Phospholipase C-ε Regulates Epidermal Morphogenesis in Caenorhabditis elegans

Migration of cells within epithelial sheets is an important feature of embryogenesis and other biological processes. Previous work has demonstrated a role for inositol 1,4,5-trisphosphate (IP₃)-mediated calcium signalling in the rearrangement of epidermal cells (also known as hypodermal cells) during embryonic morphogenesis in Caenorhabditis elegans. However the mechanism by which IP₃ production is stimulated is unknown. IP₃ is produced by the action of phospholipase C (PLC). We therefore surveyed the PLC family of C. elegans using RNAi and mutant strains, and found that depletion of PLC-1/PLC-ε produced substantial embryonic lethality. We used the epithelial cell marker ajm-1::gfp to follow the behaviour of epidermal cells and found that 96% of the arrested embryos have morphogenetic defects. These defects include defective ventral enclosure and aberrant dorsal intercalation. Using time-lapse confocal microscopy we show that the migration of the ventral epidermal cells, especially of the leading cells, is slower and often fails in plc-1(tm753) embryos. As a consequence plc-1 loss of function results in ruptured embryos with a Gex phenotype (gut on exterior) and lumpy larvae. Thus PLC-1 is involved in the regulation of morphogenesis. Genetic studies using gain- and loss-of-function alleles of itr-1, the gene encoding the IP₃ receptor in C. elegans, demonstrate that PLC-1 acts through ITR-1. Using RNAi and double mutants to deplete the other PLCs in a plc-1 background, we show that PLC-3/PLC-γ and EGL-8/PLC-β can compensate for reduced PLC-1 activity. Our work places PLC-ε into a pathway controlling epidermal cell migration, thus establishing a novel role for PLC-ε.     

Calcium Release at Fertilization in Starfish Eggs Is Mediated by Phospholipase Cγ

Although inositol trisphosphate (IP₃) functions in releasing Ca²⁺ in eggs at fertilization, it is not known how fertilization activates the phospholipase C that produces IP₃. To distinguish between a role for PLCγ, which is activated when its two src homology-2 (SH2) domains bind to an activated tyrosine kinase, and PLCβ, which is activated by a G protein, we injected starfish eggs with a PLCγ SH2 domain fusion protein that inhibits activation of PLCγ. In these eggs, Ca²⁺ release at fertilization was delayed, or with a high concentration of protein and a low concentration of sperm, completely inhibited. The PLCγSH2 protein is a specific inhibitor of PLCγ in the egg, since it did not inhibit PLCβ activation of Ca²⁺ release initiated by the serotonin 2c receptor, or activation of Ca²⁺ release by IP₃ injection. Furthermore, injection of a PLCγ SH2 domain protein mutated at its phosphotyrosine binding site, or the SH2 domains of another protein (the phosphatase SHP2), did not inhibit Ca²⁺ release at fertilization. These results indicate that during fertilization of starfish eggs, activation of phospholipase Cγ by an SH2 domain-mediated process stimulates the production of IP₃ that causes intracellular Ca²⁺ release.     

Phospholipase C-�� Regulates Epidermal Morphogenesis in Caenorhabditis elegans

Migration of cells within epithelial sheets is an important feature of embryogenesis and other biological processes. Previous work has demonstrated a role for inositol 1,4,5-trisphosphate (IP₃)-mediated calcium signalling in the rearrangement of epidermal cells (also known as hypodermal cells) during embryonic morphogenesis in Caenorhabditis elegans. However the mechanism by which IP₃ production is stimulated is unknown. IP₃ is produced by the action of phospholipase C (PLC). We therefore surveyed the PLC family of C. elegans using RNAi and mutant strains, and found that depletion of PLC-1/PLC-ε produced substantial embryonic lethality. We used the epithelial cell marker ajm-1::gfp to follow the behaviour of epidermal cells and found that 96% of the arrested embryos have morphogenetic defects. These defects include defective ventral enclosure and aberrant dorsal intercalation. Using time-lapse confocal microscopy we show that the migration of the ventral epidermal cells, especially of the leading cells, is slower and often fails in plc-1(tm753) embryos. As a consequence plc-1 loss of function results in ruptured embryos with a Gex phenotype (gut on exterior) and lumpy larvae. Thus PLC-1 is involved in the regulation of morphogenesis. Genetic studies using gain- and loss-of-function alleles of itr-1, the gene encoding the IP₃ receptor in C. elegans, demonstrate that PLC-1 acts through ITR-1. Using RNAi and double mutants to deplete the other PLCs in a plc-1 background, we show that PLC-3/PLC-γ and EGL-8/PLC-β can compensate for reduced PLC-1 activity. Our work places PLC-ε into a pathway controlling epidermal cell migration, thus establishing a novel role for PLC-ε.     

In vivo imaging of C. elegans ASH neurons: cellular response and adaptation to chemical repellents

ASH sensory neurons are required in Caenorhabditis elegans for a wide range of avoidance behaviors in response to chemical repellents, high osmotic solutions and nose touch. The ASH neurons are therefore hypothesized to be polymodal nociceptive neurons. To understand the nature of polymodal sensory response and adaptation at the cellular level, we expressed the calcium indicator protein cameleon in ASH and analyzed intracellular Ca(2+) responses following stimulation with chemical repellents, osmotic shock and nose touch. We found that a variety of noxious stimuli evoked strong responses in ASH including quinine, denatonium, detergents, heavy metals, both hyper- and hypo-osmotic shock and nose touch. We observed that repeated chemical stimulation led to a reversible reduction in the magnitude of the sensory response, indicating that adaptation occurs within the ASH sensory neuron. A key component of ASH adaptation is GPC-1, a G-protein gamma-subunit expressed specifically in chemosensory neurons. We hypothesize that G-protein gamma-subunit heterogeneity provides a mechanism for repellent-specific adaptation, which could facilitate discrimination of a variety of repellents by these polymodal sensory neurons.     

Structural Studies and Protein Engineering of Inositol Phosphate Multikinase

Inositol phosphates (IPs) regulate vital processes in eukaryotes, and their production downstream of phospholipase C activation is controlled through a network of evolutionarily conserved kinases and phosphatases. Inositol phosphate multikinase (IPMK, also called Ipk2 and Arg82) accounts for phosphorylation of IP₃ to IP₅, as well as production of several other IP molecules. Here, we report the structure of Arabidopsis thaliana IPMKα at 2.9 Å and find it is similar to the yeast homolog Ipk2, despite 17% sequence identity, as well as the active site architecture of human IP₃ 3-kinase. Structural comparison and substrate modeling were used to identify a putative basis for IPMK selectivity. To test this model, we re-engineered binding site residues predicted to have restricted substrate specificity. Using steady-state kinetics and in vivo metabolic labeling studies in modified yeast strains, we observed that K117W and K117W:K121W mutants exhibited nearly normal 6-kinase function but harbored significantly reduced 3-kinase activity. These mutants complemented conditional nutritional growth defects observed in ipmk null yeast and, remarkably, suppressed lethality observed in ipmk null flies. Our data are consistent with the hypothesis that IPMK 6-kinase activity and production of Ins(1,4,5,6)P₄ are critical for cellular signaling. Overall, our studies provide new insights into the structure and function of IPMK and utilize a synthetic biological approach to redesign inositol phosphate signaling pathways.     

Odorant-stimulated phosphoinositide signaling in mammalian olfactory receptor neurons

Recent evidence has revived interest in the idea that phosphoinositides (PIs) may play a role in signal transduction in mammalian olfactory receptor neurons (ORNs). To provide direct evidence that odorants indeed activate PI signaling in ORNs, we used adenoviral vectors carrying two different fluorescently tagged probes, the pleckstrin homology (PH) domains of phospholipase Cδ1 (PLCδ1) and the general receptor of phosphoinositides (GRP1), to monitor PI activity in the dendritic knobs of ORNs in vivo. Odorants mobilized PI(4,5)P₂/IP₃ and PI(3,4,5)P₃, the substrates and products of PLC and PI3K. We then measured odorant activation of PLC and PI3K in olfactory ciliary-enriched membranes in vitro using a phospholipid overlay assay and ELISAs. Odorants activated both PLC and PI3K in the olfactory cilia within 2 s of odorant stimulation. Odorant-dependent activation of PLC and PI3K in the olfactory epithelium could be blocked by enzyme-specific inhibitors. Odorants activated PLC and PI3K with partially overlapping specificity. These results provide direct evidence that odorants indeed activate PI signaling in mammalian ORNs in a manner that is consistent with the idea that PI signaling plays a role in olfactory transduction.     

Type 1 IP3 receptors activate BKCa channels via local molecular coupling in arterial smooth muscle cells

Plasma membrane large-conductance Ca²⁺-activated K⁺ (BK_Ca) channels and sarcoplasmic reticulum inositol 1,4,5-trisphosphate (IP₃) receptors (IP₃Rs) are expressed in a wide variety of cell types, including arterial smooth muscle cells. Here, we studied BK_Ca channel regulation by IP₃ and IP₃Rs in rat and mouse cerebral artery smooth muscle cells. IP₃ activated BK_Ca channels both in intact cells and in excised inside-out membrane patches. IP₃ caused concentration-dependent BK_Ca channel activation with an apparent dissociation constant (K_d) of ∼4 µM at physiological voltage (−40 mV) and intracellular Ca²⁺ concentration ([Ca²⁺]_i; 10 µM). IP₃ also caused a leftward-shift in BK_Ca channel apparent Ca²⁺ sensitivity and reduced the K_d for free [Ca²⁺]_i from ∼20 to 12 µM, but did not alter the slope or maximal P_o. BAPTA, a fast Ca²⁺ buffer, or an elevation in extracellular Ca²⁺ concentration did not alter IP₃-induced BK_Ca channel activation. Heparin, an IP₃R inhibitor, and a monoclonal type 1 IP₃R (IP₃R1) antibody blocked IP₃-induced BK_Ca channel activation. Adenophostin A, an IP₃R agonist, also activated BK_Ca channels. IP₃ activated BK_Ca channels in inside-out patches from wild-type (IP₃R1^+/+) mouse arterial smooth muscle cells, but had no effect on BK_Ca channels of IP₃R1-deficient (IP₃R1^−/−) mice. Immunofluorescence resonance energy transfer microscopy indicated that IP₃R1 is located in close spatial proximity to BK_Ca α subunits. The IP₃R1 monoclonal antibody coimmunoprecipitated IP₃R1 and BK_Ca channel α and β1 subunits from cerebral arteries. In summary, data indicate that IP₃R1 activation elevates BK_Ca channel apparent Ca²⁺ sensitivity through local molecular coupling in arterial smooth muscle cells.     

Selective down-regulation of IP3receptor subtypes by caspases and calpain during TNF α -induced apoptosis of human T-lymphoma cells

There are at least three types of inositol 1,4,5-trisphosphate receptor (IP₃R) [IP₃-gated Ca²⁺channels], which are expressed in different cell types and mammalian tissues. In this study, we have identified three IP₃R subtypes in human Jurkat T-lymphoma cells. All three subtypes have a molecular mass of about 260 kDa, and display Ca²⁺channel properties in an IP₃-dependent manner. We have also demonstrated that TNFα promotes the activity of different proteases (e.g. caspase-8, caspase-3 and calpain), alters the TCR-mediated Ca²⁺response and subsequently induces apoptosis in Jurkat cells. During the first 6 h of incubation with TNFα, several IP₃R subtype-related changes occur (e.g. proteolysis of IP₃R subtypes, inhibition of IP₃binding and impairment of IP₃-mediated Ca²⁺flux) concomitantly with an elevation of protease (caspase-8, caspase-3 and calpain) activity. Furthermore, the caspase inhibitor, Z-VAD-fmk, significantly reduces TNFα-mediated perturbation of IP₃R1 and IP₃R2 (but not IP₃R3) function; whereas the calpain inhibitor I, ALLN, is capable of blocking the inhibitory effect of TNFα on IP₃R3 function. These findings suggest that IP₃R1 and IP₃R2 serve as cellular substrates for caspases, and IP₃R3 is a substrate for calpain. We propose that the selective down-regulation of IP₃R subtype-mediated Ca²⁺function by caspase-dependent and calpain-sensitive mechanisms may be responsible for the early onset of the apoptotic signal by TNFα in human T-cells.     

Phospholipase C-β1 and β4 Contribute to Non-Genetic Cell-to-Cell Variability in Histamine-Induced Calcium Signals in HeLa Cells

A uniform extracellular stimulus triggers cell-specific patterns of Ca²⁺ signals, even in genetically identical cell populations. However, the underlying mechanism that generates the cell-to-cell variability remains unknown. We monitored cytosolic inositol 1,4,5-trisphosphate (IP₃) concentration changes using a fluorescent IP₃ sensor in single HeLa cells showing different patterns of histamine-induced Ca²⁺ oscillations in terms of the time constant of Ca²⁺ spike amplitude decay and the Ca²⁺ oscillation frequency. HeLa cells stimulated with histamine exhibited a considerable variation in the temporal pattern of Ca²⁺ signals and we found that there were cell-specific IP₃ dynamics depending on the patterns of Ca²⁺ signals. RT-PCR and western blot analyses showed that phospholipase C (PLC)-β1, -β3, -β4, -γ1, -δ3 and -ε were expressed at relatively high levels in HeLa cells. Small interfering RNA-mediated silencing of PLC isozymes revealed that PLC-β1 and PLC-β4 were specifically involved in the histamine-induced IP₃ increases in HeLa cells. Modulation of IP₃ dynamics by knockdown or overexpression of the isozymes PLC-β1 and PLC-β4 resulted in specific changes in the characteristics of Ca²⁺ oscillations, such as the time constant of the temporal changes in the Ca²⁺ spike amplitude and the Ca²⁺ oscillation frequency, within the range of the cell-to-cell variability found in wild-type cell populations. These findings indicate that the heterogeneity in the process of IP₃ production, rather than IP₃-induced Ca²⁺ release, can cause cell-to-cell variability in the patterns of Ca²⁺ signals and that PLC-β1 and PLC-β4 contribute to generate cell-specific Ca²⁺ signals evoked by G protein-coupled receptor stimulation.     

Apical Vesicles Bearing Inositol 1,4,5-trisphosphate Receptors in the Ca2+Initiation Site of Ductal Epithelium of Submandibular Gland

In polarized epithelial cells, agonists trigger Ca²⁺ waves and oscillations. These patterns may be caused by the compartmentalization of inositol 1,4,5-trisphosphate (IP₃)-sensitive Ca²⁺ pools into specific regions. We have investigated the relationship between the distribution of IP₃ receptors (IP₃Rs) and the spatiotemporal pattern of Ca²⁺ signaling in the duct cells of the rat submandibular gland (SMG). Using immunofluorescence, although labeling was somewhat heterogeneous, the IP₃Rs were colocalized to the apical pole of the duct cells. Immunoelectron microscopy identified small apical vesicles bearing IP₃R2 in some types of duct cells. Real-time confocal imaging of intact ducts demonstrated that, after carbachol stimulation, an initial Ca²⁺ spike occurred in the apical region. Subsequently, repetitive Ca²⁺ spikes spread from the apical to the middle cytoplasm. These apical Ca²⁺ initiation sites were found only in some “pioneer cells,” rather than in all duct cells. We performed both Ca²⁺ imaging and immunofluorescence on the same ducts and detected the strongest immunosignals of IP₃R2 in the Ca²⁺ initiation sites of the pioneer cells. The subcellular localization and expression level of IP₃Rs correlated strongly with the spatiotemporal nature of the intracellular Ca²⁺ signal and distinct Ca²⁺ responses among the rat SMG duct cells.     

Dual Functions for the Schizosaccharomyces pombe Inositol Kinase Ipk1 in Nuclear mRNA Export and Polarized Cell Growth

The inositol 1,3,4,5,6-pentakisphosphate (IP₅) 2-kinase (Ipk1) catalyzes the production of inositol hexakisphosphate (IP₆) in eukaryotic cells. Previous studies have shown that IP₆ is required for efficient nuclear mRNA export in the budding yeast Saccharomyces cerevisiae. Here, we report the first functional analysis of ipk1⁺ in Schizosaccharomyces pombe. S. pombe Ipk1 (SpIpk1) is unique among Ipk1 orthologues in that it harbors a novel amino (N)-terminal domain with coiled-coil structural motifs similar to those of BAR (Bin-amphiphysin-Rvs) domain proteins. Mutants with ipk1⁺ deleted (ipk1Δ) had mRNA export defects as well as pleiotropic defects in polarized growth, cell morphology, endocytosis, and cell separation. The SpIpk1 catalytic carboxy-terminal domain was required to rescue these defects, and the mRNA export block was genetically linked to SpDbp5 function and, likely, IP₆ production. However, the overexpression of the N-terminal domain alone also inhibited these functions in wild-type cells. This revealed a distinct noncatalytic function for the N-terminal domain. To test for connections with other inositol polyphosphates, we also analyzed whether the loss of asp1⁺ function, encoding an IP₆ kinase downstream of Ipk1, had an effect on ipk1Δ cells. The asp1Δ mutant alone did not block mRNA export, and its cell morphology, polarized growth, and endocytosis defects were less severe than those of ipk1Δ cells. Moreover, ipk1Δ asp1Δ double mutants had altered inositol polyphosphate levels distinct from those of the ipk1Δ mutant. This suggested novel roles for asp1⁺ upstream of ipk1⁺. We propose that IP₆ production is a key signaling linchpin for regulating multiple essential cellular processes.     

Regulation of Drosophila TRPC channels by protein and lipid interactions

The Drosophila TRPC channels TRP and TRPL are the founding members of the TRP superfamily of ion channels, proteins likely to be important components of calcium influx pathways. The activation of these channels in the context of fly phototransduction is one of the few in vivo models for TRPC channel activation and has served as a paradigm for understanding TRPC function. TRP and TRPL are activated by G-protein coupled PI(4,5)P₂ hydrolysis through a mechanism in which IP₃ receptor mediated calcium release seems dispensable. Recent analysis has provided compelling evidence that the accurate turnover of PI(4,5)P₂ generated lipid messengers in essential for regulating TRP and TRPL activity. TRP channels also appear to exist in the context of a macromolecular complex containing key components involved in activation such as phospholipase Cβ and protein kinase C. This complex may be important for activation. The role of these protein and lipid elements in regulating TRP and TRPL activity is discussed in this review.     

'Trigger' events precede calcium puffs in Xenopus oocytes

The liberation of calcium ions sequestered in the endoplasmic reticulum through inositol 1,4,5-trisphosphate receptors/channels (IP₃Rs) results in a spatiotemporal hierarchy of calcium signaling events that range from single-channel openings to local Ca²⁺ puffs believed to arise from several to tens of clustered IP₃Rs to global calcium waves. Using high-resolution confocal linescan imaging and a sensitive Ca²⁺ indicator dye (fluo-4-dextran), we show that puffs are often preceded by small, transient Ca²⁺ elevations that we christen “trigger events”. The magnitude of triggers is consistent with their arising from the opening of a single IP₃ receptor/channel, and we propose that they initiate puffs by recruiting neighboring IP₃Rs within the cluster by a regenerative process of Ca²⁺-induced Ca²⁺ release. Puff amplitudes (fluorescence ratio change) are on average ~6 times greater than that of the triggers, suggesting that at least six IP₃Rs may simultaneously be open during a puff. Trigger events have average durations of ~12 ms, as compared to 19 ms for the mean rise time of puffs, and their spatial extent is ~3 times smaller than puffs (respective widths at half peak amplitude 0.6 and 1.6 μm). All these parameters were relatively independent of IP₃ concentration, although the proportion of puffs showing resolved triggers was greatest (~80%) at low [IP₃]. Because Ca²⁺ puffs constitute the building blocks from which cellular IP₃-mediated Ca²⁺ signals are constructed, the events that initiate them are likely to be of fundamental importance for cell signaling. Moreover, the trigger events provide a useful yardstick by which to derive information regarding the number and spatial arrangement of IP₃Rs within clusters.     

Gene Knock-Outs of Inositol 1,4,5-Trisphosphate Receptors Types 1 and 2 Result in Perturbation of Cardiogenesis

Background

Inositol 1,4,5-trisphosphate receptors (IP₃R1, 2, and 3) are intracellular Ca²⁺ release channels that regulate various vital processes. Although the ryanodine receptor type 2, another type of intracellular Ca²⁺ release channel, has been shown to play a role in embryonic cardiomyocytes, the functions of the IP₃Rs in cardiogenesis remain unclear.

Methodology/Principal Findings

We found that IP₃R1^−/−-IP₃R2^−/− double-mutant mice died in utero with developmental defects of the ventricular myocardium and atrioventricular (AV) canal of the heart by embryonic day (E) 11.5, even though no cardiac defect was detectable in IP₃R1^−/− or IP₃R2^−/− single-mutant mice at this developmental stage. The double-mutant phenotype resembled that of mice deficient for calcineurin/NFATc signaling, and NFATc was inactive in embryonic hearts from the double knockout-mutant mice. The double mutation of IP₃R1/R2 and pharmacologic inhibition of IP₃Rs mimicked the phenotype of the AV valve defect that result from the inhibition of calcineurin, and it could be rescued by constitutively active calcineurin.

Conclusions/Significance

Our results suggest an essential role for IP₃Rs in cardiogenesis in part through the regulation of calcineurin-NFAT signaling.     

Changes in phosphoinositide metabolism with days in culture affect signal transduction pathways in galdieria sulphuraria

The metabolism of phosphatidylinositol-4,5-bisphosphate (PIP₂) changed during the culture period of the thermoacidophilic red alga Galdieria sulphuraria. Seven days after inoculation, the amount of PIP₂ in the cells was 910 ± 100 pmol g⁻¹ fresh weight; by 12 d, PIP₂ levels increased to 1200 ± 150 pmol g⁻¹ fresh weight. In vitro assays indicated that phosphatidylinositol monophosphate (PIP) kinase specific activity increased from 75 to 230 pmol min⁻¹ mg⁻¹ protein between d 7 and 12. When G. sulphuraria cells were osmostimulated, transient increases of up to 4-fold could be observed in inositol-1,4,5-trisphosphate (IP₃) levels within 90 s, regardless of the age of the cells. In d-12 cells, the increase in IP₃ was preceded by a transient increase of up to 5-fold in specific PIP kinase activity, whereas no such increase was detected after osmostimulation of d-7 cells. The increase in PIP kinase activity before IP₃ signaling in d-12 cells indicates that there is an additional pathway for regulation of phosphoinositide metabolism after stimulation other than an initial activation of phospholipase C. Also, the rapid activation of PIP₂ biosynthesis in cells with already-high PIP₂ levels suggests that the PIP₂ present was not available for signal transduction. By comparing the response of the cells at d 7 and 12, we have identified two potentially distinct pools of PIP₂.     

IP3R2 levels dictate the apoptotic sensitivity of diffuse large B-cell lymphoma cells to an IP3R-derived peptide targeting the BH4 domain of Bcl-2

Disrupting inositol 1,4,5-trisphosphate (IP₃) receptor (IP₃R)/B-cell lymphoma 2 (Bcl-2) complexes using a cell-permeable peptide (stabilized TAT-fused IP₃R-derived peptide (TAT-IDP^S)) that selectively targets the BH4 domain of Bcl-2 but not that of B-cell lymphoma 2-extra large (Bcl-Xl) potentiated pro-apoptotic Ca²⁺ signaling in chronic lymphocytic leukemia cells. However, the molecular mechanisms rendering cancer cells but not normal cells particularly sensitive to disrupting IP₃R/Bcl-2 complexes are poorly understood. Therefore, we studied the effect of TAT-IDP^S in a more heterogeneous Bcl-2-dependent cancer model using a set of ‘primed to death'' diffuse large B-cell lymphoma (DL-BCL) cell lines containing elevated Bcl-2 levels. We discovered a large heterogeneity in the apoptotic responses of these cells to TAT-IDP^S with SU-DHL-4 being most sensitive and OCI-LY-1 being most resistant. This sensitivity strongly correlated with the ability of TAT-IDP^S to promote IP₃R-mediated Ca²⁺ release. Although total IP₃R-expression levels were very similar among SU-DHL-4 and OCI-LY-1, we discovered that the IP₃R2-protein level was the highest for SU-DHL-4 and the lowest for OCI-LY-1. Strikingly, TAT-IDP^S-induced Ca²⁺ rise and apoptosis in the different DL-BCL cell lines strongly correlated with their IP₃R2-protein level, but not with IP₃R1-, IP₃R3- or total IP₃R-expression levels. Inhibiting or knocking down IP₃R2 activity in SU-DHL-4-reduced TAT-IDP^S-induced apoptosis, which is compatible with its ability to dissociate Bcl-2 from IP₃R2 and to promote IP₃-induced pro-apoptotic Ca²⁺ signaling. Thus, certain chronically activated B-cell lymphoma cells are addicted to high Bcl-2 levels for their survival not only to neutralize pro-apoptotic Bcl-2-family members but also to suppress IP₃R hyperactivity. In particular, cancer cells expressing high levels of IP₃R2 are addicted to IP₃R/Bcl-2 complex formation and disruption of these complexes using peptide tools results in pro-apoptotic Ca²⁺ signaling and cell death.     

Structural Studies of Inositol 1,4,5-Trisphosphate Receptor: COUPLING LIGAND BINDING TO CHANNEL GATING*

The three isoforms of the inositol 1,4,5-trisphosphate receptor (IP₃R) exhibit distinct IP₃ sensitivities and cooperativities in calcium (Ca²⁺) channel function. The determinants underlying this isoform-specific channel gating mechanism have been localized to the N-terminal suppressor region of IP₃R. We determined the 1.9 Å crystal structure of the suppressor domain from type 3 IP₃R (IP₃R3_SUP, amino acids 1–224) and revealed structural features contributing to isoform-specific functionality of IP₃R by comparing it with our previously determined structure of the type 1 suppressor domain (IP₃R1_SUP). The molecular surface known to associate with the ligand binding domain (amino acids 224–604) showed marked differences between IP₃R3_SUP and IP₃R1_SUP. Our NMR and biochemical studies showed that three spatially clustered residues (Glu-20, Tyr-167, and Ser-217 in IP₃R1 and Glu-19, Trp-168, and Ser-218 in IP₃R3) within the N-terminal suppressor domains of IP₃R1_SUP and IP₃R3_SUP interact directly with their respective C-terminal fragments. Together with the accompanying paper (Yamazaki, H., Chan, J., Ikura, M., Michikawa, T., and Mikoshiba, K. (2010) J. Biol. Chem. 285, 36081–36091), we demonstrate that the single aromatic residue in this region (Tyr-167 in IP₃R1 and Trp-168 in IP₃R3) plays a critical role in the coupling between ligand binding and channel gating.     

Ca-DEPENDENT CHANGES OF ACETYLCHOLINE RELEASE AND IP3 MASS IN TORPEDO CHOLINERGIC SYNAPTOSOMES

The aim of the present study was to investigate possible changes of inositol 1,4,5-trisphosphate (IP₃) mass in Torpedo cholinergic synaptosomes in conditions promoting stimulated acetylcholine (ACh) release. For this purpose, we used a radioreceptor IP₃ mass assay and a chemiluminescent method for ACh detection. Torpedo cholinergic synaptosomes have consistent IP₃ mass levels under resting conditions. The IP₃ mass was neither modified by changes in external Ca²⁺ nor by a Ca²⁺-free medium containing EGTA. IP₃ mass and ACh release, measured in the same conditions and in parallel, were increased by depolarization with high K⁺ and by the ionophores A-23187 and gramicidin-D in a manner dependent on external Ca²⁺ emphasizing that Ca²⁺ entry, independently of the influx mechanism involved, leads to an IP₃ increase. The phospholipase C_β inhibitors U-73122 and U-73343 reduced K⁺-stimulated IP₃ levels while K⁺-evoked ACh release was almost completely blocked suggesting an additional effect of these drugs on depolarization-neurotransmitter secretion coupling. The effect reported showing an increase of IP₃ by agents that stimulate ACh release may suggest a possible link between IP₃ metabolism and the neurotransmitter release mechanism. However, such a link is probably not a direct one as implied by the results obtained with the inhibitors of phospholipase C. Copyright © 1996 Elsevier Science Ltd     

A function for tyrosine phosphorylation of type 1 inositol 1,4,5-trisphosphate receptor in lymphocyte activation

Sustained elevation of intracellular calcium by Ca²⁺ release–activated Ca²⁺ channels is required for lymphocyte activation. Sustained Ca²⁺ entry requires endoplasmic reticulum (ER) Ca²⁺ depletion and prolonged activation of inositol 1,4,5-trisphosphate receptor (IP₃R)/Ca²⁺ release channels. However, a major isoform in lymphocyte ER, IP3R1, is inhibited by elevated levels of cytosolic Ca²⁺, and the mechanism that enables the prolonged activation of IP₃R1 required for lymphocyte activation is unclear. We show that IP₃R1 binds to the scaffolding protein linker of activated T cells and colocalizes with the T cell receptor during activation, resulting in persistent phosphorylation of IP₃R1 at Tyr353. This phosphorylation increases the sensitivity of the channel to activation by IP₃ and renders the channel less sensitive to Ca²⁺-induced inactivation. Expression of a mutant IP3R1-Y353F channel in lymphocytes causes defective Ca²⁺ signaling and decreased nuclear factor of activated T cells activation. Thus, tyrosine phosphorylation of IP3R1-Y353 may have an important function in maintaining elevated cytosolic Ca²⁺ levels during lymphocyte activation.     

Cell Surface Sialylation and Fucosylation Are Regulated by L1 via Phospholipase Cγ and Cooperate to Modulate Neurite Outgrowth,Cell Survival and Migration

Background

Cell surface glycosylation patterns are markers of cell type and status. However, the mechanisms regulating surface glycosylation patterns remain unknown.

Methodology/Principal Findings

Using a panel of carbohydrate surface markers, we have shown that cell surface sialylation and fucosylation were downregulated in L1^−/y neurons versus L1^+/y neurons. Consistently, mRNA levels of sialyltransferase ST6Gal1, and fucosyltransferase FUT9 were significantly reduced in L1^−/y neurons. Moreover, treatment of L1^+/y neurons with L1 antibodies, triggering signal transduction downstream of L1, led to an increase in cell surface sialylation and fucosylation compared to rat IgG-treated cells. ShRNAs for both ST6Gal1 and FUT9 blocked L1 antibody-mediated enhancement of neurite outgrowth, cell survival and migration. A phospholipase Cγ (PLCγ) inhibitor and shRNA, as well as an Erk inhibitor, reduced ST6Gal1 and FUT9 mRNA levels and inhibited effects of L1 on neurite outgrowth and cell survival.

Conclusions

Neuronal surface sialylation and fucosylation are regulated via PLCγ by L1, modulating neurite outgrowth, cell survival and migration.