Activation Of Galpha Research Articles

RGS2 Modulation of Angiotensin II Receptor Type 1 Trafficking and Signaling in Neuroblastoma Cells

IntroductionAltered expression and function of brain angiotensin II receptor type 1 (AT1R) has been implicated in the pathophysiology of various neuropsychiatric diseases such as drug addiction and Alzheimer's disease. However, there is a knowledge gap in understanding the molecular mechanisms of altered AT1R trafficking and function. Regulators of G‐protein signaling (RGS) proteins are negative modulators of G‐protein coupled receptors (GPCRs). Mechanistically, RGS proteins terminate signaling by accelerating the hydrolysis of GTP bound to active G‐alpha subunits. Among all the RGS subtypes, our lab found that RGS2 co‐localizes with AT1R in the brain, suggesting a potential role of RGS2 regulation of AT1R signaling. Additionally, our lab previously found that RGS2 regulates the constitutive and agonist‐induced trafficking of dopamine D2 receptors. Therefore, this project assessed the role of RGS2 in regulation of AT1R trafficking and signaling using a neuroblastoma 2a (N2A) cell model. Knowledge learned from this project may be relevant to the physiological function of RGS2 in the brain.MethodsWe generated stable expression of the human Angiotensin Type I receptor (HA‐AT1R) in N2A cells. N2A‐AT1R cells were then transiently transfected with RGS2 siRNA to knock down RGS2 expression. Cells were then treated with vehicle or angiotensin II (AngII, 10 μM) for 2, 5, or 15 minutes for western blotting of phosphorylated Akt and total Akt. Immunocytochemistry and confocal microscopy were performed on cells treated with AngII for various times to measure the trafficking of AT1R.ResultsWe found that the knockdown of RGS2 enhanced AT1R‐mediated signaling by increasing Akt phosphorylation. RGS2 knockdown also showed to increase AngII‐induced internalization of AT1R. This is the first report on the role of RGS2 in regulation of AT1R trafficking. Further study is necessary to investigate the molecular mechanisms underlying RGS2 modulation of the signaling and trafficking of AT1R in neuronal cells.Support or Funding InformationThis work is supported by NIH R01DA042862, P50DA006634, F31AA025532This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

P.1.g.032 - Receptor-mediated activation of Galpha(q) and Galpha(i-3) assessed by [35S]GTPgammaS binding/immunoprecipitation assay in postmortem human brain membranes

The recent discovery of allosteric potentiators and agonists of the muscarinic M1 receptor represents a significant advance in the muscarinic receptor pharmacology. In the current study we describe the receptor pharmacology and pro-cognitive action of the allosteric agonist AC-260584. Using in vitro cell-based assays with cell proliferation, phosphatidylinositol hydrolysis or calcium mobilization as endpoints, AC-260584 was found to be a potent (pEC50 7.6–7.7) and efficacious (90–98% of carbachol) muscarinic M1 receptor agonist. Furthermore, as compared to orthosteric binding agonists, AC-260584 showed functional selectivity for the M1 receptor over the M2, M3, M4 and M5 muscarinic receptor subtypes. Using GTPγS binding assays, its selectivity was found to be similar in native tissues expressing mAChRs to its profile in recombinant systems. In rodents, AC-260584 activated extracellular signal-regulated kinase 1 and 2 (ERK1/2) phosphorylation in the hippocampus, prefrontal cortex and perirhinal cortex. The ERK1/2 activation was dependent upon muscarinic M1 receptor activation since it was not observed in M1 knockout mice. AC-260584 also improved the cognitive performance of mice in the novel object recognition assay and its action is blocked by the muscarinic receptor antagonist pirenzepine. Taken together these results indicate for the first time that a M1 receptor agonist selective over the other mAChR subtypes can have a symptomatically pro-cognitive action. In addition, AC-260584 was found to be orally bioavailable in rodents. Therefore, AC-260584 may serve as a lead compound in the development of M1 selective drugs for the treatment of cognitive impairment associated with schizophrenia and Alzheimer's disease.

Cardiac overexpression of constitutively active Galpha q causes angiotensin II type1 receptor activation, leading to progressive heart failure and ventricular arrhythmias in transgenic mice.

BackgroundTransgenic mice with transient cardiac expression of constitutively active Galpha q (Gαq-TG) exhibt progressive heart failure and ventricular arrhythmias after the initiating stimulus of transfected constitutively active Gαq becomes undetectable. However, the mechanisms are still unknown. We examined the effects of chronic administration of olmesartan on heart failure and ventricular arrhythmia in Gαq-TG mice.Methodology/Principal FindingsOlmesartan (1 mg/kg/day) or vehicle was chronically administered to Gαq-TG from 6 to 32 weeks of age, and all experiments were performed in mice at the age of 32 weeks. Chronic olmesartan administration prevented the severe reduction of left ventricular fractional shortening, and inhibited ventricular interstitial fibrosis and ventricular myocyte hypertrophy in Gαq-TG. Electrocardiogram demonstrated that premature ventricular contraction (PVC) was frequently (more than 20 beats/min) observed in 9 of 10 vehicle-treated Gαq-TG but in none of 10 olmesartan-treated Gαq-TG. The collected QT interval and monophasic action potential duration in the left ventricle were significantly shorter in olmesartan-treated Gαq-TG than in vehicle-treated Gαq-TG. CTGF, collagen type 1, ANP, BNP, and β-MHC gene expression was increased and olmesartan significantly decreased the expression of these genes in Gαq-TG mouse ventricles. The expression of canonical transient receptor potential (TRPC) 3 and 6 channel and angiotensin converting enzyme (ACE) proteins but not angiotensin II type 1 (AT1) receptor was increased in Gαq-TG ventricles compared with NTG mouse ventricles. Olmesartan significantly decreased TRPC6 and tended to decrease ACE expressions in Gαq-TG. Moreover, it increased AT1 receptor in Gαq-TG.Conclusions/SignificanceThese findings suggest that angiotensin II type 1 receptor activation plays an important role in the development of heart failure and ventricular arrhythmia in Gαq-TG mouse model of heart failure.

Abstract 037: Transient Cardiac Expression Of Constitutively Active G alpha Q Activates Renin-angiotensin System, Leading To Progressive Heart Failure And Ventricular Arrhythmias In Transgenic Mice

Introduction: Transgenic mice with transient cardiac expression of constitutively active Galpha q (Gαq-TG) caused progressive heart failure and ventricular arrhythmias after the initiating stimulus becomes undetectable. However, the mechanisms are still unknown. Renin-angiotensin system plays a critical role in the development of cardiac hypertrophy and heart failure. We examined the effects of chronic administration of olmesartan on ventricular function, the number of premature ventricular contractions (PVC), and ventricular remodeling in Gαq-TG mice. Methods and Results: Olmesartan (1 mg/kg/day) or vehicle was chronically administered to Gαq-TG from 6 to 32 weeks of age, and all experiments were performed in mice at the age of 32 weeks. Chronic olmesartan treatment prevented the severe reduction of left ventricular fractional shortening and inhibited ventricular interstitial fibrosis and ventricular myocyte hypertrophy in Gαq-TG. Electrocardiogram demonstrated that premature ventricular contraction (PVC) was frequently (more than 20 beats/min) observed in 9 of 10 vehicle-treated Gαq-TG but in none of 10 olmesartan -treated Gαq-TG. The QT interval was significantly shorter in olmesartan-treated Gαq-TG than vehicle-treated Gαq-TG. CTGF, collagen type 1, ANP, BNP, and β-MHC gene expression was increased in vehicle-treated Gαq-TG. Olmesartan significantly decreased these gene expressions in Gαq-TG. Moreover, protein expressions of canonical transient receptor potential (TRPC) channels 3 and 6 increased in vehicle-treated Gαq-TG hearts. Olmesartan significantly decreased TRPC6 expressions in Gαq-TG. Angiotensin converting enzyme (ACE) 1 and 2 gene expressions were also increased in vehicle-treated Gαq-TG and was not decreased to the control level in olmesartan-treated Gαq-TG. Conclusions: These findings suggest that renin-angiotensin system has an important role in the development of cardiac hypertrophy and heart failure even if the initiating stimulus is different from the activation of renin-angiotensin system.

New insights into the mechanism of light modulated signaling by heterotrimeric G-proteins: ENVOY acts on gna1 and gna3 and adjusts cAMP levels in Trichoderma reesei (Hypocrea jecorina)

Sensing of environmental signals is often mediated by G-protein coupled receptors and their cognate heterotrimeric G-proteins. In Trichoderma reesei (Hypocrea jecorina) the signals transmitted via the G-protein alpha subunits GNA1 and GNA3 cause considerable modulation of cellulase transcript levels and the extent of this adjustment is dependent on the light status. We therefore intended to elucidate the underlying mechanism connecting light response and heterotrimeric G-protein signaling.Analysis of double mutant strains showed that constitutive activation of GNA1 or GNA3 in the absence of the PAS/LOV domain protein ENVOY (ENV1) leads to the phenotype of constitutive G-alpha activation in darkness. In light, however the deletion-phenotype of Δenv1 was observed with respect to growth, conidiation and cellulase gene transcription. Additionally deletion of env1 causes decreased intracellular cAMP accumulation, even upon constitutive activation of GNA1 or GNA3. While supplementation of cAMP caused an even more severe growth phenotype of all strains lacking env1 in light, addition of the phosphodiesterase inhibitor caffeine rescued the growth phenotype of these strains.ENV1 is consequently suggested to connect the light response pathway with nutrient signaling by the heterotrimeric G-protein cascade by adjusting transcript levels of gna1 and gna3 and action on cAMP levels – presumably through inhibition of a phosphodiesterase.

A screen for genes involved in respiration control and longevity in Schizosaccharomyces pombe

We present results showing that glucose signaling has proaging effects in the yeast Schizosaccharomyces pombe. Deletion of the receptor that senses extracellular glucose (Git3) increases the life span of S. pombe, while constitutive activation of the Galpha subunit acting downstream of this receptor (Gpa2) shortens its life span. The latter mutant is also impaired for growth under respiration conditions. We have used this phenotype in a selection strategy to identify genes that when overexpressed can rescue the respiratory defect of constitutively active Galpha subunit mutants. Here, we report an extended version of the work we presented at the IABG meeting and the results of this screen. This strategy allowed us to isolate four genes: psp1(+)/moc1(+), cka1(+), adh1(+), and rpb10(+). Interestingly, the overexpression of these genes was also capable of increasing the chronological life span of wild-type yeast cells.

CCK activates RhoA and Rac1 differentially through Gα13 and Gαq in mouse pancreatic acini

Cholecystokinin (CCK) has been shown to activate RhoA and Rac1, as well as reorganize the actin cytoskeleton and, thereby, modify acinar morphology and amylase secretion in mouse pancreatic acini. The aim of the present study was to determine which heterotrimeric G proteins activate RhoA and Rac1 upon CCK stimulation. Galpha(13), but not Galpha(12), was identified in mouse pancreatic acini by RT-PCR and Western blotting. Using specific assays for RhoA and Rac1 activation, we showed that only active Galpha(13) activated RhoA. By contrast, active Galpha(13) and Galpha(q), but not Galpha(s), slightly increased GTP-bound Rac1 levels. A greater increase in Rac1 activation was observed when active Galpha(13) and active Galpha(q) were coexpressed. Galpha(i) was not required for CCK-induced RhoA or Rac1 activation. The regulator of G protein signaling (RGS) domain of p115-Rho guanine nucleotide exchange factor (p115-RGS), a specific inhibitor of Galpha(12/13)-mediated signaling, abolished CCK-stimulated RhoA activation. By contrast, both RGS-2, an inhibitor of Galpha(q), and p115-RGS abolished CCK-induced Rac1 activation, which was PLC pathway-independent. Active Galpha(q) and Galpha(13), but not Galpha(s), induced morphological changes and actin redistribution similar to 1 nM CCK. CCK-induced actin cytoskeletal reorganization was inhibited by RGS-2, but not by p115-RGS, whereas CCK-induced amylase secretion was blocked by both inhibitors. Together, these findings indicate that, in mouse pancreatic acini, Galpha(13) links CCK stimulation to the activation of RhoA, whereas both Galpha(13) and Galpha(q) link CCK stimulation to the activation of Rac1. CCK-induced actin cytoskeletal reorganization is mainly mediated by Galpha(q). By contrast, Galpha(13) and Galpha(q) signaling are required for CCK-induced amylase secretion.

C-terminal deletion of metabotropic glutamate receptor 1 selectively abolishes coupling to Gαq

Recent studies indicate that the intracellular C-terminus of Group I metabotropic glutamate receptors (mGlu(1) and mGlu(5) receptor) is important in G protein coupling. To determine the necessity of the C-tail, a deletion mutant of mGlu(1) receptor was constructed, which included the first 840 amino acids of the rat mGlu(1a) receptor (mGlu(1)-dCT). G protein coupling of the receptors was assessed by measuring glutamate mediated inhibition of native calcium currents when each receptor was expressed in isolated sympathetic neurons from the rat superior cervical ganglion. Wild type mGlu(1) receptor activates both the Galpha(i/o) and Galpha(q/11) protein families. Each pathway can be detected in superior cervical ganglion neurons as voltage dependent and voltage independent inhibition of the calcium currents, respectively. While wild type mGlu(1) receptor gave rise to a strong, mixed voltage dependent and independent calcium current inhibition, mGlu(1)-dCT exhibited a weaker inhibition that was strongly voltage dependent, indicating activation of Galpha(i/o) was predominant. Further, pertussis toxin treatment reduced the inhibition by wild type mGlu(1) receptor to a smaller, voltage independent inhibition as expected, but completely abolished signaling through mGlu(1)-dCT. Finally, to test whether mGlu(1)-dCT could produce any activation of Galpha(q/11), inhibition of the native superior cervical ganglion M-type potassium currents was examined. M-channels, inhibited by PIP(2) depletion, were strongly inhibited by glutamate in cells expressing wild type mGlu(1) receptor, but no inhibition was detectable in neurons expressing mGlu(1)-dCT. These data indicate that C-terminal deletion of mGlu(1) receptor selectively abolishes Galpha(q/11) coupling.

Functional Selectivity of Natural and Synthetic Prostaglandin EP4Receptor Ligands

Classically, the prostaglandin E(2) (PGE(2)) receptor EP(4) has been classified as coupling to the Galpha(s) subunit, leading to intracellular cAMP increases. However, EP(4) signaling has been revealed to be more complex and also involves coupling to pertussis toxin-sensitive Galpha(i) proteins and beta-arrestin-mediated effects. There are now many examples of selective activation of independent pathways by G protein-coupled receptor (GPCR) ligands, a concept referred to as functional selectivity. Because most EP(4) ligands had thus far only been functionally characterized by their ability to stimulate cAMP production, we systematically determined the potencies and efficacies of a panel of EP(4) ligands for activation of Galpha(s), Galpha(i), and beta-arrestin relative to the endogenous ligand PGE(2). For this purpose, we adapted three bioluminescence resonance energy transfer (BRET) assays to evaluate the respective pathways in living cells. Our results suggest considerable functional selectivity among the tested, structurally related agonists. PGE(2) was the most selective in activating Galpha(s), whereas PGF(2alpha) and PGE(1) alcohol were the most biased for activating Galpha(i1) and beta-arrestin, respectively. We observed reversal in order of potencies between beta-arrestin 2 and Galpha(i1) functional assays comparing PGE(1) alcohol and either PGF(2alpha), PGD(2), or 7-[(1R,2R)-2-[(E,3R)-3-hydroxy-4-(phenoxy)but-1-enyl]-5-oxocyclopentyl]heptanoic acid (M&B28767). Most ligands were full agonists for the three pathways tested. Our results have implications for the use of PGE(2) analogs in experimental and possibly clinical settings, because their activity spectra on EP(4) differ from that of the native agonist. The BRET-based methodology used for this first systematic assessment of a set of EP(4) agonists should be applicable for the study of other GPCRs.

Effect of a heterotrimeric G protein alpha subunit on conidia germination, stress response, and roquefortine C production in Penicillium roqueforti.

Heterotrimeric G protein signaling regulates many processes in fungi, such as development, pathogenicity, and secondary metabolite biosynthesis. For example, the Galpha subunit Pga1 from Penicillium chrysogenum regulates conidiation and secondary metabolite production in this fungus. The dominant activating allele, pga1G42R, encoding a constitutively active Pga1 Galpha subunit, was introduced in Penicillium roqueforti by transformation, resulting in a phenotype characterized by low sporulation and slow growth. In this work, the effect of the constitutively active Pga1G42R Galpha subunit on conidial germination, stress tolerance, and roquefortine C production of P. roqueforti was studied. Pga1G42R triggered germination in the absence of a carbon source, in addition to negatively regulating thermal and osmotic stress tolerance. The presence of the Pga1G42R Galpha subunit also had an important effect on roquefortine C biosynthesis, increasing production and maintaining high levels of the mycotoxin throughout a culture period of 30 days. Together, the results suggest that G protein-mediated signaling participates in the regulation of these three processes in P. roqueforti.

Haematopoietic stem cells depend on Gαs-mediated signalling to engraft bone marrow

Hematopoietic stem/progenitor cells (HSPC) transition in location during development1 and circulate in mammals throughout life2, moving into and out of the bloodstream to engage bone marrow (BM) niches in sequential steps of homing, engraftment and retention3–5. We show here that HSPC engraftment of BM in fetal development is dependent upon the guanine nucleotide binding protein stimulatory alpha subunit (Gsα). Adult Gsα−/− HSPCs differentiate and undergo chemotaxis, but also do not home to or engraft in the BM in adult mice and demonstrate marked inability to engage the marrow microvasculature. If deleted after engraftment, Gsα did not lead to lack of retention in the marrow, rather cytokine-induced mobilization into the blood was impaired. Testing whether activation of Gsα affects HSPC, pharmacologic activators enhanced homing and engraftment in vivo. Gsα governs specific aspects of HSPC localization under physiologic conditions in vivo and may be pharmacologically targeted to improve transplantation efficiency.

Histone H3 Phosphorylation is Under the Opposite Tonic Control of Dopamine D2 and Adenosine A2A Receptors in Striatopallidal Neurons

The antipsychotic agent haloperidol regulates gene transcription in striatal medium spiny neurons (MSNs) by blocking dopamine D2 receptors (D2Rs). We examined the mechanisms by which haloperidol increases the phosphorylation of histone H3, a key step in the nucleosomal response. Using bacterial artificial chromosome (BAC)-transgenic mice that express EGFP under the control of the promoter of the dopamine D1 receptor (D1R) or the D2R, we found that haloperidol induced a rapid and sustained increase in the phosphorylation of histone H3 in the striatopallidal MSNs of the dorsal striatum, with no change in its acetylation. This effect was mimicked by raclopride, a selective D2R antagonist, and prevented by the blockade of adenosine A2A receptors (A2ARs), or genetic attenuation of the A2AR-associated G protein, Galpha(olf). Mutation of the cAMP-dependent phosphorylation site (Thr34) of the 32-kDa dopamine and cAMP-regulated phosphoprotein (DARPP-32) decreased the haloperidol-induced H3 phosphorylation, supporting the role of cAMP in H3 phosphorylation. Haloperidol also induced extracellular signal-regulated kinase (ERK) phosphorylation in striatopallidal MSNs, but this effect was not implicated in H3 phosphorylation. The levels of mitogen- and stress-activated kinase 1 (MSK1), which has been reported to mediate ERK-induced H3 phosphorylation, were lower in striatopallidal than in striatonigral MSNs. Moreover, haloperidol-induced H3 phosphorylation was unaltered in MSK1-knockout mice. These data indicate that, in striatopallidal MSNs, H3 phosphorylation is controlled by the opposing actions of D2Rs and A2ARs. Thus, blockade of D2Rs promotes histone H3 phosphorylation through the A2AR-mediated activation of Galpha(olf) and inhibition of protein phosphatase-1 (PP-1) through the PKA-dependent phosphorylation of DARPP-32.

CXCL12-Mediated Induction of Plasminogen Activator Inhibitor-1 Expression in Human CXCR4 Positive Astroglioma Cells

Glioblastoma is the most malignant and common brain tumor. To promote their growth, these glioma cells secrete a variety of soluble factors including plasminogen activator inhibitor-1 (PAI-1), which functions as an inhibitor of plasminogen activators. We report here with the basis of microarray gene expression analysis that CXCR4 expressing glioma cells are capable of expressing PAI-1 mRNA and protein upon CXCL12 stimulation. Pretreatment with U0126, an inhibitor of mitogen activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) kinase (MEK) 1/2, abrogated CXCL12-induced PAI-1 expression. Pertussis toxin (PTX), an inhibitor of Galpha(i) proteins, also had inhibitory effects, indicating that the activation of Galpha(i) and ERK MAPK are required for this response. Interestingly, CXCL12 showed additive effects with another PAI-1 inducers, tumor necrosis factor (TNF)-alpha and/or tumor growth factor (TGF)-beta1, in increasing PAI-1 expression. These results indicate that CXCL12/CXCR4 signaling in glioma cells may be another mechanism for these cells to express PAI-1, which may be involved in angiogenesis and tumor invasion in brain tumors.

Evidence for a Direct and Functional Interaction between the Regulators of G Protein Signaling-2 and Phosphorylated C Terminus of Cholecystokinin-2 Receptor

Signaling of G protein-coupled receptors (GPCRs) is regulated by different mechanisms. One of these involves regulators of G protein signaling (RGS), which are diverse and multifunctional proteins that bind to active Galpha subunits of G proteins and act as GTPase-activating proteins. Little is known about the molecular mechanisms that govern the selective use of RGS proteins in living cells. We first demonstrated that CCK2R-mediated inositol phosphate production, known to be G(q)-dependent, is more sensitive to RGS2 than to RGS4 and is insensitive to RGS8. Both basal and agonist-stimulated activities of the CCK2R are regulated by RGS2. By combining biochemical, functional, and in silico structural approaches, we demonstrate that a direct and functional interaction occurs between RGS2 and agonist-stimulated cholecystokinin receptor-2 (CCK2R) and identified the precise residues involved: phosphorylated Ser434 and Thr439 located in the C-terminal tail of CCK2R and Lys62, Lys63, and Gln67, located in the N-terminal domain of RGS2. These findings confirm previous reports that RGS proteins can interact with GPCRs to modulate their signaling and provide a molecular basis for RGS2 recognition by the CCK2R.

P2Y6 receptor-Gα12/13 signalling in cardiomyocytes triggers pressure overload-induced cardiac fibrosis

Cardiac fibrosis, characterized by excessive deposition of extracellular matrix proteins, is one of the causes of heart failure, and it contributes to the impairment of cardiac function. Fibrosis of various tissues, including the heart, is believed to be regulated by the signalling pathway of angiotensin II (Ang II) and transforming growth factor (TGF)-beta. Transgenic expression of inhibitory polypeptides of the heterotrimeric G12 family G protein (Galpha(12/13)) in cardiomyocytes suppressed pressure overload-induced fibrosis without affecting hypertrophy. The expression of fibrogenic genes (TGF-beta, connective tissue growth factor, and periostin) and Ang-converting enzyme (ACE) was suppressed by the functional inhibition of Galpha(12/13). The expression of these fibrogenic genes through Galpha(12/13) by mechanical stretch was initiated by ATP and UDP released from cardiac myocytes through pannexin hemichannels. Inhibition of G-protein-coupled P2Y6 receptors suppressed the expression of ACE, fibrogenic genes, and cardiac fibrosis. These results indicate that activation of Galpha(12/13) in cardiomyocytes by the extracellular nucleotides-stimulated P2Y(6) receptor triggers fibrosis in pressure overload-induced cardiac fibrosis, which works as an upstream mediator of the signalling pathway between Ang II and TGF-beta.

Ghrelin signalling in guinea‐pig femoral artery smooth muscle cells

Our aim was to study the new signalling pathway of ghrelin in the guinea-pig femoral artery using the outward I(K) as a sensor. Whole-cell patch-clamp experiments were performed on single smooth muscle cells, freshly isolated from the guinea-pig femoral artery. The contractile force of isometric preparations of the same artery was measured using a wire-myograph. In a Ca2+- and nicardipine-containing external solution, 1 mmol L(-1) tetraethylammonium reduced the net I(K) by 49 +/- 7%. This effect was similar and not additive to the effect of the specific BK(Ca) channel inhibitor iberiotoxin. Ghrelin (10(-7) mol L(-1)) quickly and significantly reduced the amplitudes of tetraethylammonium- and iberiotoxin-sensitive currents through BK(Ca) channels. The application of 5 x 10(-6) mol L(-1) desacyl ghrelin did not affect the amplitude of the control I(K) but it successfully prevented the ghrelin-induced I(K) decrease. The effect of ghrelin on I(K) was insensitive to selective inhibitors of cAMP-dependent protein kinase, soluble guanylyl cyclase, cGMP-dependent protein kinase or a calmodulin antagonist, but was effectively antagonized by blockers of BK(Ca) channels, phosphatidylinositol-phospholipase C, phosphatidylcholine-phospholipase C, protein kinase C, SERCA, IP(3)-induced Ca2+ release and by pertussis toxin. The ghrelin-induced increase in the force of contractions was blocked when iberiotoxin (10(-7) mol L(-1)) was present in the bath solution. Ghrelin reduces I(K(Ca)) in femoral artery myocytes by a mechanism that requires activation of Galpha(i/o)-proteins, phosphatidylinositol phospholipase C, phosphatidylcholine phospholipase C, protein kinase C and IP(3)-induced Ca2+ release.

Signal Amplification Between Gβγ Release and PI3Kγ-Mediated PI(3,4,5)P3Formation Monitored by a Fluorescent Gβγ Biosensor Protein and Repetitive Two Component Total Internal Reflection/Fluorescence Redistribution after Photobleaching Analysis

Phosphoinositide 3-kinase gamma (PI3Kgamma) is activated by Gbetagamma release after stimulation of Galpha i -coupled receptors, involving a recruitment of the enzyme to the plasma membrane via interaction of the regulatory subunit p101 or p87 with Gbetagamma. The receptor-mediated release of Gbetagamma was, however, insufficient to elicit a translocation of p101 observable by classical fluorescence microscopy approaches. Since the mobilities of plasma membrane-associated and cytosolic proteins differ strongly, small changes in the amount of plasma membrane association should be detectable by an altered diffusional behavior. Here, changes in mobility were monitored by fluorescence redistribution after photobleaching (FRAP) which was repetitively applied before and after stimulation of cells. To combine the advantages of total internal reflection (TIR) illumination, which preferentially excites fluorophors located at or near the plasma membrane, with that provided by the mobility information, we developed a combined TIR/FRAP setup which enabled us to point bleach parts of an image that was observed under TIR illumination. For FRAP data analysis, we introduce a convolution-based method and a global two component model. Using this TIR/FRAP approach, an increased plasma membrane association of the fluorescent Gbetagamma-binding domain of p101 after Gbetagamma release by G protein-coupled receptor stimulation could be detected and quantified. By comparing the translocation efficiency of this domain with that of YFP-GRP1(PH), a biosensor for the PI3Kgamma product PI(3,4,5)P3, we evaluate the signal amplification between Gbetagamma release and PI(3,4,5)P3 formation after activation of Galpha i -coupled receptors.

Lysophosphatidic acid‐induced membrane ruffling and brain‐derived neurotrophic factor gene expression are mediated by ATP release in primary microglia

We examined the effects of lysophosphatidic acid (LPA) on microglia, which may play an important role in the development and maintenance of neuropathic pain. LPA caused membrane ruffling as detected by scanning electron microscopy, and increased the expression of brain-derived neurotrophic factor (BDNF) in a primary culture of rat microglia, which express LPA(3), but not LPA(1) or LPA(2) receptors. These actions were inhibited by a Galpha(q/11)-antisense oligodeoxynucleotide (AS-ODN), U73122, an inhibitor of phospholipase C (PLC), and apyrase, which specifically degrades ATP and ADP. When ATP release was measured using a luciferin-luciferase bioluminescence assay, LPA was shown to increase it in an LPA(3) and PLC inhibitor-reversible manner. However, LPA-induced ATP release was also blocked by the Galpha(q/11) AS-ODN, but not by pertussis toxin. These results suggest that LPA induces the release of ATP from rat primary cultured microglia via the LPA(3) receptor, Galpha(q/11) and PLC, and that the released ATP or ectopically converted ADP may in turn cause membrane ruffling via P2Y(12) receptors and Galpha(i/o) activation, and BDNF expression via activation of P2X(4) receptors.

Excitability of pontine startle processing neurones is regulated by the two‐pore‐domain K+ channel TASK‐3 coupled to 5‐HT2C receptors

The mammalian startle reflex is a fast response to sudden intense sensory stimuli that can be increased by anxiety or decreased by reward. The cellular integration of sensory and modulatory information takes place in giant neurones of the caudal pontine reticular formation (PnC). The startle reflex is known to be enhanced by 5-hydroxytryptamine (5-HT); however, signalling mechanisms that change the excitability of the PnC giant neurones are poorly understood. Possible molecular candidates are two-pore-domain K(+) (K(2)P) channels that generate a variable K(+) background conductance and control neuronal excitability upon activation of G-protein-coupled receptors. We demonstrate by in situ hybridization that the K(2)P channel TASK-3 is substantially expressed in PnC giant neurones. Brain slice recordings revealed a corresponding background K(+) current in these cells that forms about 30% of the outward current at -30 mV. Inactivation of TASK-3 at pH 6.4 and by ruthenium red depolarized the cells by about 7 mV and increased the action potential frequency as well as duration. Specific activation of Galpha(q)-coupled 5-HT(2) receptors with alpha-methyl 5-HT evoked a similar increase of neuronal excitability. Consistently, we measured afferent synaptic inputs from serotonergic raphe neurones and detected 5-HT(2C) receptors in PnC giant neurones by immunohistochemistry. Thus, neuronal excitability of PnC giant neurones in vivo is most likely increased by serotonergic projections via the K(2)P channel TASK-3.

Activation of Gαi and Subsequent Uncoupling of Receptor-Gαi Signaling by Pasteurella multocida Toxin

Bacterial protein toxins are powerful tools for elucidating signaling mechanisms in eukaryotic cells. A number of bacterial protein toxins, e.g. cholera toxin, pertussis toxin (PTx), or Pasteurella multocida toxin (PMT), target heterotrimeric G proteins and have been used to stimulate or block specific signaling pathways or to demonstrate the contribution of their target proteins in cellular effects. PMT is a major virulence factor of P. multocida causing pasteurellosis in man and animals and is responsible for atrophic rhinitis in pigs. PMT modulates various signaling pathways, including phospholipase Cbeta and RhoA, by acting on the heterotrimeric G proteins Galpha(q) and Galpha(12/13), respectively. Here we report that PMT is a powerful activator of G(i) protein. We show that PMT decreases basal isoproterenol and forskolin-stimulated cAMP accumulation in intact Swiss 3T3 cells, inhibits adenylyl cyclase activity in cell membrane preparations, and enhances the inhibition of cAMP accumulation caused by lysophosphatidic acid via endothelial differentiation gene receptors. PMT-mediated inhibition of cAMP production is independent of toxin activation of Galpha(q) and/or Galpha(12/13). Although the effects of PMT are not inhibited by PTx, PMT blocks PTx-catalyzed ADP-ribosylation of G(i). PMT also inhibits steady-state GTPase activity and GTP binding of G(i) in Swiss 3T3 cell membranes stimulated by lysophosphatidic acid. The data indicate that PMT is a novel activator of G(i), modulating its GTPase activity and converting it into a PTx-insensitive state.