3, PtdIns3P distributed in an equivalent density in the 2 leaflets of the autophagosome membrane, suggesting that the asymmetry in wild-type yeast is generated as a result of unilateral PtdIns3P hydrolysis. The contrasting PtdIns3P distribution revealed in the present study suggested that formation of autophagic membranes may proceed in different ways in yeast and mammals. 相似文献
9.
Tensin is a protein confined at those discrete and specialized regions of the plasma membrane, known as focal adhesions. It contains, at the C-terminus, a phosphotyrosine binding (PTB) domain that can interact with the cytoplasmic tail of beta-integrins and is necessary for localization of the protein to cell-matrix adhesions. Here, we present the NMR solution structure of the PTB domain of tensin1. Moreover, through NMR binding studies, we demonstrate that the PTB domain of tensin1 is able to interact with phosphatidylinositol 4, 5-diphosphate (PtIns(4,5)P2) and phosphatidylinositol 4-phosphate (PtIns(4)P), presenting higher affinity for the diphosphorylated inositide. Chemical shift mapping studies reveal a putative PtIns(4,5)P2 binding region that is distinct from the predicted integrin beta-tail recognition site. Heteronuclear NOE experiments, recorded in absence and presence of PtIns(4,5)P2, indicate that the interaction with lipids decreases the flexibility of loop regions, predicted to be important for integrin binding, and thus, proposes a possible correlation between the two distinct binding events. Therefore, our studies suggest that capture of lipids by the PTB domain of tensin1 may play a role for the protein function at focal adhesions. 相似文献
10.
Shimamura M 《Carbohydrate research》2008,343(12):2010-2017
A novel invariant Valpha19-Jalpha33 T cell receptor alpha chain, first found in mammalian blood cells, was primarily expressed by natural killer T cell repertoire (Valpha19 NKT cell). Attempts have been made to find specific antigens for Valpha19 NKT cells. A series of alpha- and beta-glycosyl ceramides were synthesized and tested whether they had potential to stimulate the cells isolated from invariant Valpha19-Jalpha33 TCR transgenic mice (where the development of Valpha19 NKT cells is facilitated). Comprehensive examinations revealed substantial antigenic activity in alpha-ManCer that was presented by MR1, one of the MHC class Ib molecules. Next, naturally occurring and synthetic alpha-mannosyl glycolipids were further analyzed to determine structural requirements for natural ligands for Valpha19 NKT cells. As a result, alpha-mannosyl phosphatidyl inositols (PI) such as (alpha-Man)(2)-PI and alpha-Man-alpha-GlcNH(2)-PI (a partial structure of mycobacterial lipoarabinomannan and GPI-anchors) as well as alpha-ManCer derivatives were found to activate Valpha19 NKT cells in vivo and in vitro. Thus, Valpha19 NKT cells are possibly responsive to certain alpha-mannosyl glycolipids and may have roles in the innate and adaptative immune systems to protect against various antigens expressing alpha-mannosyl glycolipids and to regulate the adaptive immune system responding to the intracellular ligands. 相似文献
11.
In rat cardiac sarcolemmal membranes a phosphoinositide-specific phospholipase C (PLC) was found to be present. The enzyme hydrolysed exogenous [3H-]phosphatidylinositol 4,5-biphosphate ([3H-]PtdIns(4,5)P
2) in an optimized assay mixture containing 15 leg SL protein, 100 mM NaCl, 1 mM free Ca2+,14 mM Na-cholate and 20 AM [3H-]PtdIns (4,5)P
2 (400–500 dpm/gm-l) in 30 mM HEPES-Tris buffer (pH 7.0). The average specific activity was 9.14±0.55 nmol-mg–1·2.5 min–1. The addition of Mg2+ to the assay mixture did not change PLC activity but increased the relative amounts of dephosphorylated inositol products. In the absence of Na+ and at a low Ca2+ concentration (0.3 M), Mg2+ also enhanced the intraSL levels of PtdIns4P and PtdIns, and, moreover, inhibited PLC activity (IC500.07 mM). PtdIns4P seemd to be a good substrate for the rat SL PLC (23.07 ± 1.57 nmol·mg–1·2.5 min–1) whereas PtdIns was hydrolysed at a very low rate (0.36 ± 0.08 nmol·mg–1·2.5 min–1). Unlike PtdIns(4,5)P
2, PLC-dependent PtdIns4P and PtdIns hydrolysis was not inhibited by Ca2+ concentrations over 1 mM. The possibility of distinct isozymes being responsible for the different hydrolytic activities is discussed. (Mol Cell Biochem116: 27–31, 1992).Abbreviations DAG
sn-1,2-diacylglycerol
- EGTA
ethyleneglycol-O,O-bis(aminoethyl)-N,N,N,N,-tetraacetic acid
- Ins(1,4,5)P
3
inositol 1,4,5-trisphosphate
- InsP
inositol monophosphate (unidentified isomer)
- InsP
2
inositol bisphosphate (unidentified isomer)
- InsP
3
inositol trisphosphate (unidentified isomer)
- InsP
x
any inositol phosphate
- PLC
phospholipase C
- PtdIns
phosphatidylinositol
- PtdIns(4,5)P
2
phosphatidylinositol 4,5-bisphosphate
- PtdIns4P
phosphatidylinositol 4-monophosphate
- SL
sarcolemma 相似文献
12.
13.
Phosphatidylinositol 4,5-bisphosphate induces actin stress-fiber formation and inhibits membrane ruffling in CV1 cells 总被引:5,自引:0,他引:5
Yamamoto M Hilgemann DH Feng S Bito H Ishihara H Shibasaki Y Yin HL 《The Journal of cell biology》2001,152(5):867-876
Phosphatidylinositol 4,5 bisphosphate (PIP(2)) is widely implicated in cytoskeleton regulation, but the mechanisms by which PIP(2) effect cytoskeletal changes are not defined. We used recombinant adenovirus to infect CV1 cells with the mouse type I phosphatidylinositol phosphate 5-kinase alpha (PIP5KI), and identified the players that modulate the cytoskeleton in response to PIP(2) signaling. PIP5KI overexpression increased PIP(2) and reduced phosphatidylinositol 4 phosphate (PI4P) levels. It promoted robust stress-fiber formation in CV1 cells and blocked PDGF-induced membrane ruffling and nucleated actin assembly. Y-27632, a Rho-dependent serine/threonine protein kinase (ROCK) inhibitor, blocked stress-fiber formation and inhibited PIP(2) and PI4P synthesis in cells. However, Y-27632 had no effect on PIP(2) synthesis in lysates, although it inhibited PI4P synthesis. Thus, ROCK may regulate PIP(2) synthesis by controlling PI4P availability. PIP5KI overexpression decreased gelsolin, profilin, and capping protein binding to actin and increased that of ezrin. These changes can potentially account for the increased stress fiber and nonruffling phenotype. Our results establish the physiological role of PIP(2) in cytoskeletal regulation, clarify the relation between Rho, ROCK, and PIP(2) in the activation of stress-fiber formation, and identify the key players that modulate the actin cytoskeleton in response to PIP(2). 相似文献
14.
We previously found that pathophysiological concentrations (< or = 10 nm) of an amyloid beta protein (Abeta25-35) reduced the plasma membrane phosphatidylinositol monophosphate level in cultured rat hippocampal neurons with a decrease in phosphatidylinositol 4-monophosphate-dependent Cl- -ATPase activity. As this suggested an inhibitory effect of Abeta25-35 on plasma membrane phosphatidylinositol 4-kinase (PI4K) activity, in vitro effects of Abetas on PI4K activity was examined using rat brain subcellular fractions and recombinant human type II PI4K (PI4KII). Abeta25-35 (10 nm) inhibited PI4KII activity, but neither PI 3-kinase (PI3K) nor type III PI4K (PI4KIII) activity, in microsomal fractions, while 100 nm Abeta25-35 inhibited PI3K activity in mitochondrial fractions. In plasma membrane-rich fractions, Abetas (> 0.5 nm) dose-dependently inhibited PI4KII activity, the maximal inhibition to 77-87% of control being reached around 10 nm of Abetas without significant changes in apparent Km values for ATP and PI, suggesting non-competitive inhibition by Abetas. The inhibition by 10 nm Abeta25-35 was reversible. In recombinant human PI4KIIalpha, inhibition profiles of Abetas were similar to those in rat brain plasma membranes. Therefore, pathophysiological concentrations of Abetas directly and reversibly inhibited plasma membrane PI4KII activity, suggesting that plasma membrane PI4KII is a target of Abetas in the pathogenesis of Alzheimer's disease. 相似文献
15.
Kir6.2 channels linked to the green fluorescent protein (GFP) (Kir6. 2-GFP) have been expressed alone or with the sulfonylurea receptor SUR1 in HEK293 cells to study the regulation of K(ATP) channels by adenine nucleotides, phosphatidylinositol bisphosphate (PIP(2)), and phosphorylation. Upon excision of inside-out patches into a Ca(2+)- and MgATP-free solution, the activity of Kir6.2-GFP+SUR1 channels spontaneously ran down, first quickly within a minute, and then more slowly over tens of minutes. In contrast, under the same conditions, the activity of Kir6.2-GFP alone exhibited only slow rundown. Thus, fast rundown is specific to Kir6.2-GFP+SUR1 and involves SUR1, while slow rundown is a property of both Kir6.2-GFP and Kir6.2-GFP+SUR1 channels and is due, at least in part, to Kir6.2 alone. Kir6. 2-GFP+SUR1 fast phase of rundown was of variable amplitude and led to increased ATP sensitivity. Excising patches into a solution containing MgADP prevented this phenomenon, suggesting that fast rundown involves loss of MgADP-dependent stimulation conferred by SUR1. With both Kir6.2-GFP and Kir6.2-GFP+SUR1, the slow phase of rundown led to further increase in ATP sensitivity. Ca(2+) accelerated this process, suggesting a role for PIP(2) hydrolysis mediated by a Ca(2+)-dependent phospholipase C. PIP(2) could reactivate channel activity after a brief exposure to Ca(2+), but not after prolonged exposure. However, in both cases, PIP(2) reversed the increase in ATP sensitivity, indicating that PIP(2) lowers the ATP sensitivity by increasing P(o) as well as by decreasing the channel affinity for ATP. With Kir6.2-GFP+SUR1, slow rundown also caused loss of MgADP stimulation and sulfonylurea inhibition, suggesting functional uncoupling of SUR1 from Kir6.2-GFP. Ca(2+) facilitated the loss of sensitivity to MgADP, and thus uncoupling of the two subunits. The nonselective protein kinase inhibitor H-7 and the selective PKC inhibitor peptide 19-36 evoked, within 5-15 min, increased ATP sensitivity and loss of reactivation by PIP(2) and MgADP. Phosphorylation of Kir6.2 may thus be required for the channel to remain PIP(2) responsive, while phosphorylation of Kir6.2 and/or SUR1 is required for functional coupling. In summary, short-term regulation of Kir6.2+SUR1 channels involves MgADP, while long-term regulation requires PIP(2) and phosphorylation. 相似文献
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17.
The compartmentalization of cAMP by specifically targeted phosphodiesterases (PDEs) contributes to signal regulation in defined regions of cells. We previously demonstrated that the 20 N-terminal amino acids of Aplysia PDE4 (ApPDE4) long-form (L(N20)) and the two mutants of L(N20) were localized to the Golgi complex. However, the molecular mechanisms underlying the Golgi complex targeting of ApPDE4 long-form and its mutated forms are not clear. In the present study, we show that the Golgi complex targeting of L(N20/C14,15S)-enhanced green fluorescent protein (EGFP) was antimycin A-, phenylarsine oxide (PAO)-, and adenosine-sensitive, but insensitive to high concentrations of wortmannin. On the other hand, the Golgi complex targeting of L(N20)-EGFP and L(N20/C3,14S)-EGFP was antimycin A- and PAO-insensitive. These results suggest that the Golgi-localized lipid kinase protein, phosphatidylinositol 4-kinase type II alpha (PI4KIIα), the activity of which is inhibited by PAO and adenosine, but not by high concentrations of wortmannin, is likely involved in the Golgi complex targeting of L(N20/C14,15S)-EGFP. In addition, subcellular localization of L(N20/C14,15S)-EGFP, but not L(N20)-EGFP or L(N20/C3,14S)-EGFP, was changed from the Golgi complex only to both the endoplasmic reticulum (ER) and the Golgi complex following treatment with a palmitoylation inhibitor, 2-bromo palmitate. Taken together, our results suggest that L(N20/C14,15S)-EGFP, but not L(N20)-EGFP or L(N20/C3,14S)-EGFP, is localized to the Golgi complex in a PI4KII activity- and palmitoylation-dependent manner. Therefore, phosphatidylinositol 4-phosphate (PI4P) generated by PI4KIIα at the Golgi complex might play a key role in the Golgi complex targeting of L(N20/C14,15S)-EGFP. 相似文献
18.
19.
Summary The effect of pituitary human growth hormone (hGH) on the 32P-labelling of phosphoinositides and phosphatidic acid was studied in noradrenaline-stimulated rat adipocytes which were either responsive or non-responsive to the antilipolytic (insulin-like) effect of hGH. In cells responsive to the insulin-like effect of hGH, hormone treatment resulted in a marked increase of the 32P-labelling of phosphatidic acid and phosphatidyl inositol in the plasma membrane, high density microsomal, and low density microsomal fractions. The increased 32P-labelling most likely reflects an activation of phosphoinositide phospholipase C.Abbreviations IP3
Myo-Inositol 1,4,5-Triphosphate
- BSA
Bovine Serum Albumin
- HEPES
N-2Hydroxyethylpiperazine-N-2-Ethanesulphonic Acid
- hGH
Human Pituitary Growth Hormone
- LPC
Lysophosphatidylcholine
- LPE
Lysophosphatidylethanolamine
- LPI
Lysophosphatidylinositol
- PA
Phosphatidic Acid
- PC
Phosphatidylcholine
- PE
Phosphatidylethanolamine
- PI
Phosphatidylinositol
- PI
Phosphatidylinositol-4-Phosphate
- PIPZ
Phosphatidylinositol-4,5-Diphosphate
- PS
Phosphatidylserine 相似文献
20.
Lipid associated calcium ionophores in islet cell plasma membrane following glucose stimulation 总被引:1,自引:0,他引:1
The effect of glucose exposure on lipid associated calcium ionophoretic activity was measured in cultured neonatal rat pancreatic islet cells using two model systems. The first measured the ability of a lipid extract of islet cells to facilitate calcium transfer from an aqueous to organic phase and thus detected lipids which transfer calcium in the manner of authentic ionophores or which chelate the ion. In this system glucose stimulation was followed by an increase in total cell ionophoretic activity and a decrease in the activity associated with the plasma membrane. The second system measured the transfer of calcium across an artificial phospholipid membrane and detected authentic ionophoretic activity. In this model an increase in total ionophoretic activity was again seen following glucose but there was no change in the ionophoretic activity of a plasma membrane extract. The results indicate that the lipid modifications which accompany glucose-induced insulin release may alter cellular calcium stores by decreasing lipid bound calcium at the plasma membrane and increasing the capacity for calcium ionophoresis at intracellular sites. 相似文献