Phosphoinositide Signaling Cascade Research Articles

Functional Expression of Adenosine Receptors in Mesenchymal Stromal Cells

Extracellular adenosine is a signaling molecule involved in the regulation of physiological processes in a variety of cells, including mesenchymal stromal cells (MSCs). We have shown using microphotometry and Ca2+ probes that the population of MSCs isolated from human adipose tissue contained a small (5–10%) subpopulation of adenosinergic cells. In these cells, A1, A2A, and A2B adenosine receptors functioned; they were coupled with a phosphoinositide signaling cascade. Stimulation of adenosinergic MSCs by adenosine initiated the mobilization of intracellular Ca2+; Ca2+ responses to adenosine were generated according to the “all or nothing” principle: in small doses adenosine did not change the level of intracellular Ca2+, but it evoked Ca2+ responses of almost identical shape and amplitude at any concentration exceeding a threshold. It is noteworthy that the duration of the response delay from the moment of application of adenosine decreased as its concentration increased. Key stages of the generation of the Ca2+ response to adenosine were studied using the inhibitor analysis. The obtained data indicated that the adenosine receptor activated phospholipase C, which stimulated the production of IP3; the latter activated IP3 receptors and initiated the release of stored Ca2+, which most likely was dependent on the concentration of the agonist. This initial Ca2+ signal stimulated a large-scale release of Ca2+ from intracellular stores by the mechanism of Ca2+-induced Ca2+ release, which formed a secondary Ca2+ response of universal shape and amplitude.

Receptor- and store-operated mechanisms of calcium entry during the nanosecond electric pulse-induced cellular response

Nanosecond electric pulses have been shown to open nanopores in the cell plasma membrane by fluorescent imaging of calcium uptake and fluorescent dyes, including propidium (Pr) iodide and YO-PRO-1 (YP1). Recently, we demonstrated that nsEPs also induce the phosphoinositide intracellular signaling cascade by phosphatidylinositol-4,5-bisphosphate (PIP2) depletion resulting in physiological responses similar to those observed following stimulation of Gq11-coupled receptors. In this paper, we explore the role of receptor- and store-operated calcium entry (ROCE/SOCE) mechanisms in the observed response of cells to nsEP. We show that addition of the ROCE/SOCE and transient receptor potential channel (TRPC) blocker gadolinium (Gd3+, 300 μM) slows PIP2 depletion following 1 and 20 nsEP exposures. Lipid rafts, regions of the plasma membrane rich in PIP2 and TRPC, are also disrupted by nsEP exposure suggesting that ROCE/SOCE mechanisms are likely impacted. Reducing the expression of stromal interaction molecule 1 (STIM1) protein, a key protein in ROCE and SOCE, in cells exposure to nsEP resulted in a reduction in induced intracellular calcium rise. Additionally, after exposure to 1 and 20 nsEPs (16.2 kV/cm, 5 Hz), intracellular calcium rises were significantly reduced by the addition of GD3+ and SKF-96365 (1-[2-(4-methoxyphenyl)-2-[3-(4-methoxyphenyl) propoxy] ethyl-1H-imidazole hydrochloride, 100 μM), a blocker of STIM1 interaction. However, using similar nsEP exposure parameters, SKF-96365 was less effective at reducing YP1 uptake compared to Gd3+. Thus, it is possible that SKF-96365 could block STIM1 interactions within the cell, while Gd3+ could acts on TRPC/nanopores from outside of the cell. Our results present evidence of nsEP induces ROCE and SOCE mechanisms and demonstrate that YP1 and Ca2+ cannot be used solely as markers of nsEP-induced nanoporation.

The Drosophila light-activated TRP and TRPL channels - Targets of the phosphoinositide signaling cascade.

The Drosophila light-activated Transient Receptor Potential (TRP) channel is the founding member of a large and diverse family of channel proteins. It is now established that TRP channels are evolutionarily conserved and are found in many organisms and tissues. This review outlines the progress made in our understanding of Drosophila phototransduction with a focus on the light sensitive TRP channels. The visual system of Drosophila has remarkable capabilities, such as single photon sensitivity, low dark noise, wide dynamic range of responses to changing ambient light intensities and an unusually wide range of frequency responses to modulated lights. These capabilities are obtained by a unique cellular structure called rhabdomere, which contains ∼40,000 microvilli, harboring a sophisticated molecular machinery performing phototransduction. The phototransduction cascade was discovered mainly by using the power of Drosophila molecular genetics and the ability to generate mutations in virtually every gene of the cascade. This allowed a detailed functional analysis and mechanistic description of the phototransduction cascade. Drosophila phototransduction has been a model system, instrumental for studying phosphoinositide signaling and its participation in TRP channel activation. Accordingly, the phosphoinositide signaling cascade activates the TRP/TRPL channels via Gq-protein-mediated PLCβ, while the gating mechanism of the channels following PLC activation is still under debate. Detailed studies of the single photon response (quantum bump) and the spontaneous dark bump has given important tools to investigate critical features of channel activation and regulation including: synchronization in channel activity, the existence of a Ca2+ regulated threshold of channel activation, positive and negative feedback and refractory period in bump generation. We anticipate that studies in Drosophila photoreceptors will continue shed light on mechanisms that operate in mammalian TRP channels.

INPP5E Preserves Genomic Stability through Regulation of Mitosis.

ABSTRACTThe partially understood phosphoinositide signaling cascade regulates multiple aspects of cellular metabolism. Previous studies revealed that INPP5E, the inositol polyphosphate-5-phosphatase that is mutated in the developmental disorders Joubert and MORM syndromes, is essential for the function of the primary cilium and maintenance of phosphoinositide balance in nondividing cells. Here, we report that INPP5E further contributes to cellular homeostasis by regulating cell division. We found that silencing or genetic knockout of INPP5E in human and murine cells impairs the spindle assembly checkpoint, centrosome and spindle function, and maintenance of chromosomal integrity. Consistent with a cell cycle regulatory role, we found that INPP5E expression is cell cycle dependent, peaking at mitotic entry. INPP5E localizes to centrosomes, chromosomes, and kinetochores in early mitosis and shuttles to the midzone spindle at mitotic exit. Our findings identify the previously unknown, essential role of INPP5E in mitosis and prevention of aneuploidy, providing a new perspective on the function of this phosphoinositide phosphatase in health and development.

Expression and localization of the diacylglycerol kinase family and of phosphoinositide signaling molecules in adrenal gland.

Adrenal glands play a central role in the secretion of steroid hormones and catecholamines. Previous studies have revealed that molecules engaged in phosphoinositide (PI) turnover are expressed in the adrenal gland, suggesting the importance of PI signaling in adrenal signal transduction. Diacylglycerol kinase (DGK) catalyzes the phosphorylation of diacylglycerol (DG), a major second messenger in the PI signaling cascade. The DGK family is expressed in distinct patterns in endocrine organs at the mRNA and protein levels. Nevertheless, little is known about the characteristics and morphological aspects of DGKs in the adrenal gland. We have performed immunohistochemical analyses to investigate the expression and localization of DGK isozymes, together with PI signaling molecules, in the adrenal gland at the protein level. Our results show that the DGK family and a set of PI signaling molecules are expressed intensely in zona glomerulosa cells and medullary chromaffin cells in the adrenal gland. In adrenal cells, DGKγ localizes to the Golgi complex, DGKε to the plasma membrane, and DGKζ to the nucleus. These findings show the distinct expression and subcellular localization of DGK isozymes and PI signaling molecules in the adrenal gland, suggesting that each DGK isozyme has a role in signal transduction in adrenal cells, especially in the zona glomerulosa and medulla.

Electroretinogram (ERG) Recordings from Drosophila.

Phototransduction is a process in which light is converted into electrical signals used by the central nervous system. Invertebrate phototransduction is a process mediated by the phosphoinositide signaling cascade, characterized by Phospholipase C (PLC) as the effector enzyme and the Transient Receptor Potential (TRP) channels as its target. The great advantage of using invertebrate photoreceptors is the simplicity of the preparation, the ease of light stimulation, the robust expression of key molecular components, and most importantly, the ability to apply the power of molecular genetics. This last feature is mainly attributed to Drosophila melanogaster as a preferred animal model. The Electroretinogram (ERG) is an extracellular voltage recording from the entire eye, which reflects the total electrical activity arising from the retina in response to a light stimulation. The Drosophila ERG light response is robust and easily obtained, thus making it a convenient method to identify defects in the light response as a result of mutations. The Prolonged Depolarizing Afterpotential (PDA) is a useful ERG phenomenon that can be recorded from white-eyed flies following intense blue light. It is induced by a massive photo-conversion of the photopigment rhodopsin to its dark stable state called metarhodopsin, due to failure of light response termination. Unlike the light coincident ERG recording, which declines quickly to the dark baseline after the cessation of the light stimulus, the PDA response continues long (hours) after light offset. However, this response can be suppressed to the dark baseline at any time by photo-conversion of metarhodopsin back to rhodopsin, by application of an intense orange light stimulus (see Figure 7; Minke, 2012). The PDA has been used as an important tool to screen for visual defective mutant (Minke, 2012).

Conserved Gating Elements in TRPC4 and TRPC5 Channels

TRPC4 and TRPC5 proteins share 65% amino acid sequence identity and form Ca(2+)-permeable nonselective cation channels. They are activated by stimulation of receptors coupled to the phosphoinositide signaling cascade. Replacing a conserved glycine residue within the cytosolic S4-S5 linker of both proteins by a serine residue forces the channels into an open conformation. Expression of the TRPC4G503S and TRPC5G504S mutants causes cell death, which could be prevented by buffering the Ca(2+) of the culture medium. Current-voltage relationships of the TRPC4G503S and TRPC5G504S mutant ion channels resemble that of fully activated TRPC4 and TRPC5 wild-type channels, respectively. Modeling the structure of the transmembrane domains and the pore region (S4-S6) of TRPC4 predicts a conserved serine residue within the C-terminal sequence of the predicted S6 helix as a potential interaction site. Introduction of a second mutation (S623A) into TRPC4G503S suppressed the constitutive activation and partially rescued its function. These results indicate that the S4-S5 linker is a critical constituent of TRPC4/C5 channel gating and that disturbance of its sequence allows channel opening independent of any sensor domain.

Activation of intracellular phosphoinositide signaling after a single 600 nanosecond electric pulse

Exposure to nanosecond pulsed electrical fields (nsPEFs) results in a myriad of observable effects in mammalian cells. While these effects are often attributed to the direct permeabilization of both the plasma and organelle membranes, the underlying mechanism(s) are not well understood. We hypothesize that nsPEF-induced membrane disturbance will initiate complex intracellular lipid signaling pathways, which ultimately lead to the observed multifarious effects. In this article, we show activation of one of these pathways — phosphoinositide signaling cascade. Here we demonstrate that nsPEF initiates phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) hydrolysis or depletion from the plasma membrane, accumulation of inositol-1,4,5-trisphosphate (IP3) in the cytoplasm and increase of diacylglycerol (DAG) on the inner surface of the plasma membrane. All of these events are initiated by a single 16.2kV/cm, 600ns pulse exposure. To further this claim, we show that the nsPEF-induced activation mirrors the response of M1-acetylcholine Gq/11-coupled metabotropic receptor (hM1). This demonstration of PIP2 hydrolysis by nsPEF exposure is an important step toward understanding the mechanisms underlying this unique stimulus for activation of lipid signaling pathways and is critical for determining the potential for nsPEFs to modulate mammalian cell functions.

DGKζ is involved in LPS-activated phagocytosis through IQGAP1/Rac1 pathway

Diacylglycerol kinase (DGK) plays an important role in phosphoinositide signaling cascade by regulating the intracellular level of diacylglycerol and phosphatidic acid. The DGK family is involved in various pathophysiological responses that are mediated through unique binding partners in different tissues and cells. In this study, we identified a small GTPase effector protein, IQGAP1, as a novel DGKζ-associated complex protein. A bacterial endotoxin, lipopolysaccharide (LPS), facilitated the complex formation in macrophages. Both proteins co-localized at the edge and phagocytic cup of the cell. Furthermore, RNA interference-mediated knockdown of DGKζ or IQGAP1 impaired LPS-induced Rac1 activation. Primary macrophages derived from DGKζ−/− mice attenuated LPS-induced phagocytosis of bacteria. These results suggest that DGKζ is involved in IQGAP1/Rac1-mediated phagocytosis upon LPS stimulation in macrophages.

Phototransduction mechanisms in Drosophila microvillar photoreceptors

AbstractPhototransduction in Drosophila is mediated by a G‐protein coupled phospholipase C (PLC) cascade leading to graded membrane depolarization. It is the preferred model for phototransduction in microvillar photoreceptors and an important model for the ubiquitous phosphoinositide signaling cascade. The photoreceptors respond sensitively to single photons 10–100× more rapidly than vertebrate rods, yet still light adapt to signal under full sunlight. Incident light is absorbed and transduced in the rhabdomere, a 1–2 µm diameter, 100 µm long waveguide composed of ∼30,000 tightly packed microvilli, which contain the visual pigment rhodopsin and all the major components of the cascade. PLC hydrolyzes phosphatidyl‐inositol (4,5) bisphosphate (PIP2) to generate diacylglycerol (DAG), inositol (1,4,5) trisphosphate (InsP3), and also a proton. This results in activation of two classes of Ca2+ permeable cation channels, TRP and TRPL—the prototypical members of the transient receptor potential (TRP) superfamily of cation channels. Recent evidence suggests that the channels may be activated in a combinatorial manner by two neglected consequences of PLC activity, namely simultaneous depletion of PIP2 and acidification. Several components of the cascade, including TRP, PLC, and protein kinase C are assembled into multimolecular signaling complexes by the PDZ‐domain scaffolding protein INAD. Ca2+ influx via TRP channels is essential for rapid kinetics, amplification, and light adaptation and mediates both positive and negative feedback via multiple downstream targets, including the channels, rhodopsin, and PLC. This Ca2+‐dependent feedback along with the ultracompartmentalization provided by the microvillar design and molecular scaffolding is critical for the combination of sensitivity, rapid kinetics, and broad dynamic range. WIREs Membr Transp Signal 2012, 1:162–187. doi: 10.1002/wmts.20For further resources related to this article, please visit the WIREs website.

Signaling Cascade of Diacylglycerol Kinase β in the Pituitary Intermediate Lobe: Dopamine D2 Receptor/Phospholipase Cβ4/Diacylglycerol Kinase β/Protein Kinase Cα

The pituitary gland dynamically changes its hormone output under various pathophysiological conditions. One of the pathways implicated in the regulatory mechanism of this gland is a dopaminergic system that operates the phosphoinositide (PI) cycle to transmit downstream signal through second messengers. We have previously shown that diacylglycerol kinase β (DGKβ) is coexpressed with dopamine D1 and D2 receptors in medium spiny neurons of the striatum, suggesting a plausible implication of DGKβ in dopaminergic transmission. However, it remains elusive whether DGKβ is involved in the dopaminergic system in the pituitary gland. The aim of this study is to investigate the expression and localization of DGK in the pituitary gland, together with the molecular components involved in the PI signaling cascade, including dopamine receptors, phospholipase C (PLC), and a major downstream molecule, protein kinase C (PKC). Here we show that DGKβ and the dopamine D2 receptor are coexpressed in the intermediate lobe and localize to the plasma membrane side by side. In addition, we reveal that PLCβ4 and PKCα are the subtypes expressed in the intermediate lobe among those families. These findings will substantiate and further extend our understanding of the molecular-anatomical pathway of PI signaling and the functional roles of DGK in the pituitary intermediate lobe.

Calmodulin and Calcium Interplay in the Modulation of TRPC5 Channel Activity: IDENTIFICATION OF A NOVEL C-TERMINAL DOMAIN FOR CALCIUM/CALMODULIN-MEDIATED FACILITATION

TRPC5 forms Ca2+-permeable nonselective cation channels important for neurite outgrowth and growth cone morphology of hippocampal neurons. Here we studied the activation of mouse TRPC5 expressed in Chinese hamster ovary and human embryonic kidney 293 cells by agonist stimulation of several receptors that couple to the phosphoinositide signaling cascade and the role of calmodulin (CaM) on the activation. We showed that exogenous application of 10 microM CaM through patch pipette accelerated the agonist-induced channel activation by 2.8-fold, with the time constant for half-activation reduced from 4.25 +/- 0.4 to 1.56 +/- 0.85 min. We identified a novel CaM-binding site located at the C terminus of TRPC5, 95 amino acids downstream from the previously determined common CaM/IP3R-binding (CIRB) domain for all TRPC proteins. Deletion of the novel CaM-binding site attenuated the acceleration in channel activation induced by CaM. However, disruption of the CIRB domain from TRPC5 rendered the channel irresponsive to agonist stimulation without affecting the cell surface expression of the channel protein. Furthermore, we showed that high (>5 microM) intracellular free Ca2+ inhibited the current density without affecting the time course of TRPC5 activation by receptor agonists. These results demonstrated that intracellular Ca2+ has dual and opposite effects on the activation of TRPC5. The novel CaM-binding site is important for the Ca2+/CaM-mediated facilitation, whereas the CIRB domain is critical for the overall response of receptor-induced TRPC5 channel activation.

The phosphoinositide signaling cascade is involved in photoreception in the leech Hirudo medicinalis.

The effects of BAPTA, heparin, and neomycin on electrical light responses were studied in the photoreceptors of Hirudo medicinalis. Light activation produces a fast increase in intracellular Ca2+ concentration (Cai) as detected with the fluorescent Ca2+ indicator calcium green-5N. Chelating intracellular calcium by injections of 10 mmol(-1) BAPTA suppresses spontaneous quantum bumps, reduces light sensitivity by more than 2 log(10) units, and substantially increases the latent period of light responses. BAPTA strongly inhibits the plateau phase of responses to long steps of light. Injections of 45-100 mg ml(-1) of heparin act in a similar manner to BAPTA, affecting the latency of the light responses even more. De-N-sulfated heparin, an inactive analog, is almost ineffective at the same concentration compared with heparin. Heparin diminishes the light-induced Cai elevation significantly, whereas de-N-sulfated heparin does not. Intracellular injections of 50-100 mmol l(-1) of the aminoglycoside neomycin, which inhibits phospholipase-C-mediated inositol 1,4,5-trisphosphate formation, acts similar to BAPTA and heparin. Pressure injections of the hydrolysis resistant analog of inositol 1,4,5-trisphosphate, inositol 2,4,5-trisphosphate, strongly depolarize leech photoreceptors and mimic an effect of light adaptation. These results suggest a close similarity between phototransduction mechanisms in leech photoreceptors and existing models for visual transduction in other invertebrate microvillar photoreceptors.

Intracellular translocation of PKC isoforms in canine pulmonary artery smooth muscle cells by ANG II.

Our goals were to identify the isoforms of protein kinase C (PKC) present in primary cultures of canine pulmonary artery smooth muscle cells (PASMCs) and to determine whether angiotensin II (ANG II) triggers translocation of specific PKC isoforms to discreet intracellular locations. Isoform-specific antibodies and Western blot analysis were utilized to identify the isoforms of PKC in PASMCs. Indirect immunofluorescence and confocal microscopy were used to examine the subcellular distribution of PKC isoforms. Inositol phosphate production was used to assess phospholipase C activation, and fura 2 was utilized to monitor intracellular Ca2+ concentration in response to ANG II. Six isoforms (alpha, delta, epsilon, zeta, iota/lambda, and mu) of PKC were identified by Western blot analysis. Immunolocalization of 5 isoforms (alpha, delta, zeta, iota/lambda, and mu) revealed a unique pattern of staining for each individual isoform. ANG II caused translocation of PKC-alpha from the cytosol to the nuclear envelope and of PKC-delta to the myofilaments. In contrast, cytosolic PKC-zeta did not translocate, but nuclear PKC-zeta was upregulated. Translocation of PKC-alpha and PKC-delta and upregulation of PKC-zeta in response to ANG II were blocked by the ANG II type 1-receptor antagonist losartan. In addition, ANG II stimulated inositol phosphate production and intracellular Ca2+ concentration oscillations, which were blocked by losartan. Thus activation of ANG II type 1 receptors triggers the phosphoinositide signaling cascade, resulting in translocation or upregulation of specific PKC isoforms at discreet intracellular sites. The alpha and zeta isoforms may act to regulate nuclear events, whereas PKC-delta may be involved in modulating contraction via actions on the myofilaments.

Novel insights into histamine H2 receptor biology.

Histamine exerts multiple biological actions through one of three receptor subtypes (H1, H2, and H3). This review focuses on new developments regarding the structure and function of the H2 receptor. In addition to the important role this receptor plays in stimulating gastric acid secretion, recent studies have demonstrated that it is also involved in regulating gastrointestinal motility and intestinal secretion. The potential role of the H2 receptor in regulating cell growth and differentiation has also been added to the list of actions this biogenic amine may exert in both normal and transformed tissues. Molecular cloning of the gene indicates that it has the structural characteristics of a heptahelical G protein-linked receptor. Site-directed mutagenesis studies of this receptor reveal the presence of key amino acids within the third and fifth transmembrane domains that are critical for ligand recognition. Molecular approaches have also shed light on the structural components of the H2 receptor important in regulating desensitization and internalization. Although the H2 receptor was classically thought to couple to the adenylate cyclase pathway, recent work with the cloned receptor indicates that it can also activate the phosphoinositide signaling cascade through an independent G protein-dependent mechanism. The novel observation that histamine may stimulate c-fos gene expression lends further support to the possible role of this receptor in regulating cell growth and differentiation.

Cyclic-GMP enhances light-induced excitation and induces membrane currents in Drosophila retinal photoreceptors

Phototransduction in the Drosophila retina appears to require the phosphoinositide signaling cascade following receptor/G-protein activation. Subsequent opening of membrane cationic channels causes excitation. The biochemical events underlying channel opening and regulation of sensitivity remain largely unknown. Evidence is mounting that phototransduction in Drosophila and other invertebrate species may additionally involve the second messenger, cyclic-GMP (cGMP). We report that exogenous cGMP influenced Drosophila retinal phototransduction in two ways. In whole cell tight-seal voltage-clamp experiments, membrane permeant cGMP analog, 8-bromo-cyclic-GMP (8-Br-cGMP), induced membrane currents and dramatically enhanced light-induced currents. The currents induced by 8-Br-cGMP possessed reversal potentials similar to those induced by light. The magnitudes of cGMP-induced currents exhibited marked dependence on intensity of background illumination. Potential direct or modulatory roles of cGMP in Drosophila phototransduction are discussed.

Challenge with high concentrations of cyclic AMP induces transient changes in the cytosolic free calcium concentration in Dictyostelium discoideum.

Dictyostelium discoideum cells use cyclic AMP (cAMP) for chemotactic signaling as well as for differentiation. The precise regulation of the cytosolic Ca2+ concentration ([Ca2+]i) seems to play a key role for both processes. We performed single cell measurements of [Ca2+]i in amoebae that were starved in suspension for various times and scrape-loaded with the Ca2+ indicator fura-2. Stimulation of cells with cAMP at the concentration required to induce gene expression (> or = 100 microM) elicited a global transient increase in [Ca2+]i that depended on the presence of external Ca2+. Both vegetative and aggregation-competent cells displayed a rise in [Ca2+]i, with aggregation-competent cells responding more often than vegetative cells. Basal [Ca2+]i in the presence of Ca2+ was high in vegetative cells and declined during development; the cAMP-induced rise in [Ca2+]i was higher and lasted longer in vegetative cells than in aggregative cells. The addition of 2'-deoxy-cAMP, which binds to the cAMP receptor, induced an increase in [Ca2+]i, whereas the membrane-permeant analogue 8-bromo-cAMP that has a low affinity for the receptor but activates cAMP-dependent protein kinase had no effect. This indicates that the change in [Ca2+]i is mediated by the cell surface cAMP receptor. Since HC85 mutant cells, which lack the G alpha 2 subunit of the G-protein that couples the receptor to phospholipase C, also responded to stimulation with cAMP, the Ca2+ influx does not seem to be triggered by the phosphoinositide signaling cascade.(ABSTRACT TRUNCATED AT 250 WORDS)