Inositol 1,4,5-Trisphosphate Receptor Subtype-Specific Regulation of Calcium Oscillations

Oscillatory fluctuations in the cytosolic concentration of free calcium ions (Ca²⁺) are considered a ubiquitous mechanism for controlling multiple cellular processes. Inositol 1,4,5-trisphosphate (IP₃) receptors (IP₃R) are intracellular Ca²⁺ release channels that mediate Ca²⁺ release from endoplasmic reticulum (ER) Ca²⁺ stores. The three IP₃R subtypes described so far exhibit differential structural, biophysical, and biochemical properties. Subtype specific regulation of IP₃R by the endogenous modulators IP₃, Ca²⁺, protein kinases and associated proteins have been thoroughly examined. In this article we will review the contribution of each IP₃R subtype in shaping cytosolic Ca²⁺ oscillations.     

Increased Phosphorylation of a 26-kD Pollen Protein Is Induced by the Self-Incompatibility Response in Papaver rhoeas

We have investigated whether specific protein phosphorylation events are induced in Papaver rhoeas pollen as a consequence of the self-incompatibility (SI) response. Pollen grown in vitro in the presence of 32P-orthophosphate was challenged with biologically active recombinant S proteins, and pollen proteins were extracted and analyzed. The results provide strong evidence that the increased phosphorylation of a 26-kD protein of pl 6.2, p26, is specifically induced by the SI response. This phosphorylation event occurs in living pollen tubes and was observed specifically when pollen was challenged with S proteins that are incompatible with the S alleles carried by the pollen and not when pollen was challenged with compatible or incompatible heat-denatured S proteins. Further characterization demonstrated that p26 comprises two phosphoproteins, p26.1 and p26.2, that are found in soluble and microsomal fractions, respectively. Increased phosphorylation of p26.1 is implicated in the SI response and appears to be Ca2+ and calmodulin dependent. These data argue for the involvement of a Ca2+-dependent protein kinase requiring calmodulin-like domains, whose activation comprises an intracellular signal mediating the SI response in P. rhoeas pollen.     

Apical Localization of Inositol 1,4,5-Trisphosphate Receptors Is Independent of Extended Synaptotagmins in Hepatocytes

Extended synaptotagmins (E-Syts) are a recently identified family of proteins that tether the endoplasmic reticulum (ER) to the plasma membrane (PM) in part by conferring regulation of cytosolic calcium (Ca²⁺) at these contact sites (Cell, 2013). However, the mechanism by which E-Syts link this tethering to Ca²⁺ signaling is unknown. Ca²⁺ waves in polarized epithelia are initiated by inositol 1,4,5-trisphosphate receptors (InsP3Rs), and these waves begin in the apical region because InsP3Rs are targeted to the ER adjacent to the apical membrane. In this study we investigated whether E-Syts are responsible for this targeting. Primary rat hepatocytes were used as a model system, because a single InsP3R isoform (InsP3R-II) is tethered to the peri-apical ER in these cells. Additionally, it has been established in hepatocytes that the apical localization of InsP3Rs is responsible for Ca²⁺ waves and secretion and is disrupted in disease states in which secretion is impaired. We found that rat hepatocytes express two of the three identified E-Syts (E-Syt1 and E-Syt2). Individual or simultaneous siRNA knockdown of these proteins did not alter InsP3R-II expression levels, apical localization or average InsP3R-II cluster size. Moreover, apical secretion of the organic anion 5-chloromethylfluorescein diacetate (CMFDA) was not changed in cells lacking E-Syts but was reduced in cells in which cytosolic Ca²⁺ was buffered. These data provide evidence that E-Syts do not participate in the targeting of InsP3Rs to the apical region. Identifying tethers that bring InsP3Rs to the apical region remains an important question, since mis-targeting of InsP3Rs leads to impaired secretory activity.     

Glucocorticoid-mediated Inhibition of Lck Modulates the Pattern of T Cell Receptor-induced Calcium Signals by Down-regulating Inositol 1,4,5-Trisphosphate Receptors

Glucocorticoids are potent immunosuppressive agents that block upstream signaling events required for T cell receptor (TCR) activation. However, the mechanism by which glucocorticoids inhibit downstream responses, such as inositol 1,4,5-trisphosphate (IP₃)-induced calcium signals, is not completely understood. Here we demonstrate that low concentrations of dexamethasone rapidly convert transient calcium elevations to oscillations after strong TCR stimulation. Dexamethasone converted the pattern of calcium signaling by inhibiting the Src family kinase Lck, which was shown to interact with and positively regulate Type I IP₃ receptor. In addition, low concentrations of dexamethasone were sufficient to inhibit calcium oscillations and interleukin-2 mRNA after weak TCR stimulation. Together, these findings indicate that by inhibiting Lck and subsequently down-regulating IP₃ receptors, glucocorticoids suppress immune responses by weakening the strength of the TCR signal.     

Post-translational Regulation of the Type III Inositol 1,4,5-Trisphosphate Receptor by miRNA-506

The type III isoform of the inositol 1,4,5-trisphosphate receptor (InsP₃R3) is apically localized and triggers Ca²⁺ waves and secretion in a number of polarized epithelia. However, nothing is known about epigenetic regulation of this InsP3R isoform. We investigated miRNA regulation of InsP₃R3 in primary bile duct epithelia (cholangiocytes) and in the H69 cholangiocyte cell line, because the role of InsP₃R3 in cholangiocyte Ca²⁺ signaling and secretion is well established and because loss of InsP₃R3 from cholangiocytes is responsible for the impairment in bile secretion that occurs in a number of liver diseases. Analysis of the 3′-UTR of human InsP₃R3 mRNA revealed two highly conserved binding sites for miR-506. Transfection of miR-506 mimics into cell lines expressing InsP₃R3–3′UTR-luciferase led to decreased reporter activity, whereas co-transfection with miR-506 inhibitors led to enhanced activity. Reporter activity was abrogated in isolated mutant proximal or distal miR-506 constructs in miR-506-transfected HEK293 cells. InsP₃R3 protein levels were decreased by miR-506 mimics and increased by inhibitors, and InsP₃R3 expression was markedly decreased in H69 cells stably transfected with miR-506 relative to control cells. miR-506-H69 cells exhibited a fibrotic signature. In situ hybridization revealed elevated miR-506 expression in vivo in human-diseased cholangiocytes. Histamine-induced, InsP₃-mediated Ca²⁺ signals were decreased by 50% in stable miR-506 cells compared with controls. Finally, InsP₃R3-mediated fluid secretion was significantly decreased in isolated bile duct units transfected with miR-506, relative to control IBDU. Together, these data identify miR-506 as a regulator of InsP₃R3 expression and InsP₃R3-mediated Ca²⁺ signaling and secretion.     

Nerve Growth Factor Stimulates the Production of Inositol 1,3,4-and 1,4,5-Trisphosphate and Inositol 1,3,4,5-Tetrakisphosphate in PC 12 Cells

Abstract: In PC12 cells, preincubated with [³H]inositol, nerve growth factor (NGF) stimulated an ～ 100% increase in the levels of [³H]inositol 1,3,4-trisphosphate {[³H]-Ins(1,3,4)P₃}, [³H]inositol 1,4,5-trisphosphate {[³H]lns(1,4,5)P₃}, and [³H]inositol 1,3,4,5-tetrakisphosphate {[³H]-Ins(1,3,4,5)P₄} as early as 5–15 s after addition of NGF. This NGF-mediated response was apparent only when the cells had been cultured in the absence of fetal bovine serum (FBS). PC12 cells cultured in FBS-containing medium did not display NGF-mediated increases in [³H]-Ins(1,3,4)P₃, [³H]-Ins(1,4,5)P₃, and [³H]-Ins(1,3,4,5)P₄ levels. Using cells cultured in the absence of FBS, epidermal growth factor (EGF) and fibroblast growth factor also stimulated production of [³H]lns(1,3,4)P₃, [³H]-Ins(1,4,5)P₃, and [³H]lns(1,3,4,5)P₄. Lavendustin A, a tyrosine kinase inhibitor, inhibited both the EGF-and NGF-stimulated increases in the levels of these tritiated inositol phosphates. These results suggest that NGF stimulates the production of lns(1,3,4)P₃, lns(1,4,5)P₃, and lns(1,3,4,5)P₄ and that this response is dependent on tyrosine kinase activity. Furthermore, although the production of lns(1,3,4)P₃, lns(1,4,5)P₃, and lns(1,3,4,5)P₄ may be a common response to factors stimulating neuronal differentiation, it is not sufficient for stimulation of neuronal differentiation.     

Inositol 1,4,5-Trisphosphate Receptor Immunoreactivity in SH-SY5Y Human Neuroblastoma Cells Is Reduced by Chronic Muscarinic Receptor Activation

Inositol 1,4,5-trisphosphate (InsP3) receptor immunoreactivity in SH-SY5Y human neuroblastoma cells was monitored with a monoclonal antibody raised against the mouse cerebellar InsP3 receptor. Recognition of a protein corresponding to the InsP3 receptor (molecular mass, approximately 275 kDa) was inhibited markedly following exposure of cells to 0.1 mM carbachol. This effect was half-maximal and maximal at approximately 2 and approximately 6 h, respectively; was blocked by atropine; but was not mimicked by thapsigargin, K+, or phorbol 12-myristate 13-acetate. However, the decrease in immunoreactivity following exposure of cells to carbachol for 5 h was blocked if the extracellular Ca2+ concentration was reduced from 1.3 mM to 200 nM. This manipulation also reduced markedly carbachol-induced increases in InsP3 concentration at 5 h. These data indicate that chronic muscarinic stimulation of phosphoinositide hydrolysis reduces InsP3 receptor concentration in SH-SY5Y cells, perhaps via a mechanism that involves prolonged elevation of InsP3 levels.     

Inositol 1,4,5-Trisphosphate (InsP3) and Calcium Interact to Increase the Dynamic Range of InsP3 Receptor-dependent Calcium Signaling

The inositol 1,4,5-trisphosphate (InsP₃)-gated Ca channel in cerebellum is tightly regulated by Ca (Bezprozvanny, I., J. Watras, and B.E. Ehrlich. 1991. Nature (Lond.). 351:751–754; Finch, E.A., T.J. Turner, and S.M. Goldin. 1991. Science (Wash. DC). 252:443–446; Hannaert-Merah, Z., J.F. Coquil, L. Combettes, M. Claret, J.P. Mauger, and P. Champeil. 1994. J. Biol. Chem. 269:29642–29649; Iino, M. 1990. J. Gen. Physiol. 95:1103–1122; Marshall, I., and C. Taylor. 1994. Biochem. J. 301:591–598). In previous single channel studies, the Ca dependence of channel activity, monitored at 2 μM InsP₃, was described by a bell-shaped curve (Bezprozvanny, I., J. Watras, and B.E. Ehrlich. 1991. Nature (Lond.). 351:751–754). We report here that, when we used lower InsP₃ concentrations, the peak of the Ca-dependence curve shifted to lower Ca concentrations. Unexpectedly, when we used high InsP₃ concentrations, channel activity persisted at Ca concentrations as high as 30 μM. To explore this unexpected response of the channel, we measured InsP₃ binding over a broad range of InsP₃ concentrations. We found the well-characterized high affinity InsP₃ binding sites (with K _d < 1 and 50 nM) (Maeda, N., M. Niinobe, and K. Mikoshiba. 1990. EMBO (Eur. Mol. Biol. Organ.) J. 9:61–67; Mignery, G., T.C. Sudhof, K. Takei, and P. De Camilli. 1989. Nature (Lond.). 342:192–195; Ross, C.A., J. Meldolesi, T.A. Milner, T. Satoh, S. Supattapone, and S.H. Snyder. 1989. Nature (Lond.). 339:468–470) and a low affinity InsP₃ binding site (K _d = 10 μM). Using these InsP₃ binding sites, we developed a new model that accounts for the shift in the Ca-dependence curve at low InsP₃ levels and the maintained channel activity at high Ca and InsP₃ levels. The observed Ca dependence of the InsP₃-gated Ca channel allows the cell to abbreviate the rise of intracellular Ca in the presence of low levels of InsP₃, but also provides a means of maintaining high intracellular Ca during periods of prolonged stimulation.     

Polycystin-2 Activation by Inositol 1,4,5-Trisphosphate-induced Ca2+ Release Requires Its Direct Association with the Inositol 1,4,5-Trisphosphate Receptor in a Signaling Microdomain

Autosomal dominant polycystic kidney disease is characterized by the loss-of-function of a signaling complex involving polycystin-1 and polycystin-2 (TRPP2, an ion channel of the TRP superfamily), resulting in a disturbance in intracellular Ca²⁺ signaling. Here, we identified the molecular determinants of the interaction between TRPP2 and the inositol 1,4,5-trisphosphate receptor (IP₃R), an intracellular Ca²⁺ channel in the endoplasmic reticulum. Glutathione S-transferase pulldown experiments combined with mutational analysis led to the identification of an acidic cluster in the C-terminal cytoplasmic tail of TRPP2 and a cluster of positively charged residues in the N-terminal ligand-binding domain of the IP₃R as directly responsible for the interaction. To investigate the functional relevance of TRPP2 in the endoplasmic reticulum, we re-introduced the protein in TRPP2^−/− mouse renal epithelial cells using an adenoviral expression system. The presence of TRPP2 resulted in an increased agonist-induced intracellular Ca²⁺ release in intact cells and IP₃-induced Ca²⁺ release in permeabilized cells. Using pathological mutants of TRPP2, R740X and D509V, and competing peptides, we demonstrated that TRPP2 amplified the Ca²⁺ signal by a local Ca²⁺-induced Ca²⁺-release mechanism, which only occurred in the presence of the TRPP2-IP₃R interaction, and not via altered IP₃R channel activity. Moreover, our results indicate that this interaction was instrumental in the formation of Ca²⁺ microdomains necessary for initiating Ca²⁺-induced Ca²⁺ release. The data strongly suggest that defects in this mechanism may account for the altered Ca²⁺ signaling associated with pathological TRPP2 mutations and therefore contribute to the development of autosomal dominant polycystic kidney disease.     

Muscarinic Receptor-Mediated Inositol 1,4,5-Trisphosphate Formation in SH-SY5Y Neuroblastoma Cells Is Regulated Acutely by Cytosolic Ca2+ and by Rapid Desensitization

Abstract: Stimulation of muscarinic receptors expressed in SH-SY5Y human neuroblastoma cells resulted in a complex profile of inositol 1,4,5-trisphosphate (InsP₃) accumulation, with a dramatic increase (six- to eightfold) over the first 10 s (the “peak” phase) and subsequently, from ～60 s onward, maintained at a lower but sustained level (the “plateau” phase). Chelation of extracellular Ca²⁺ with EGTA or inhibition of Ca²⁺ channels with Ni²⁺ showed that the plateau phase was dependent upon Ca²⁺ entry. Furthermore, use of thapsigargin and EGTA to discharge and sequester Ca²⁺ from intracellular stores revealed that Ca²⁺ from this source was capable of supporting the peak phase of the InsP₃ response. Carbachol-stimulated phosphoinositidase C activity in permeabilized SH-SY5Y cells was also shown to be highly dependent on free Ca²⁺ concentration (20–100 nM) and suggests that under normal conditions, InsP₃ formation is enhanced by increases in cytosolic free Ca²⁺ concentration that accompany muscarinic receptor activation. Measurement of carbachol-stimulated total inositol phosphate accumulation in the presence of Li⁺ indicated that the initial rate of phosphoinositide hydrolysis (from 0 to 30 s) was about fivefold greater than that from 30 to 300 s. This rapid but partial desensitization of receptor-mediated phosphoinositide hydrolysis provides strong evidence for the mechanism underlying the changes in InsP₃ accumulation over this time. Because very similar data were obtained in Chinese hamster ovary cells transfected with human m3 receptor cDNA, we suggest that although increases in cytosolic free Ca²⁺ concentration amplify InsP₃ formation during stimulation of m3 muscarinic receptors, the primary factor that governs the profile of InsP₃ accumulation is rapid, but partial, desensitization. Such desensitization does not appear to be mediated by changes in cytosolic Ca²⁺ or protein kinase C activity.     

Unique Regulatory Properties of Heterotetrameric Inositol 1,4,5-Trisphosphate Receptors Revealed by Studying Concatenated Receptor Constructs

The ability of inositol 1,4,5-trisphosphate receptors (IP₃R) to precisely initiate and generate a diverse variety of intracellular Ca²⁺ signals is in part mediated by the differential regulation of the three subtypes (R1, R2, and R3) by key functional modulators (IP₃, Ca²⁺, and ATP). However, the contribution of IP₃R heterotetramerization to Ca²⁺ signal diversity has largely been unexplored. In this report, we provide the first definitive biochemical evidence of endogenous heterotetramer formation. Additionally, we examine the contribution of individual subtypes within defined concatenated heterotetramers to the shaping of Ca²⁺ signals. Under conditions where key regulators of IP₃R function are optimal for Ca²⁺ release, we demonstrate that individual monomers within heteromeric IP₃Rs contributed equally toward generating a distinct ''blended'' sensitivity to IP₃ that is likely dictated by the unique IP₃ binding affinity of the heteromers. However, under suboptimal conditions where [ATP] were varied, we found that one subtype dictated the ATP regulatory properties of heteromers. We show that R2 monomers within a heterotetramer were both necessary and sufficient to dictate the ATP regulatory properties. Finally, the ATP-binding site B in R2 critical for ATP regulation was mutated and rendered non-functional to address questions relating to the stoichiometry of IP₃R regulation. Two intact R2 monomers were sufficient to maintain ATP regulation in R2 homotetramers. In summary, we demonstrate that heterotetrameric IP₃R do not necessarily behave as the sum of the constituent subunits, and these properties likely extend the versatility of IP₃-induced Ca²⁺ signaling in cells expressing multiple IP₃R isoforms.     

Inositol 1,4,5-Trisphosphate 3-Kinase-A Is a New Cell Motility-promoting Protein That Increases the Metastatic Potential of Tumor Cells by Two Functional Activities

Cellular migration is an essential prerequisite for metastatic dissemination of cancer cells. This study demonstrates that the neuron/testis-specific F-actin-targeted inositol 1,4,5-trisphosphate 3-kinase-A (ITPKA) is ectopically expressed in different human tumor cell lines and during tumor progression in the metastatic tumor model Balb-neuT. High expression of ITPKA increases invasive migration in vitro and metastasis in a xenograft SCID mouse model. Mechanistic studies show that ITPKA promotes migration of tumor cells by two different mechanisms as follows: growth factor independently high levels of ITPKA induce the formation of large cellular protrusions by directly modulating the actin cytoskeleton. The F-actin binding activity of ITPKA stabilizes and bundles actin filaments and thus increases the levels of cellular F-actin. In growth factor-stimulated cells, the catalytically active domain enhances basal ITPKA-induced migration by activating store-operated calcium entry through production of inositol 1,3,4,5-tetrakisphosphate and subsequent inhibition of inositol phosphate 5-phosphatase. These two functional activities of ITPKA stimulating tumor cell migration place the enzyme among the potential targets of anti-metastatic therapy.     

Protein Kinase A Increases Type-2 Inositol 1,4,5-Trisphosphate Receptor Activity by Phosphorylation of Serine 937

Protein kinase A (PKA) phosphorylation of inositol 1,4,5-trisphosphate receptors (InsP₃Rs) represents a mechanism for shaping intracellular Ca²⁺ signals following a concomitant elevation in cAMP. Activation of PKA results in enhanced Ca²⁺ release in cells that express predominantly InsP₃R2. PKA is known to phosphorylate InsP₃R2, but the molecular determinants of this effect are not known. We have expressed mouse InsP₃R2 in DT40-3KO cells that are devoid of endogenous InsP₃R and examined the effects of PKA phosphorylation on this isoform in unambiguous isolation. Activation of PKA increased Ca²⁺ signals and augmented the single channel open probability of InsP₃R2. A PKA phosphorylation site unique to the InsP₃R2 was identified at Ser⁹³⁷. The enhancing effects of PKA activation on this isoform required the phosphorylation of Ser⁹³⁷, since replacing this residue with alanine eliminated the positive effects of PKA activation. These results provide a mechanism responsible for the enhanced Ca²⁺ signaling following PKA activation in cells that express predominantly InsP₃R2.Hormones, neurotransmitters, and growth factors stimulate the production of InsP₃³ and Ca²⁺ signals in virtually all cell types (1). The ubiquitous nature of this mode of signaling dictates that this pathway does not exist in isolation; indeed, a multitude of additional signaling pathways can be activated simultaneously. A prime example of this type of “cross-talk” between independently activated signaling systems results from the parallel activation of cAMP and Ca²⁺ signaling pathways (2, 3). Interactions between these two systems occur in numerous distinct cell types with various physiological consequences (3–6). Given the central role of InsP₃R in Ca²⁺ signaling, a major route of modulating the spatial and temporal features of Ca²⁺ signals following cAMP production is potentially through PKA phosphorylation of the InsP₃R isoform(s) expressed in a particular cell type.There are three InsP₃R isoforms (InsP₃R1, InsP₃R2, and InsP₃R3) expressed to varying degrees in mammalian cells (7, 8). InsP₃R1 is the major isoform expressed in the nervous system, but it is less abundant compared with other subtypes in non-neuronal tissues (8). Ca²⁺ release via InsP₃R2 and InsP₃R3 predominate in these tissues. InsP₃R2 is the major InsP₃R isoform in many cell types, including hepatocytes (7, 8), astrocytes (9, 10), cardiac myocytes (11), and exocrine acinar cells (8, 12). Activation of PKA has been demonstrated to enhance InsP₃-induced Ca²⁺ signaling in hepatocytes (13) and parotid acinar cells (4, 14). Although PKA phosphorylation of InsP₃R2 is a likely causal mechanism underlying these effects, the functional effects of phosphorylation have not been determined in cells unambiguously expressing InsP₃R2 in isolation. Furthermore, the molecular determinants of PKA phosphorylation of this isoform are not known.PKA-mediated phosphorylation is an efficient means of transiently and reversibly regulating the activity of the InsP₃R. InsP₃R1 was identified as a major substrate of PKA in the brain prior to its identification as the InsP₃R (15, 16). However, until recently, the functional consequences of phosphorylation were unresolved. Initial conflicting results were reported indicating that phosphoregulation of InsP₃R1 could result in either inhibition or stimulation of receptor activity (16, 17). Mutagenic strategies were employed by our laboratory to clarify this discrepancy. These studies unequivocally assigned phosphorylation-dependent enhanced Ca²⁺ release and InsP₃R1 activity at the single channel level, through phosphorylation at canonical PKA consensus motifs at Ser¹⁵⁸⁹ and Ser¹⁷⁵⁵. The sites responsible were also shown to be specific to the particular InsP₃R1 splice variant (18). These data were also corroborated by replacing the relevant serines with glutamates in a strategy designed to construct “phosphomimetic” InsP₃R1 by mimicking the negative charge added by phosphorylation (19, 20). Of particular note, however, although all three isoforms are substrates for PKA, neither of the sites phosphorylated by PKA in InsP₃R1 are conserved in the other two isoforms (21). Recently, three distinct PKA phosphorylation sites were identified in InsP₃R3 that were in different regions of the protein when compared with InsP₃R1 (22). To date, no PKA phosphorylation sites have been identified in InsP₃R2.Interactions between Ca²⁺ and cAMP signaling pathways are evident in exocrine acinar cells of the parotid salivary gland. In these cells, both signals are important mediators of fluid and protein secretion (23). Multiple components of the [Ca²⁺]_i signaling pathway in these cells are potential substrates for modulation by PKA. Previous work from this laboratory established that activation of PKA potentiates muscarinic acetylcholine receptor-induced [Ca²⁺]_i signaling in mouse and human parotid acinar cells (4, 24, 25). A likely mechanism to explain this effect is that PKA phosphorylation increases the activity of InsP₃R expressed in these cells. Consistent with this idea, activation of PKA enhanced InsP₃-induced Ca²⁺ release in permeabilized mouse parotid acinar cells and also resulted in the phosphorylation of InsP₃R2 (4).Invariably, prior work examining the functional effects of PKA phosphorylation on InsP₃R2 has been performed using cell types expressing multiple InsP₃R isoforms. For example, AR4-2J cells are the preferred cell type for examining InsP₃R2 in relative isolation, because this isoform constitutes more than 85% of the total InsP₃R population (8). InsP₃R1, however, contributes up to ∼12% of the total InsP₃R in AR4-2J cells. An initial report using InsP₃-mediated ⁴⁵Ca²⁺ flux suggested that PKA activation increased InsP₃R activity in AR4-2J cells (21). A similar conclusion was made in a later study, which documented the effects of PKA activation on agonist stimulated Ca²⁺ signals in AR4-2J cells (26). Any effects of phosphorylation observed in these experiments could plausibly have resulted from phosphorylation of the residual InsP₃R1.Although PKA enhances InsP₃-induced calcium release in cells expressing predominantly InsP₃R2, including hepatocytes, parotid acinar cells, and AR4-2J cells (4, 13, 21, 26, 27), InsP₃R2 is not phosphorylated at stoichiometric levels by PKA (21). This observation has called into question the physiological significance of PKA phosphorylation of InsP₃R2 (28). The apparent low levels of InsP₃R2 phosphorylation are clearly at odds with the augmented Ca²⁺ release observed in cells expressing predominantly this isoform. The equivocal nature of these findings probably stems from the fact that, to date, all of the studies demonstrating positive effects of PKA activation on Ca²⁺ release were conducted in cells that also express InsP₃R1. The purpose of the current experiments was to analyze the functional effects of phosphorylation on InsP₃R2 expressed in isolation on a null background. We report that InsP₃R2 activity is increased by PKA phosphorylation under these conditions, and furthermore, we have identified a unique phosphorylation site in InsP₃R2 at Ser⁹³⁷. In total, these results provide a direct mechanism for the cAMP-induced activation of InsP₃R2 via PKA phosphorylation of InsP₃R2.     

Stimulation of Inositol 1,4,5-Trisphosphate (IP3) Receptor Subtypes by Adenophostin A and Its Analogues

Inositol 1,4,5-trisphosphate receptors (IP₃R) are intracellular Ca²⁺ channels. Most animal cells express mixtures of the three IP₃R subtypes encoded by vertebrate genomes. Adenophostin A (AdA) is the most potent naturally occurring agonist of IP₃R and it shares with IP₃ the essential features of all IP₃R agonists, namely structures equivalent to the 4,5-bisphosphate and 6-hydroxyl of IP₃. The two essential phosphate groups contribute to closure of the clam-like IP₃-binding core (IBC), and thereby IP₃R activation, by binding to each of its sides (the α- and β-domains). Regulation of the three subtypes of IP₃R by AdA and its analogues has not been examined in cells expressing defined homogenous populations of IP₃R. We measured Ca²⁺ release evoked by synthetic adenophostin A (AdA) and its analogues in permeabilized DT40 cells devoid of native IP₃R and stably expressing single subtypes of mammalian IP₃R. The determinants of high-affinity binding of AdA and its analogues were indistinguishable for each IP₃R subtype. The results are consistent with a cation-π interaction between the adenine of AdA and a conserved arginine within the IBC α-domain contributing to closure of the IBC. The two complementary contacts between AdA and the α-domain (cation-π interaction and 3″-phosphate) allow activation of IP₃R by an analogue of AdA (3″-dephospho-AdA) that lacks a phosphate group equivalent to the essential 5-phosphate of IP₃. These data provide the first structure-activity analyses of key AdA analogues using homogenous populations of all mammalian IP₃R subtypes. They demonstrate that differences in the Ca²⁺ signals evoked by AdA analogues are unlikely to be due to selective regulation of IP₃R subtypes.     

Isoform- and Species-specific Control of Inositol 1,4,5-Trisphosphate (IP3) Receptors by Reactive Oxygen Species

Reactive oxygen species (ROS) stimulate cytoplasmic [Ca²⁺] ([Ca²⁺]_c) signaling, but the exact role of the IP₃ receptors (IP₃R) in this process remains unclear. IP₃Rs serve as a potential target of ROS produced by both ER and mitochondrial enzymes, which might locally expose IP₃Rs at the ER-mitochondrial associations. Also, IP₃Rs contain multiple reactive thiols, common molecular targets of ROS. Therefore, we have examined the effect of superoxide anion (O₂^⨪) on IP₃R-mediated Ca²⁺ signaling. In human HepG2, rat RBL-2H3, and chicken DT40 cells, we observed [Ca²⁺]_c spikes and frequency-modulated oscillations evoked by a O₂^⨪ donor, xanthine (X) + xanthine oxidase (XO), dose-dependently. The [Ca²⁺]_c signal was mediated by ER Ca²⁺ mobilization. X+XO added to permeabilized cells promoted the [Ca²⁺]_c rise evoked by submaximal doses of IP₃, indicating that O₂^⨪ directly sensitizes IP₃R-mediated Ca²⁺ release. In response to X+XO, DT40 cells lacking two of three IP₃R isoforms (DKO) expressing either type 1 (DKO1) or type 2 IP₃Rs (DKO2) showed a [Ca²⁺]_c signal, whereas DKO expressing type 3 IP₃R (DKO3) did not. By contrast, IgM that stimulates IP₃ formation, elicited a [Ca²⁺]_c signal in every DKO. X+XO also facilitated the Ca²⁺ release evoked by submaximal IP₃ in permeabilized DKO1 and DKO2 but was ineffective in DKO3 or in DT40 lacking every IP₃R (TKO). However, X+XO could also facilitate the effect of suboptimal IP₃ in TKO transfected with rat IP₃R3. Although in silico studies failed to identify a thiol missing in the chicken IP₃R3, an X+XO-induced redox change was documented only in the rat IP₃R3. Thus, ROS seem to specifically sensitize IP₃Rs through a thiol group(s) within the IP₃R, which is probably inaccessible in the chicken IP₃R3.     

Differential Regulation of Multiple Steps in Inositol 1,4,5-Trisphosphate Signaling by Protein Kinase C Shapes Hormone-stimulated Ca2+ Oscillations

How Ca²⁺ oscillations are generated and fine-tuned to yield versatile downstream responses remains to be elucidated. In hepatocytes, G protein-coupled receptor-linked Ca²⁺ oscillations report signal strength via frequency, whereas Ca²⁺ spike amplitude and wave velocity remain constant. IP₃ uncaging also triggers oscillatory Ca²⁺ release, but, in contrast to hormones, Ca²⁺ spike amplitude, width, and wave velocity were dependent on [IP₃] and were not perturbed by phospholipase C (PLC) inhibition. These data indicate that oscillations elicited by IP₃ uncaging are driven by the biphasic regulation of the IP₃ receptor by Ca²⁺, and, unlike hormone-dependent responses, do not require PLC. Removal of extracellular Ca²⁺ did not perturb Ca²⁺ oscillations elicited by IP₃ uncaging, indicating that reloading of endoplasmic reticulum stores via plasma membrane Ca²⁺ influx does not entrain the signal. Activation and inhibition of PKC attenuated hormone-induced Ca²⁺ oscillations but had no effect on Ca²⁺ increases induced by uncaging IP₃. Importantly, PKC activation and inhibition differentially affected Ca²⁺ spike frequencies and kinetics. PKC activation amplifies negative feedback loops at the level of G protein-coupled receptor PLC activity and/or IP₃ metabolism to attenuate IP₃ levels and suppress the generation of Ca²⁺ oscillations. Inhibition of PKC relieves negative feedback regulation of IP₃ accumulation and, thereby, shifts Ca²⁺ oscillations toward sustained responses or dramatically prolonged spikes. PKC down-regulation attenuates phenylephrine-induced Ca²⁺ wave velocity, whereas responses to IP₃ uncaging are enhanced. The ability to assess Ca²⁺ responses in the absence of PLC activity indicates that IP₃ receptor modulation by PKC regulates Ca²⁺ release and wave velocity.     

Activation of the Inositol (1,4,5)-Triphosphate Calcium Gate Receptor Is Required for HIV-1 Gag Release

The structural precursor polyprotein, Gag, encoded by all retroviruses, including the human immunodeficiency virus type 1 (HIV-1), is necessary and sufficient for the assembly and release of particles that morphologically resemble immature virus particles. Previous studies have shown that the addition of Ca²⁺ to cells expressing Gag enhances virus particle production. However, no specific cellular factor has been implicated as mediator of Ca²⁺ provision. The inositol (1,4,5)-triphosphate receptor (IP3R) gates intracellular Ca²⁺ stores. Following activation by binding of its ligand, IP3, it releases Ca²⁺ from the stores. We demonstrate here that IP3R function is required for efficient release of HIV-1 virus particles. Depletion of IP3R by small interfering RNA, sequestration of its activating ligand by expression of a mutated fragment of IP3R that binds IP3 with very high affinity, or blocking formation of the ligand by inhibiting phospholipase C-mediated hydrolysis of the precursor, phosphatidylinositol-4,5-biphosphate, inhibited Gag particle release. These disruptions, as well as interference with ligand-receptor interaction using antibody targeted to the ligand-binding site on IP3R, blocked plasma membrane accumulation of Gag. These findings identify IP3R as a new determinant in HIV-1 trafficking during Gag assembly and introduce IP3R-regulated Ca²⁺ signaling as a potential novel cofactor in viral particle release.Assembly of the human immunodeficiency virus (HIV) is determined by a single gene that encodes a structural polyprotein precursor, Gag (71), and may occur at the plasma membrane or within late endosomes/multivesicular bodies (LE/MVB) (7, 48, 58; reviewed in reference 9). Irrespective of where assembly occurs, the assembled particle is released from the plasma membrane of the host cell. Release of Gag as virus-like particles (VLPs) requires the C-terminal p6 region of the protein (18, 19), which contains binding sites for Alix (60, 68) and Tsg101 (17, 37, 38, 41, 67, 68). Efficient release of virus particles requires Gag interaction with Alix and Tsg101. Alix and Tsg101 normally function to sort cargo proteins to LE/MVB for lysosomal degradation (5, 15, 29, 52). Previous studies have shown that addition of ionomycin, a calcium ionophore, and CaCl₂ to the culture medium of cells expressing Gag or virus enhances particle production (20, 48). This is an intriguing observation, given the well-documented positive role for Ca²⁺ in exocytotic events (33, 56). It is unclear which cellular factors might regulate calcium availability for the virus release process.Local and global elevations in the cytosolic Ca²⁺ level are achieved by ion release from intracellular stores and by influx from the extracellular milieu (reviewed in reference 3). The major intracellular Ca²⁺ store is the endoplasmic reticulum (ER); stores also exist in MVB and the nucleus. Ca²⁺ release is regulated by transmembrane channels on the Ca²⁺ store membrane that are formed by tetramers of inositol (1,4,5)-triphosphate receptor (IP3R) proteins (reviewed in references 39, 47, and 66). The bulk of IP3R channels mediate release of Ca²⁺ from the ER, the emptying of which signals Ca²⁺ influx (39, 51, 57, 66). The few IP3R channels on the plasma membrane have been shown to be functional as well (13). Through proteomic analysis, we identified IP3R as a cellular protein that was enriched in a previously described membrane fraction (18) which, in subsequent membrane floatation analyses, reproducibly cofractionated with Gag and was enriched in the membrane fraction only when Gag was expressed. That IP3R is a major regulator of cytosolic calcium concentration (Ca²⁺) is well documented (39, 47, 66). An IP3R-mediated rise in cytosolic Ca²⁺ requires activation of the receptor by a ligand, inositol (1,4,5)-triphosphate (IP3), which is produced when phospholipase C (PLC) hydrolyzes phosphatidylinositol-4,5-bisphosphate [PI(4,5)P₂] at the plasma membrane (16, 25, 54). Paradoxically, PI(4,5)P₂ binds to the matrix (MA) domain in Gag (8, 55, 59), and the interaction targets Gag to PI(4,5)P₂-enriched regions on the plasma membrane; these events are required for virus release (45). We hypothesized that PI(4,5)P₂ binding might serve to target Gag to plasma membrane sites of localized Ca²⁺ elevation resulting from PLC-mediated PI(4,5)P₂ hydrolysis and IP3R activation. This idea prompted us to investigate the role of IP3R in Gag function.Here, we show that HIV-1 Gag requires steady-state levels of IP3R for its efficient release. Three isoforms of IP3R, types 1, 2, and 3, are encoded in three independent genes (39, 47). Types 1 and 3 are expressed in a variety of cells and have been studied most extensively (22, 39, 47, 73). Depletion of the major isoforms in HeLa or COS-1 cells by small interfering RNA (siRNA) inhibited viral particle release. Moreover, we show that sequestration of the IP3R activating ligand or blocking ligand formation also inhibited Gag particle release. The above perturbations, as well as interfering with receptor expression or activation, led to reduced Gag accumulation at the cell periphery. The results support the conclusion that IP3R activation is required for efficient HIV-1 viral particle release.