Extracellular Agonists Research Articles

Gelsolin controls the release of phosphatidylserine (PS)-positive microvesicles (MVs) from platelets

Upon activation by vascular injury or extracellular agonists, platelets undergo rapid change shape, a process regulated by the actin cytoskeleton and accessory proteins. Platelet shape change is accompanied by the secretion of hemostatic factors and immunomodulatory cytokines from their intracellular granules, as well as the release of microvesicles (MVs) containing pro-inflammatory cytokines and procoagulant phosphatidylserine (PS). However, the role of actin dynamics in MV generation remains unclear. In this study, we found that blocking actin polymerization with cytochalasin D attenuated the release of PS-positive MVs in human platelets stimulated by thrombin or the calcium ionophore A23187. The actin-severing protein gelsolin (Gsn) facilitates normal actin filament turnover in activated platelets. Platelets from Gsn-deficient (Gsn−/−) mice showed reduced MV release compared to platelets from control mice. These findings indicate that the proper dynamics of the actin cytoskeleton are essential for MV generation in platelets, which has implications for their pro-inflammatory and procoagulant functions.

Loss of INPP5K attenuates IP3-induced Ca2+ responses in the glioblastoma cell line U-251 MG cells

INPP5K (inositol polyphosphate 5-phosphatase K) is an endoplasmic reticulum (ER)-resident enzyme that acts as a phosphoinositide (PI) 5-phosphatase, capable of dephosphorylating various PIs including PI 4,5-bisphosphate (PI(4,5)P2), a key phosphoinositide found in the plasma membrane. Given its ER localization and substrate specificity, INPP5K may play a role in ER-plasma membrane contact sites. Furthermore, PI(4,5)P2 serves as a substrate for phospholipase C, an enzyme activated downstream of extracellular agonists acting on Gq-coupled receptors or tyrosine-kinase receptors, leading to IP3 production and subsequent release of Ca2+ from the ER, the primary intracellular Ca2+ storage organelle. In this study, we investigated the impact of INPP5K on ER Ca2+ dynamics using a previously established INPP5K-knockdown U-251 MG glioblastoma cell model. We here describe that loss of INPP5K impairs agonist-induced, IP3 receptor (IP3R)-mediated Ca2+ mobilization in intact cells, while the ER Ca2+ content and store-operated Ca2+ influx remain unaffected. To further elucidate the underlying mechanisms, we examined Ca2+ release in permeabilized cells stimulated with exogenous IP3. Interestingly, the absence of INPP5K also disrupted IP3-induced Ca2+ release events. These results suggest that INPP5K may directly influence IP3R activity through mechanisms yet to be resolved. The findings from this study point towards role of INPP5K in modulating ER calcium dynamics, particularly in relation to IP3-mediated signaling pathways. However, further work is needed to establish the general nature of our findings and to unravel the exact molecular mechanisms underlying the interplay between INNP5K function and Ca2+ signaling.

Pharmacological potentiators of the calcium signaling cascade identified by high-throughput screening.

Pharmacological modulators of the Ca2+ signaling cascade are important research tools and may translate into novel therapeutic strategies for a series of human diseases. We carried out a screening of a maximally diverse chemical library using the Ca2+-sensitive Cl- channel TMEM16A as a functional readout. We found compounds that were able to potentiate UTP-dependent TMEM16A activation. Mechanism of action of these compounds was investigated by a panel of assays that looked at intracellular Ca2+ mobilization triggered by extracellular agonists or by caged-IP3 photolysis, PIP2 breakdown by phospholipase C, and ion channel activity on nuclear membrane. One compound appears as a selective potentiator of inositol triphosphate receptor type 1 (ITPR1) with a possible application for some forms of spinocerebellar ataxia. A second compound is instead a potentiator of the P2RY2 purinergic receptor, an activity that could promote fluid secretion in dry eye and chronic obstructive respiratory diseases.

Towards new definitions of avoidable hospital admissions

Acidosis is an important complication of AKI and CKD. Renal intercalated cells (ICs) express the proton pumping vacuolar H⁺-ATPase (V-ATPase) and are extensively involved in acid-base homeostasis. H⁺ secretion in type A intercalated cells (A-ICs) is regulated by apical vesicle recycling and stimulated by cAMP. In other cell types, cAMP is increased by extracellular agonists, including adenosine, through purinergic receptors. Adenosine is a Food and Drug Administration–approved drug, but very little is known about the effect of adenosine on IC function. Therefore, we investigated the role of adenosine in the regulation of V-ATPase in ICs. Intravenous treatment of mice with adenosine or agonists of ADORA2A and ADORA2B purinergic P1 receptors induced V-ATPase apical membrane accumulation in medullary A-ICs but not in cortical A-ICs or other IC subtypes. Both receptors are located in A-IC apical membranes, and adenosine injection increased urine adenosine concentration and decreased urine pH. Cell fractionation showed that adenosine or an ADORA2A or ADORA2B agonist induced V-ATPase translocation from vesicles to the plasma membrane and increased protein kinase A (PKA)–dependent protein phosphorylation in purified medullary ICs that were isolated from mice. Either ADORA2A or ADORA2B antagonists or the PKA inhibitor mPKI blocked these effects. Finally, a fluorescence pH assay showed that adenosine activates V-ATPase in isolated medullary ICs. Our study shows that medullary A-ICs respond to luminal adenosine through ADORA2A and ADORA2B receptors in a cAMP/PKA pathway–dependent mechanism to induce V-ATPase–dependent H⁺ secretion.

Using cAMP Sensors to Study Cardiac Nanodomains.

3′,5′-cyclic adenosine monophosphate (cAMP) signalling plays a major role in the cardiac myocyte response to extracellular stimulation by hormones and neurotransmitters. In recent years, evidence has accumulated demonstrating that the cAMP response to different extracellular agonists is not uniform: depending on the stimulus, cAMP signals of different amplitudes and kinetics are generated in different subcellular compartments, eliciting defined physiological effects. In this review, we focus on how real-time imaging using fluorescence resonance energy transfer (FRET)-based reporters has provided mechanistic insight into the compartmentalisation of the cAMP signalling pathway and allowed for the precise definition of the regulation and function of subcellular cAMP nanodomains.

Extracellular Adenosine Stimulates Vacuolar ATPase-Dependent Proton Secretion in Medullary Intercalated Cells.

Acidosis is an important complication of AKI and CKD. Renal intercalated cells (ICs) express the proton pumping vacuolar H+-ATPase (V-ATPase) and are extensively involved in acid-base homeostasis. H+ secretion in type A intercalated cells (A-ICs) is regulated by apical vesicle recycling and stimulated by cAMP. In other cell types, cAMP is increased by extracellular agonists, including adenosine, through purinergic receptors. Adenosine is a Food and Drug Administration-approved drug, but very little is known about the effect of adenosine on IC function. Therefore, we investigated the role of adenosine in the regulation of V-ATPase in ICs. Intravenous treatment of mice with adenosine or agonists of ADORA2A and ADORA2B purinergic P1 receptors induced V-ATPase apical membrane accumulation in medullary A-ICs but not in cortical A-ICs or other IC subtypes. Both receptors are located in A-IC apical membranes, and adenosine injection increased urine adenosine concentration and decreased urine pH. Cell fractionation showed that adenosine or an ADORA2A or ADORA2B agonist induced V-ATPase translocation from vesicles to the plasma membrane and increased protein kinase A (PKA)-dependent protein phosphorylation in purified medullary ICs that were isolated from mice. Either ADORA2A or ADORA2B antagonists or the PKA inhibitor mPKI blocked these effects. Finally, a fluorescence pH assay showed that adenosine activates V-ATPase in isolated medullary ICs. Our study shows that medullary A-ICs respond to luminal adenosine through ADORA2A and ADORA2B receptors in a cAMP/PKA pathway-dependent mechanism to induce V-ATPase-dependent H+ secretion.

A Chinese Perspective on Receptors and Receptor Regulation

A receptor is a protein molecule that receives chemical signals from outside a cell, which enables the cell to respond to the signal molecule. Receptors mediate numerous important physiologic effects upon binding extracellular agonists. However, sustained activation of the receptor may lead to pathologic effects. Cells can regulate the number and function of receptors to alter their sensitivity to different molecules by a feedback mechanism, such as change in the receptor conformation, uncoupling of the receptor effector molecules, receptor sequestration, etc. In this special issue, some Chinese scientists were invited to contribute impactful discoveries and insightful reviews in the field of molecular pharmacology, especially receptor and receptor regulation.

Comparison of Ca2+ puffs evoked by extracellular agonists and photoreleased IP3

The inositol trisphosphate (IP3) signaling pathway evokes local Ca2+ signals (Ca2+ puffs) that arise from the concerted openings of clustered IP3 receptor/channels in the ER membrane. Physiological activation is triggered by binding of agonists to G-protein-coupled receptors (GPCRs) on the cell surface, leading to cleavage of phosphatidyl inositol bisphosphate and release of IP3 into the cytosol. Photorelease of IP3 from a caged precursor provides a convenient and widely employed means to study the final stage of IP3-mediated Ca2+ liberation, bypassing upstream signaling events to enable more precise control of the timing and relative concentration of cytosolic IP3. Here, we address whether Ca2+ puffs evoked by photoreleased IP3 fully replicate those arising from physiological agonist stimulation. We imaged puffs in individual SH-SY5Y neuroblastoma cells that were sequentially stimulated by picospritzing extracellular agonist (carbachol, CCH or bradykinin, BK) followed by photorelease of a poorly-metabolized IP3 analog, i-IP3. The centroid localizations of fluorescence signals during puffs evoked in the same cells by agonists and photorelease substantially overlapped (within ∼1μm), suggesting that IP3 from both sources accesses the same, or closely co-localized clusters of IP3Rs. Moreover, the time course and spatial spread of puffs evoked by agonists and photorelease matched closely. Because photolysis generates IP3 uniformly throughout the cytoplasm, our results imply that IP3 generated in SH-SY5Y cells by activation of receptors to CCH and BK also exerts broadly distributed actions, rather than specifically activating a subpopulation of IP3Rs that are scaffolded in close proximity to cell surface receptors to form a signaling nanodomain.

Allosteric coupling from G protein to the agonist-binding pocket in GPCRs.

G protein-coupled receptors (GPCRs) remain the primary conduit by which cells detect environmental stimuli and communicate with each other1. Upon activation by extracellular agonists, these seven transmembrane domain (7TM)-containing receptors interact with heterotrimeric G proteins to regulate downstream second messenger and/or protein kinase cascades1. Crystallographic evidence from a prototypic GPCR, the β2-adrenergic receptor (β2AR), in complex with its cognate G protein, Gs, has provided a model for how agonist binding promotes conformational changes that propagate through the GPCR and into the nucleotide binding pocket of the G protein α-subunit to catalyze GDP release, the key step required for GTP binding and activation of G proteins2. The structure also offers hints on how G protein binding may, in turn, allosterically influence ligand binding. Here we provide functional evidence that G protein coupling to β2AR stabilizes a ‘closed’ receptor conformation characterized by restricted access to and egress from the hormone binding site. Surprisingly, the effects of G protein on the hormone binding site can be observed in the absence of a bound agonist, where G protein coupling driven by basal receptor activity impedes the association of agonists, partial agonists, antagonists and inverse agonists. The ability of bound ligands to dissociate from the receptor is also hindered, providing a structural explanation for the G protein-mediated enhancement of agonist affinity, which has been observed for many GPCR-G protein pairs. Our studies also suggest that in contrast to agonist binding alone, coupling of a G protein in the absence of an agonist stabilizes large structural changes in a GPCR. The effects of nucleotide-free G protein on ligand binding kinetics are shared by other members of the superfamily of GPCRs, suggesting that a common mechanism may underlie G protein-mediated enhancement of agonist affinity.

PI3K/Akt in platelet integrin signaling and implications in thrombosis.

Blood platelets are anucleated circulating cells that play a critical role in hemostasis and are also implicated in arterial thrombosis, a major cause of death worldwide. The biological function of platelets strongly relies in their reactiveness to a variety of extracellular agonists that regulate their adhesion to extracellular matrix at the site of vascular injury and their ability to form rapidly growing cell aggregates. Among the membrane receptors expressed on the cell surface, integrins are crucial for both platelet activation, adhesion and aggregation. Integrin affinity for specific ligands is regulated by intracellular signaling pathways activated in stimulated platelets, and, once engaged, integrins themselves generate and propagate signals inside the cells to reinforce and consolidate platelet response and thrombus formation. Phosphatidylinositol 3-Kinases (PI3Ks) have emerged as crucial players in platelet activation, and they are directly implicated in the regulation of integrin function. This review will discuss the contribution of PI3Ks in platelet integrin signaling, focusing on the role of specific members of class I PI3Ks and their downstream effector Akt on both integrin inside-out and outside-in signaling. The contribution of the PI3K/Akt pathways stimulated by integrin engagement and platelet activation in thrombus formation and stabilization will also be discussed in order to highlight the possibility to target these enzymes in effective anti-thrombotic therapeutic strategies.

Role of Cytosolic Phospholipase A2 in Oxidative and Inflammatory Signaling Pathways in Different Cell Types in the Central Nervous System

Phospholipases A(2) (PLA(2)s) are important enzymes for the metabolism of fatty acids in membrane phospholipids. Among the three major classes of PLA(2)s in the mammalian system, the group IV calcium-dependent cytosolic PLA(2) alpha (cPLA(2)α) has received the most attention because it is widely expressed in nearly all mammalian cells and its active participation in cell metabolism. Besides Ca(2+) binding to its C2 domain, this enzyme can undergo a number of cell-specific post-translational modifications, including phosphorylation by protein kinases, S-nitrosylation through interaction with nitric oxide (NO), as well as interaction with other proteins and lipid molecules. Hydrolysis of phospholipids by cPLA(2) yields two important lipid mediators, arachidonic acid (AA) and lysophospholipids. While AA is known to serve as a substrate for cyclooxygenases and lipoxygenases, which are enzymes for the synthesis of eicosanoids and leukotrienes, lysophospholipids are known to possess detergent-like properties capable of altering microdomains of cell membranes. An important feature of cPLA(2) is its link to cell surface receptors that stimulate signaling pathways associated with activation of protein kinases and production of reactive oxygen species (ROS). In the central nervous system (CNS), cPLA(2) activation has been implicated in neuronal excitation, synaptic secretion, apoptosis, cell-cell interaction, cognitive and behavioral function, oxidative-nitrosative stress, and inflammatory responses that underline the pathogenesis of a number of neurodegenerative diseases. However, the types of extracellular agonists that target intracellular signaling pathways leading to cPLA(2) activation among different cell types and under different physiological and pathological conditions have not been investigated in detail. In this review, special emphasis is given to metabolic events linking cPLA(2) to activation in neurons, astrocytes, microglial cells, and cerebrovascular cells. Understanding the molecular mechanism(s) for regulation of this enzyme is deemed important in the development of new therapeutic targets for the treatment and prevention of neurodegenerative diseases.

Gene Expression Modulation of Two Biosynthesis Pathways via Signal Transduction in <i>Cochliobolus heterostrophus</i>

G-protein-linked pathways have evolved to allow responses to extracellular agonists (hormones, neurotransmitters, odors, chemoattractants, light and nutrients) in eukaryotic cells, ranging from simpler systems, including yeasts, filamentous fungi and slime molds, to more complex organisms, such as mammals. Although the role of G-protein and mitogen-activated protein kinase (MAPK) in filamentous fungi has been studied for over a decade, downstream elements are less known, and the study of target genes has evolved mainly in recent years. Here, we examined the involvement of G-protein subunits and MAPK in controlling the expression of two distinct target genes. These genes were selected from an array database according to their unique expression profile and the role of closely related genes found in other Ascomycetes. One of these genes is BPH, which encodes the enzyme responsible for cytochrome P450-dependent benzoate hydroxylation in microsomes. The other gene is CIPA, which encodes isoflavone reductase (IfR), an enzyme involved in the synthesis of phytoalexin, which catalyzes an intermediate step in pisatin biosynthesis. The expression profile of these two genes was determined in a series of signaling deficiency mutants that were grown on different media using a DNA microarray. Comparison of the expression profile in the two wild type strains and mutants deficient in the G-protein α or β subunits or in MAPK, revealed a unique control mechanism for the BPH and CIPA genes. The two genes are highly expressed during the infection of the host plant leaves and may associate with the fungal response to the host. Signaling via G-protein or MAPK was shown to be related to cascades that altered the expression of these genes in response to the growth condition. This work demonstrates that signal transduction pathways are controlling genes that, although sharing an environmental dependent response, participate in distinct biosynthesis pathways. Moreover, the transcriptional profile may point to distinct and shared roles of the signaling components.

Exposure of MC4R to agonist in the endoplasmic reticulum stabilizes an active conformation of the receptor that does not desensitize

Melanocortin-4 receptor (MC4R) is a G protein-coupled receptor expressed in neurons of the hypothalamus where it regulates food intake. MC4R responds to an agonist, α-melanocyte-stimulating hormone (α-MSH) and to an antagonist/inverse agonist, agouti-related peptide (AgRP), which are released by upstream neurons. Binding to α-MSH leads to stimulation of receptor activity and suppression of food intake, whereas AgRP has opposite effects. MC4R cycles constantly between the plasma membrane and endosomes and undergoes agonist-mediated desensitization by being routed to lysosomes. MC4R desensitization and increased AgRP expression are thought to decrease the effectiveness of MC4R agonists as an antiobesity treatment. In this study, α-MSH, instead of being delivered extracellularly, is targeted to the endoplasmic reticulum (ER) of neuronal cells and cultured hypothalamic neurons. We find that the ER-targeted agonist associates with MC4R at this location, is transported to the cell surface, induces constant cAMP and AMP kinase signaling at maximal amplitude, abolishes desensitization of the receptor, and promotes both cell-surface expression and constant signaling by an obesity-linked MC4R variant, I316S, that otherwise is retained in the ER. Formation of the MC4R/agonist complex in the ER stabilizes the receptor in an active conformation that at the cell surface is insensitive to antagonism by AgRP and at the endosomes is refractory to routing to the lysosomes. The data indicate that targeting agonists to the ER can stabilize an active conformation of a G protein-coupled receptor that does not become desensitized, suggesting a target for therapy.

Dueling nucleosides: cross-regulation of extracellular adenosine by guanosine. Focus on “Extracellular guanosine regulates extracellular adenosine levels”

<i>Dueling nucleosides: cross-regulation of extracellular adenosine by guanosine</i>. Focus on “Extracellular guanosine regulates extracellular adenosine levels”

Generation and characterization of a lysosomally targeted, genetically encoded Ca2+-sensor

Distinct spatiotemporal Ca2+ signalling events regulate fundamental aspects of eukaryotic cell physiology. Complex Ca2+ signals can be driven by release of Ca2+ from intracellular organelles that sequester Ca2+ such as the ER (endoplasmic reticulum) or through the opening of Ca2+-permeable channels in the plasma membrane and influx of extracellular Ca2+. Late endocytic pathway compartments including late-endosomes and lysosomes have recently been observed to sequester Ca2+ to levels comparable with those found within the ER lumen. These organelles harbour ligand-gated Ca2+-release channels and evidence indicates that they can operate as Ca2+-signalling platforms. Lysosomes sequester Ca2+ to a greater extent than any other endocytic compartment, and signalling from this organelle has been postulated to provide ‘trigger’ release events that can subsequently elicit more extensive Ca2+ signals from stores including the ER. In order to investigate lysosomal-specific Ca2+ signalling a simple method for measuring lysosomal Ca2+ release is essential. In the present study we describe the generation and characterization of a genetically encoded, lysosomally targeted, cameleon sensor which is capable of registering specific Ca2+ release in response to extracellular agonists and intracellular second messengers. This probe represents a novel tool that will permit detailed investigations examining the impact of lysosomal Ca2+ handling on cellular physiology.

Risk of Intracranial Hemorrhage With Protease-Activated Receptor-1 Antagonists

See related article, p 3189. Acute clinical syndromes caused by atherosclerotic disease of the arterial wall are among the world’s leading causes of death and disability.1 These conditions include acute coronary syndromes, ischemic stroke/transient ischemic attack, and symptomatic peripheral arterial disease. As average life expectancy continues to rise, the burden posed by these diseases is expected to increase.2 Antiplatelet therapy plays a pivotal role in primary and secondary prevention of acute atherothrombotic events by targeting platelet activation, a mechanism that mediates both the beneficial (hemostatic) and detrimental (thrombotic) effect of platelets. This activation process occurs through several pathways, each targeted by different classes of drugs.3 Platelet activation can be triggered by three main extracellular agonists: thromboxane A2, adenosine diphosphate, and thrombin. These agonists activate the platelet integrin glycoprotein IIb/IIIa, a main receptor for both adhesion and aggregation. Endothelial-derived factors limit platelet aggregation by increasing intracellular levels of cyclic nucleotides (cyclic guanosine monophosphate and cyclic adenosine monophosphate).4 The most commonly used oral antiplatelet medications are inhibitors of the thromboxane A2 (aspirin) and adenosine diphosphate (P2Y12 inhibitors: clopidogrel, ticlopidine, and prasugrel) pathways. A third group of drugs acts on the cyclic nucleotide-dependent regulation step by inhibiting the phosphodiesterase responsible for the degradation of cyclic nucleotides (dipyridamole and cilostazol), and a fourth group directly inhibits the glycoprotein IIb/IIIa (abciximab, eptifibatide, and tirofiban).5 In …

Action of Molecular Switches in GPCRs - Theoretical and Experimental Studies

G protein coupled receptors (GPCRs), also called 7TM receptors, form a huge superfamily of membrane proteins that, upon activation by extracellular agonists, pass the signal to the cell interior. Ligands can bind either to extracellular N-terminus and loops (e.g. glutamate receptors) or to the binding site within transmembrane helices (Rhodopsin-like family). They are all activated by agonists although a spontaneous auto-activation of an empty receptor can also be observed. Biochemical and crystallographic methods together with molecular dynamics simulations and other theoretical techniques provided models of the receptor activation based on the action of so-called “molecular switches” buried in the receptor structure. They are changed by agonists but also by inverse agonists evoking an ensemble of activation states leading toward different activation pathways. Switches discovered so far include the ionic lock switch, the 3-7 lock switch, the tyrosine toggle switch linked with the nPxxy motif in TM7, and the transmission switch. The latter one was proposed instead of the tryptophan rotamer toggle switch because no change of the rotamer was observed in structures of activated receptors. The global toggle switch suggested earlier consisting of a vertical rigid motion of TM6, seems also to be implausible based on the recent crystal structures of GPCRs with agonists. Theoretical and experimental methods (crystallography, NMR, specific spectroscopic methods like FRET/BRET but also single-molecule-force-spectroscopy) are currently used to study the effect of ligands on the receptor structure, location of stable structural segments/domains of GPCRs, and to answer the still open question on how ligands are binding: either via ensemble of conformational receptor states or rather via induced fit mechanisms. On the other hand the structural investigations of homo- and heterodimers and higher oligomers revealed the mechanism of allosteric signal transmission and receptor activation that could lead to design highly effective and selective allosteric or ago-allosteric drugs.

Voltage-Dependent Modulation of α2A Adrenergic Receptor Conformations

G protein-coupled receptors (GPCRs) are proteins that span the cell membrane seven times. They are stimulated by extracellular agonists and activate heterotrimeric G proteins to elicit intracellular responses. Recent evidence suggests that GPCRs are voltage-sensitive: They exhibit gating currents similar to ion channels and respond to depolarization of the plasma membrane with changes in agonist affinity. We used a FRET-based biosensor of the α2A adrenergic receptor to analyze voltage dependence of receptor activation in HEK 293 cells by means of voltage-clamp recording. The biosensor was stimulated either with the partial agonist clonidine or with the full agonist norepinephrine (NE) and receptor activation was measured as decrease in FRET ratio. Receptor stimulation by NE was reduced by membrane depolarization and enhanced by hyperpolarization. This effect was present in wilde-type receptors and transduced to the level of G protein activation, which we determined in a FRET assay that directly detects Gαi protein activation. Depolarization-mediated inhibition of NE activated receptors was strong at low concentrations (500 nM: 60 % inhibition) but almost undetectable at saturating agonist concentrations (100 μM: 9 % inhibition). Both agonist-induced and hyperpolarization-induced receptor activation exhibited a similar monoexponential time course. For both activation modes, speed of activation was primarily dependent on agonist concentration, indicating that depolarization lowers the apparent affinity of the NE receptor interaction and thus causes receptor deactivation by means of NE release. Application of clonidine (1 μM, VM=-90 mV) resulted in a FRET response that was inhibited by 40 % at +60 mV. In contrast to NE, strong receptor-inhibition at +60 mV was present even at super-saturating concentrations of clonidine (100 μM). Therefore we conclude that negative membrane potentials promote active conformations of the α2A adrenergic receptor, increase affinity of full agonists and enhance G protein signaling.

The Small G Protein Rac1 Activates Phospholipase Cδ1 through Phospholipase Cβ2

Rac1, which is associated with cytoskeletal pathways, can activate phospholipase Cbeta2 (PLCbeta2) to increase intracellular Ca(2+) levels. This increased Ca(2+) can in turn activate the very robust PLCdelta1 to synergize Ca(2+) signals. We have previously found that PLCbeta2 will bind to and inhibit PLCdelta1 in solution by an unknown mechanism and that PLCbeta2.PLCdelta1 complexes can be disrupted by Gbetagamma subunits. However, because the major populations of PLCbeta2 and PLCdelta1 are cytosolic, their regulation by Gbetagamma subunits is not clear. Here, we have found that the pleckstrin homology (PH) domains of PLCbeta2 and PLCbeta3 are the regions that result in PLCdelta1 binding and inhibition. In cells, PLCbeta2.PLCdelta1 form complexes as seen by Förster resonance energy transfer and co-immunoprecipitation, and microinjection of PHbeta2 dissociates the complex. Using PHbeta2 as a tool to assess the contribution of PLCbeta inhibition of PLCdelta1 to Ca(2+) release, we found that, although PHbeta2 only results in a 25% inhibition of PLCdelta1 in solution, in cells the presence of PHbeta2 appears to eliminates Ca(2+) release suggesting a large threshold effect. We found that the small plasma membrane population of PLCbeta2.PLCdelta1 is disrupted by activation of heterotrimeric G proteins, and that the major cytosolic population of the complexes are disrupted by Rac1 activation. Thus, the activity of PLCdelta1 is controlled by the amount of bound PLCbeta2 that changes with displacement of the enzyme by heterotrimeric or small G proteins. Through PLCbeta2, PLCdelta1 activation is linked to surface receptors as well as signals that mediate cytoskeletal pathways.

Cross-Talk between Signaling Pathways Can Generate Robust Oscillations in Calcium and cAMP

BackgroundTo control and manipulate cellular signaling, we need to understand cellular strategies for information transfer, integration, and decision-making. A key feature of signal transduction is the generation of only a few intracellular messengers by many extracellular stimuli.Methodology/Principal FindingsHere we model molecular cross-talk between two classic second messengers, cyclic AMP (cAMP) and calcium, and show that the dynamical complexity of the response of both messengers increases substantially through their interaction. In our model of a non-excitable cell, both cAMP and calcium concentrations can oscillate. If mutually inhibitory, cross-talk between the two second messengers can increase the range of agonist concentrations for which oscillations occur. If mutually activating, cross-talk decreases the oscillation range, but can generate ‘bursting’ oscillations of calcium and may enable better filtering of noise.ConclusionWe postulate that this increased dynamical complexity allows the cell to encode more information, particularly if both second messengers encode signals. In their native environments, it is unlikely that cells are exposed to one stimulus at a time, and cross-talk may help generate sufficiently complex responses to allow the cell to discriminate between different combinations and concentrations of extracellular agonists.