NMR Analyses of the Interaction between the FYVE Domain of Early Endosome Antigen 1 (EEA1) and Phosphoinositide Embedded in a Lipid Bilayer

Phosphoinositides (PIs) are crucial lipid components of membranes and are involved in a number of cellular processes through interactions with their effector proteins. Recently, we have established a lipid-protein nanoscale bilayer (nanodisc) containing PIs, hereafter referred to as PI-nanodisc and demonstrated that it could be used for both qualitative and quantitative evaluations of protein-membrane interactions. Here, we report further NMR analyses for obtaining structural insights at the residue-specific level between PI-binding effector protein and PI-nanodisc, using the FYVE domain of early endosome antigen 1 (EEA1), denoted as EEA1 FYVE, and PI(3)P-nanodisc as a model system. We performed a combination of the NMR analyses including chemical shift perturbation, transferred cross-saturation, and paramagnetic relaxation enhancement experiments. These enabled an identification of the interaction surface, structural change, and relative orientation of EEA1 FYVE to the PI(3)P-incorporated lipid bilayer, substantiating that NMR analyses of protein-membrane interactions using nanodisc makes it possible to show the residue-specific interactions in the lipid bilayer environment.     

Investigation into the mechanism regulating MRP localization

The major PKC substrates MARCKS and MacMARCKS (MRP) are membrane-binding proteins implicated in cell spreading, integrin activation and exocytosis. According to the myristoyl-electrostatic switch model the co-operation between the myristoyl moiety and the positively charged effector domain (ED) is an essential mechanism by which proteins bind to membranes. Loss of the electrostatic interaction between the ED and phospholipids, such as Ptdins(4,5)P2, results in the translocation of such proteins to the cytoplasm. While this model has been extensively tested for the binding of MARCKS far less is known about the mechanisms regulating MRP localization. We demonstrate that after phosphorylation, MRP is relocated to the intracellular membranes of late endosomes and lysosomes. MRP binds to all membranes via its myristoyl moiety, but for its localization at the plasma membrane the ED is also required. Although the ED of MRP can bind to Ptdins(4,5)P2 in vitro, this binding is not essential for its retention at or targeting to the plasma membrane. We conclude that the co-operation between the myristoyl moiety and the ED is not required for the binding to membranes in general but that it is essential for the targeting of MRP to the plasma membrane in a Ptdins(4,5)P2-independent manner.     

Identification and Structural Characterization of a Legionella Phosphoinositide Phosphatase

Bacterial pathogen Legionella pneumophila is the causative agent of Legionnaires'' disease, which is associated with intracellular replication of the bacteria in macrophages of human innate immune system. Recent studies indicate that pathogenic bacteria can subvert host cell phosphoinositide (PI) metabolism by translocated virulence effectors. However, in which manner Legionella actively exploits PI lipids to benefit its infection is not well characterized. Here we report that L. pneumophila encodes an effector protein, named SidP, that functions as a PI-3-phosphatase specifically hydrolyzing PI(3)P and PI(3,5)P₂ in vitro. This activity of SidP rescues the growth phenotype of a yeast strain defective in PI(3)P phosphatase activity. Crystal structure of SidP orthologue from Legionella longbeachae reveals that this unique PI-3-phosphatase is composed of three distinct domains: a large catalytic domain, an appendage domain that is inserted into the N-terminal portion of the catalytic domain, and a C-terminal α-helical domain. SidP has a small catalytic pocket that presumably provides substrate specificity by limiting the accessibility of bulky PIs with multiple phosphate groups. Together, our identification of a unique family of Legionella PI phosphatases highlights a common scheme of exploiting host PI lipids in many intracellular bacterial pathogen infections.     

Noradrenaline Stimulation Unbalances the Phosphoinositide Cycle in Rat Cerebral Cortical Slices

Abstract: Muscarinic cholinergic and α₁-adrenoceptor-mediated stimulation of phosphoinositide hydrolysis in rat cerebral cortex were compared by measuring carbachol- and noradrenaline-induced accumulation of various intermediates of the phosphoinositide cycle. Unlike carbachol, noradrenaline in the presence of guanosine 5'- O -(3-thiotriphosphate) did not stimulate phospholipase C activity in brain cortical membranes. In cortical slices, the efficacy of noradrenaline to stimulate accumulation of ³H-inositol phosphates and [³²P]phosphatidic acid was 2.5 to threefold that of carbachol. However, noradrenaline was less effective than carbachol in stimulating accumulation of [³H]CDP-diacylglycerol and resynthesis of phosphatidylinositol. This was not due to calcium inhibition of CTP:phosphatidate cytidyltransferase or to different lithium requirements for carbachol- and noradrenaline-stimulated accumulation of [³H]CDP-diacylglycerol. The noradrenaline-induced unbalance of the phosphoinositide cycle, which was most apparent at relatively high concentrations of calcium (2.5 m M ) in the incubation buffer, was qualitatively reproduced with ionomycin. The use of the α_1a-subtype-selective adrenoceptor antagonists WB4101 and 5-methylurapidil revealed a single α_1a-like component mediating the effects of noradrenaline. Our results suggest that the primary mechanism for phospholipase C activation by brain α₁ adrenoceptors involves an increase in intracellular calcium concentration.     

Lithium Enhances Muscarinic Receptor–Stimulated CDP-Diacylglycerol Formation in Inositol-Depleted SK-N-SH Neuroblastoma Cells

Abstract: The psychotherapeutic action of Li⁺ in brain has been proposed to result from the depletion of cellular inositol secondary to its block of inositol monophosphatase. This action is thought to slow phosphoinositide resynthesis, thereby attenuating stimulated phosphoinositidase-mediated signal transduction in affected cells. In the present study, the effect of Li⁺ on muscarinic receptor–stimulated formation of the immediate precursor of phosphatidylinositol, CDP-diacylglycerol (CDP-DAG), has been examined in human SK-N-SH neuroblastoma cells that have been cultured under conditions that alter the cellular content of myo-inositol. Resting neuroblastoma cells, like brain cells in vivo, were found to concentrate inositol from the culture medium, achieving an intracellular level of 60.0 ± 4 nmol/mg of protein. The addition of carbachol to [³H]cytidine-prelabeled cells elicited a four- to fivefold increase in the accumulation of labeled CDP-DAG. This stimulated formation of [³H]CDP-DAG was completely blocked by the addition of 10 μM atropine, was not dependent on the presence of Li⁺, nor was it affected by co-incubation with myo-inositol. This result was in sharp contrast to findings in rat brain slices, in which carbachol-stimulated formation of [³H]CDP-DAG was potentiated ～ 10-fold by Li⁺ and substantially reduced by coincubation with inositol. The formation of [³H]CDP-DAG in labeled SK-N-SH cells by carbachol was both concentration and time dependent. The order of efficacy of muscarinic ligands in stimulating [³H]-CDP-DAG accumulation paralleled that established in these cells for inositol phosphate accumulation, i.e., carbachol ≥ oxotremorine-M > bethanecol ≥ arecoline > oxotremorine > pilocarpine. Extended culture of the SK-N-SH cells in an inositol-free chemically defined growth medium progressively reduced the intracellular inositol content to <5 nmol/mg of protein, a level comparable with that seen in cortical slices. In these inositol-depleted cells, Li⁺ potentiated carbachol-stimulated [³H]CDP-DAG formation, and this effect was completely reversed by coincubation with inositol (EC₅₀ 0.2 mM). The present study thus demonstrates, in the same cultured cell line, the effects of normal and reduced intracellular inositol levels on the ability of Li⁺ to attenuate phosphoinositide resynthesis, as inferred from [³H]CDP-DAG accumulation. The results indicate that Li⁺ can lead to a slowing of stimulated phosphoinositide turnover in neuroblastoma cells, provided that the intracellular inositol content has been significantly reduced.     

Phospholipase Cγ1 in Bovine Rod Outer Segments: Immunolocalization and Light-Dependent Binding to Membranes

Abstract: We have investigated the isozymes of a phosphoinositide-specific phospholipase C (PLC) in bovine retina using several monoclonal antisera to PLCβ₁, γ₁, and δ₁. Immunoblot analysis showed that all three isozymes were present in the retina. Immunocytochemical localization in frozen bovine retina sections showed that PLCγ₁ was present in the photoreceptor cell layer, outer plexiform cell layer, inner plexiform cell layer, and ganglion cell layer. Immunoreaction within the photoreceptor cell layer was dependent on dark/light adaptation state of retinas. Immunoblot analysis of rod outer segments (ROS) with monoclonal or polyclonal antibodies to PLCγ₁ showed the presence of an immunoreactive band of 140 kDa. ROS prepared from retinas light-adapted in vitro had more PLCγ₁ on immunoblots than ROS from dark-adapted retinas. PLC enzyme activity in ROS from light-adapted retinas was 69 and 46% higher than ROS from dark-adapted retinas, when assayed in the presence and absence of ATP, respectively. This increase in enzyme activity was observed at [Ca²⁺]_free between 0.32 and 100 µ M . These results demonstrate the presence of PLCγ₁ in bovine ROS and show that ROS prepared from light-adapted retinas are enriched in this isozyme, suggesting that light may promote the binding of this isozyme to bleached ROS membranes.     

Activation of Adenosine A2 Receptors Stimulates Phosphoinositide Metabolism in Rat Peripheral Nerve

Abstract: The effects of adenosine analogues on phosphoinositide metabolism in rat sciatic nerve were examined. Sciatic nerve segments were prelabeled with [³H]-cytidine and incubated in the presence of LiCl and varying concentrations of adenosine analogues. The formation of [³H]cytidine monophosphate phosphatidic acid ([³H]-CMP-PA) was determined as an index of phosphoinositide breakdown. Liponucleotide accumulation was elevated significantly in the presence of 5'- N -ethylcarboxamidoadenosine (NECA), a nonselective analogue, and two different A₂-selective analogues, N ⁶-[2-(3,5-dimethoxyphenyl)-2-(2-methylphenyl)ethyl]adenosine and 2- p -(2-carboxyethyl)phenethylamino-NECA (CGS 21680), but not by N ⁶-cyclopentyladenosine, an A₁-selective analogue. The stimulatory action of CGS 21680 was blocked by the A₂-selective adenosine receptor antagonists 3,7-dimethyl-1-propargylxanthine (DMPX) and 1,3-dipropyl-7-methylxanthine. Inositol phosphate formation was also stimulated to a comparable degree by CGS 21680 and this response was antagonized by DMPX. Carbamylcholine, which was previously shown to stimulate phosphoinositide breakdown, also enhanced the accumulation of CMP-PA. When adenosine analogues and carbamylcholine were simultaneously present, their effects were additive. Taken together, these data suggest that an adenosine receptor, possibly of the A₂ subtype, is coupled to enhanced phosphoinositide hydrolysis in peripheral nerve. However, adenosine-receptor activation does not appear to modulate phosphoinositide hydrolysis stimulated via muscarinic receptors.     

Cloning of CDP-Diacylglycerol Synthase from a Human Neuronal Cell Line

Abstract: A critical step in the supply of substrate for the phosphoinositide signal transduction pathway is the formation of the liponucleotide intermediate, CDP-diacylglycerol, catalyzed by CDP-diacylglycerol synthase. Further insight into the regulation of phosphoinositide biosynthesis was sought by cloning of the gene for the vertebrate enzyme. Sequence of the corresponding gene from Drosophila was used to prepare a probe for screening of a human neuronal cell cDNA library. A cDNA was isolated with a predicted open reading frame of 1,332 bases, encoding a protein of 51 kDa. The amino acid sequence showed 50% identity (75% similarity) to that of Drosophila eye CDP-diacylglycerol synthase and substantial similarity to the Saccharomyces cerevisiae and Escherichia coli homologues. Northern blot analysis, with human cDNA riboprobes, suggested that the corresponding mRNA was expressed in all human tissues examined. Expression of the human cDNA in COS cells resulted in a more than fourfold increase in CDP-diacylglycerol synthase activity. Knowledge of the sequence of vertebrate CDP-diacylglycerol synthase should facilitate further investigations into its regulation and the possible existence of distinct isoforms.     

Nerve Growth Factor Potentiates the Hormone-Stimulated Intracellular Accumulation of Inositol Phosphates and Ca2+ in Rat PC12 Pheochromocytoma Cells: Comparison with the Effect of Epidermal Growth Factor

The effects of nerve growth factor (NGF) and epidermal growth factor (EGF) on the intracellular accumulation of inositol phosphates and on cytosolic free Ca2+ concentrations were studied in rat PC12 pheochromocytoma cells. Both NGF and EGF potentiate in these cells the increase in the accumulation of inositol phosphates that is elicited by bradykinin and carbachol. A corresponding potentiation was also found for the agonist-induced increase of cytosolic Ca2+ concentrations. The effect of NGF, but not that of EGF, is abolished when the cells are preincubated with 5'-deoxy-5'-methylthioadenosine, an inhibitor of S-adenosylhomocysteine hydrolase. These results suggest that an increased response to hormones, which act via phosphoinositide-derived second messengers, may be important in the mechanism of action of NGF and EGF.     

Concerted CMP-Dependent [3H]Inositol Labeling of Phosphoinositides and Agonist Activation of Phospholipase C in Rat Brain Cortical Membranes

[3H]Inositol ([3H]Ins) labeling of phosphoinositides was studied in rat brain cortical membranes. [3H]Ins was incorporated into a common lipid pool through both CMP-dependent and independent mechanisms. These are as follows: (1) a reverse reaction catalyzed by phosphatidyl-inositol (PtdIns) synthase, and (2) the reaction performed by the PtdIns headgroup exchange enzyme, respectively. Membrane phosphoinositides prelabeled in either CMP-dependent or independent fashions were hydrolyzed by guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S)- and carbachol-stimulated phospholipase C. Unlike CMP-dependent labeling, however, CMP-independent incorporation of [3H]Ins into lipids was inhibited by 1 mM (0.04%) sodium deoxycholate. Thus, when PtdIns labeling and phospholipase C stimulation were studied in a concerted fashion, [3H]Ins was incorporated into lipids primarily through the PtdIns synthase-catalyzed reaction because of the presence of deoxycholate required to observe carbachol-stimulation of phospholipase C. Little direct breakdown of [3H]PtdIns was detected because production of myo-[3H]inositol 1-monophosphate was minimal and myo-[3H]inositol 1,4-bisphosphate was the predominant product. Although PtdIns labeling and 3H-polyphosphoinositide formation were unaffected by GTP gamma S and carbachol and had no or little lag period, GTP gamma S- and carbachol-stimulated appearance of 3H-Ins phosphates exhibited an appreciable lag (10 min). Also, flux of label from [3H]Ins to 3H-Ins phosphates was restricted to a narrow range of free calcium concentrations (10-300 nM). These results show the concerted activities of PtdIns synthase, PtdIns 4-kinase, and phospholipase C, and constitute a simple assay for guanine nucleotide-dependent agonist stimulation of phospholipase C in a brain membrane system using [3H]Ins as labeled precursor.