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Sample records for g-protein signaling mediates

  1. Signaling via G proteins mediates tumorigenic effects of GPR87

    DEFF Research Database (Denmark)

    Arfelt, Kristine Niss; Fares, Suzan; Sparre-Ulrich, Alexander H.

    2017-01-01

    G protein-coupled receptors (GPCRs) constitute a large protein family of seven transmembrane (7TM) spanning proteins that regulate multiple physiological functions. GPR87 is overexpressed in several cancers and plays a role in tumor cell survival. Here, the basal activity of GPR87 was investigated...

  2. Recent Advances on the Role of G Protein-Coupled Receptors in Hypoxia-Mediated Signaling

    OpenAIRE

    Lappano, Rosamaria; Rigiracciolo, Damiano; De Marco, Paola; Avino, Silvia; Cappello, Anna Rita; Rosano, Camillo; Maggiolini, Marcello; De Francesco, Ernestina Marianna

    2016-01-01

    G protein-coupled receptors (GPCRs) are cell surface proteins mainly involved in signal transmission; however, they play a role also in several pathophysiological conditions. Chemically heterogeneous molecules like peptides, hormones, lipids, and neurotransmitters activate second messengers and induce several biological responses by binding to these seven transmembrane receptors, which are coupled to heterotrimeric G proteins. Recently, additional molecular mechanisms have been involved in GP...

  3. Gnaz couples the circadian and dopaminergic system to G protein-mediated signaling in mouse photoreceptors.

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    Patrick Vancura

    Full Text Available The mammalian retina harbors a circadian clockwork that regulates vision and promotes healthiness of retinal neurons, mainly through directing the rhythmic release of the neurohormones dopamine-acting on dopamine D4 receptors-and melatonin-acting on MT1 and MT2 receptors. The gene Gnaz-a unique Gi/o subfamily member-was seen in the present study to be expressed in photoreceptors where its protein product Gαz shows a daily rhythm in its subcellular localization. Apart from subcellular localization, Gnaz displays a daily rhythm in expression-with peak values at night-in preparations of the whole retina, microdissected photoreceptors and photoreceptor-related pinealocytes. In retina, Gnaz rhythmicity was observed to persist under constant darkness and to be abolished in retina deficient for Clock or dopamine D4 receptors. Furthermore, circadian regulation of Gnaz was disturbed in the db/db mouse, a model of diabetic retinopathy. The data of the present study suggest that Gnaz links the circadian clockwork-via dopamine acting on D4 receptors-to G protein-mediated signaling in intact but not diabetic retina.

  4. Proteomic analysis of the signaling pathway mediated by the heterotrimeric G? protein Pga1 of Penicillium chrysogenum

    OpenAIRE

    Carrasco-Navarro, Ulises; Vera-Estrella, Rosario; Barkla, Bronwyn J.; Z??iga-Le?n, Eduardo; Reyes-Vivas, Horacio; Fern?ndez, Francisco J.; Fierro, Francisco

    2016-01-01

    Background The heterotrimeric G? protein Pga1-mediated signaling pathway regulates the entire developmental program in Penicillium chrysogenum, from spore germination to the formation of conidia. In addition it participates in the regulation of penicillin biosynthesis. We aimed to advance the understanding of this key signaling pathway using a proteomics approach, a powerful tool to identify effectors participating in signal transduction pathways. Results Penicillium chrysogenum mutants with ...

  5. Shc adaptor proteins are key transducers of mitogenic signaling mediated by the G protein-coupled thrombin receptor

    DEFF Research Database (Denmark)

    Chen, Y; Grall, D; Salcini, A E

    1996-01-01

    The serine protease thrombin activates G protein signaling systems that lead to Ras activation and, in certain cells, proliferation. Whereas the steps leading to Ras activation by G protein-coupled receptors are not well defined, the mechanisms of Ras activation by receptor tyrosine kinases have......-insensitive proteins appear to mediate this effect, since (i) pertussis toxin pre-treatment of cells does not blunt the action of thrombin and (ii) Shc phosphorylation on tyrosine can be stimulated by the muscarinic m1 receptor. Shc phosphorylation does not appear to involve protein kinase C, since the addition of 4...

  6. Neuronal Orphan G-Protein Coupled Receptor Proteins Mediate Plasmalogens-Induced Activation of ERK and Akt Signaling.

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    Md Shamim Hossain

    Full Text Available The special glycerophospholipids plasmalogens (Pls are enriched in the brain and reported to prevent neuronal cell death by enhancing phosphorylation of Akt and ERK signaling in neuronal cells. Though the activation of Akt and ERK was found to be necessary for the neuronal cells survival, it was not known how Pls enhanced cellular signaling. To answer this question, we searched for neuronal specific orphan GPCR (G-protein coupled receptor proteins, since these proteins were believed to play a role in cellular signal transduction through the lipid rafts, where both Pls and some GPCRs were found to be enriched. In the present study, pan GPCR inhibitor significantly reduced Pls-induced ERK signaling in neuronal cells, suggesting that Pls could activate GPCRs to induce signaling. We then checked mRNA expression of 19 orphan GPCRs and 10 of them were found to be highly expressed in neuronal cells. The knockdown of these 10 neuronal specific GPCRs by short hairpin (sh-RNA lentiviral particles revealed that the Pls-mediated phosphorylation of ERK was inhibited in GPR1, GPR19, GPR21, GPR27 and GPR61 knockdown cells. We further found that the overexpression of these GPCRs enhanced Pls-mediated phosphorylation of ERK and Akt in cells. Most interestingly, the GPCRs-mediated cellular signaling was reduced significantly when the endogenous Pls were reduced. Our cumulative data, for the first time, suggest a possible mechanism for Pls-induced cellular signaling in the nervous system.

  7. Regulators of G-protein signaling 4: modulation of 5-HT1A-mediated neurotransmitter release in vivo.

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    Beyer, Chad E; Ghavami, Afshin; Lin, Qian; Sung, Amy; Rhodes, Kenneth J; Dawson, Lee A; Schechter, Lee E; Young, Kathleen H

    2004-10-01

    Regulators of G-protein signaling (RGS) play a key role in the signal transduction of G-protein-coupled receptors (GPCRs). Specifically, RGS proteins function as GTPase accelerating proteins (GAPs) to dampen or "negatively regulate" GPCR-mediated signaling. Our group recently showed that RGS4 effectively GAPs Galpha(i)-mediated signaling in CHO cells expressing the serotonin-1A (5-HT(1A)) receptor. However, whether a similar relationship exists in vivo has yet to be identified. In present studies, a replication-deficient herpes simplex virus (HSV) was used to elevate RGS4 mRNA in the rat dorsal raphe nuclei (DRN) while extracellular levels of 5-HT in the striatum were monitored by in vivo microdialysis. Initial experiments conducted with noninfected rats showed that acute administration of 8-OH-DPAT (0.01-0.3 mg/kg, subcutaneous [s.c.]) dose dependently decreased striatal levels of 5-HT, an effect postulated to result from activation of somatodendritic 5-HT(1A) autoreceptors in the DRN. In control rats receiving a single intra-DRN infusion of HSV-LacZ, 8-OH-DPAT (0.03 mg/kg, s.c.) decreased 5-HT levels to an extent similar to that observed in noninfected animals. Conversely, rats infected with HSV-RGS4 in the DRN showed a blunted neurochemical response to 8-OH-DPAT (0.03 mg/kg, s.c.); however, increasing the dose to 0.3 mg/kg reversed this effect. Together, these findings represent the first in vivo evidence demonstrating that RGS4 functions to GAP Galpha(i)-coupled receptors and suggest that drug discovery efforts targeting RGS proteins may represent a novel mechanism to manipulate 5-HT(1A)-mediated neurotransmitter release.

  8. G Protein-Coupled Receptor-G-Protein βγ-Subunit Signaling Mediates Renal Dysfunction and Fibrosis in Heart Failure.

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    Kamal, Fadia A; Travers, Joshua G; Schafer, Allison E; Ma, Qing; Devarajan, Prasad; Blaxall, Burns C

    2017-01-01

    Development of CKD secondary to chronic heart failure (CHF), known as cardiorenal syndrome type 2 (CRS2), clinically associates with organ failure and reduced survival. Heart and kidney damage in CRS2 results predominantly from chronic stimulation of G protein-coupled receptors (GPCRs), including adrenergic and endothelin (ET) receptors, after elevated neurohormonal signaling of the sympathetic nervous system and the downstream ET system, respectively. Although we and others have shown that chronic GPCR stimulation and the consequent upregulated interaction between the G-protein βγ-subunit (Gβγ), GPCR-kinase 2, and β-arrestin are central to various cardiovascular diseases, the role of such alterations in kidney diseases remains largely unknown. We investigated the possible salutary effect of renal GPCR-Gβγ inhibition in CKD developed in a clinically relevant murine model of nonischemic hypertrophic CHF, transverse aortic constriction (TAC). By 12 weeks after TAC, mice developed CKD secondary to CHF associated with elevated renal GPCR-Gβγ signaling and ET system expression. Notably, systemic pharmacologic Gβγ inhibition by gallein, which we previously showed alleviates CHF in this model, attenuated these pathologic renal changes. To investigate a direct effect of gallein on the kidney, we used a bilateral ischemia-reperfusion AKI mouse model, in which gallein attenuated renal dysfunction, tissue damage, fibrosis, inflammation, and ET system activation. Furthermore, in vitro studies showed a key role for ET receptor-Gβγ signaling in pathologic fibroblast activation. Overall, our data support a direct role for GPCR-Gβγ in AKI and suggest GPCR-Gβγ inhibition as a novel therapeutic approach for treating CRS2 and AKI. Copyright © 2016 by the American Society of Nephrology.

  9. Neuronal Functions of Activators of G Protein Signaling

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    Man K. Tse

    2012-05-01

    Full Text Available G protein-coupled receptors (GPCRs are one of the most important gateways for signal transduction across the plasma membrane. Over the past decade, several classes of alternative regulators of G protein signaling have been identified and reported to activate the G proteins independent of the GPCRs. One group of such regulators is the activator of G protein signaling (AGS family which comprises of AGS1-10. They have entirely different activation mechanisms for G proteins as compared to the classic model of GPCR-mediated signaling and confer upon cells new avenues of signal transduction. As GPCRs are widely expressed in our nervous system, it is believed that the AGS family plays a major role in modulating the G protein signaling in neurons. In this article, we will review the current knowledge on AGS proteins in relation to their potential roles in neuronal regulations.

  10. Heterotrimeric G proteins-mediated resistance to necrotrophic pathogens includes mechanisms independent of salicylic acid-, jasmonic acid/ethylene- and abscisic acid-mediated defense signaling.

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    Trusov, Yuri; Sewelam, Nasser; Rookes, James Edward; Kunkel, Matt; Nowak, Ekaterina; Schenk, Peer Martin; Botella, José Ramón

    2009-04-01

    Heterotrimeric G proteins are involved in the defense response against necrotrophic fungi in Arabidopsis. In order to elucidate the resistance mechanisms involving heterotrimeric G proteins, we analyzed the effects of the Gβ (subunit deficiency in the mutant agb1-2 on pathogenesis-related gene expression, as well as the genetic interaction between agb1-2 and a number of mutants of established defense pathways. Gβ-mediated signaling suppresses the induction of salicylic acid (SA)-, jasmonic acid (JA)-, ethylene (ET)- and abscisic acid (ABA)-dependent genes during the initial phase of the infection with Fusarium oxysporum (up to 48 h after inoculation). However, at a later phase it enhances JA/ET-dependent genes such as PDF1.2 and PR4. Quantification of the Fusarium wilt symptoms revealed that Gβ- and SA-deficient mutants were more susceptible than wild-type plants, whereas JA- and ET-insensitive and ABA-deficient mutants demonstrated various levels of resistance. Analysis of the double mutants showed that the Gβ-mediated resistance to F. oxysporum and Alternaria brassicicola was mostly independent of all of the previously mentioned pathways. However, the progressive decay of agb1-2 mutants was compensated by coi1-21 and jin1-9 mutations, suggesting that at this stage of F. oxysporum infection Gβ acts upstream of COI1 and ATMYC2 in JA signaling. © 2008 The Authors. Journal compilation © 2008 Blackwell Publishing Ltd.

  11. Regulator of G-protein signaling-5 is a marker of hepatic stellate cells and expression mediates response to liver injury.

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    Arya J Bahrami

    Full Text Available Liver fibrosis is mediated by hepatic stellate cells (HSCs, which respond to a variety of cytokine and growth factors to moderate the response to injury and create extracellular matrix at the site of injury. G-protein coupled receptor (GPCR-mediated signaling, via endothelin-1 (ET-1 and angiotensin II (AngII, increases HSC contraction, migration and fibrogenesis. Regulator of G-protein signaling-5 (RGS5, an inhibitor of vasoactive GPCR agonists, functions to control GPCR-mediated contraction and hypertrophy in pericytes and smooth muscle cells (SMCs. Therefore we hypothesized that RGS5 controls GPCR signaling in activated HSCs in the context of liver injury. In this study, we localize RGS5 to the HSCs and demonstrate that Rgs5 expression is regulated during carbon tetrachloride (CCl4-induced acute and chronic liver injury in Rgs5LacZ/LacZ reporter mice. Furthermore, CCl4 treated RGS5-null mice develop increased hepatocyte damage and fibrosis in response to CCl4 and have increased expression of markers of HSC activation. Knockdown of Rgs5 enhances ET-1-mediated signaling in HSCs in vitro. Taken together, we demonstrate that RGS5 is a critical regulator of GPCR signaling in HSCs and regulates HSC activation and fibrogenesis in liver injury.

  12. Control of striatal signaling by G protein regulators

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    Keqiang eXie

    2011-08-01

    Full Text Available Signaling via heterotrimeric G proteins plays a crucial role in modulating the responses of striatal neurons that ultimately shape core behaviors mediated by the basal ganglia circuitry, such as reward valuation, habit formation and movement coordination. Activation of G-protein-coupled receptors (GPCRs by extracellular signals activates heterotrimeric G proteins by promoting the binding of GTP to their α subunits. G proteins exert their effects by influencing the activity of key effector proteins in this region, including ion channels, second messenger enzymes and protein kinases. Striatal neurons express a staggering number of GPCRs whose activation results in the engagement of downstream signaling pathways and cellular responses with unique profiles but common molecular mechanisms. Studies over the last decade have revealed that the extent and duration of GPCR signaling are controlled by a conserved protein family named Regulator of G protein Signaling (RGS. RGS proteins accelerate GTP hydrolysis by the α subunits of G proteins, thus promoting deactivation of GPCR signaling. In this review, we discuss the progress made in understanding the roles of RGS proteins in controlling striatal G protein signaling and providing integration and selectivity of signal transmission. We review evidence on the formation of a macromolecular complex between RGS proteins and other components of striatal signaling pathways, their molecular regulatory mechanisms and impacts on GPCR signaling in the striatum obtained from biochemical studies and experiments involving genetic mouse models. Special emphasis is placed on RGS9-2, a member of the RGS family that is highly enriched in the striatum and plays critical roles in drug addiction and motor control.

  13. G protein-coupled receptor kinase 2 negatively regulates chemokine signaling at a level downstream from G protein subunits

    NARCIS (Netherlands)

    Jimenez-Sainz, MC; Murga, C; Kavelaars, A; Jurado-Pueyo, M; Krakstad, BF; Heijnen, CJ; Mayor, F; Aragay, AM

    The G protein-coupled receptor kinase 2 (GRK2) phosphorylates and desensitizes ligand-activated G protein-coupled-receptors. Here, evidence is shown for a novel role of GRK2 in regulating chemokine-mediated signals. The presence of increased levels of GRK2 in human embryonic kidney (HEK) 293 cells

  14. Heterotrimeric G protein-dependent WNT-5A signaling to ERK1/2 mediates distinct aspects of microglia proinflammatory transformation

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    Halleskog Carina

    2012-05-01

    Thus, WNT-5A-induced and G protein-dependent signaling to ERK1/2 is important for the regulation of proinflammatory responses in mouse primary microglia cells. We show for the first time that WNT-5A/G protein signaling mediates physiologically important processes in primary mammalian cells with natural receptor and G protein stochiometry. Consequently, WNT-5A emerges as an important means of astrocyte-microglia communication and we, therefore, suggest WNT-5A as a new player in neuroinflammatory conditions, such as neurodegenerative disease, hypoxia, stroke, injury and infection.

  15. C-terminal of human histamine H1 receptors regulates their agonist-induced clathrin-mediated internalization and G-protein signaling.

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    Hishinuma, Shigeru; Nozawa, Hiroki; Akatsu, Chizuru; Shoji, Masaru

    2016-11-01

    It has been suggested that the agonist-induced internalization of G-protein-coupled receptors from the cell surface into intracellular compartments regulates cellular responsiveness. We previously reported that G q/11 -protein-coupled human histamine H 1 receptors internalized via clathrin-dependent mechanisms upon stimulation with histamine. However, the molecular determinants of H 1 receptors responsible for agonist-induced internalization remain unclear. In this study, we evaluated the roles of the intracellular C-terminal of human histamine H 1 receptors tagged with hemagglutinin (HA) at the N-terminal in histamine-induced internalization in Chinese hamster ovary cells. The histamine-induced internalization was evaluated by the receptor binding assay with [ 3 H]mepyramine and confocal immunofluorescence microscopy with an anti-HA antibody. We found that histamine-induced internalization was inhibited under hypertonic conditions or by pitstop, a clathrin terminal domain inhibitor, but not by filipin or nystatin, disruptors of the caveolar structure and function. The histamine-induced internalization was also inhibited by truncation of a single amino acid, Ser487, located at the end of the intracellular C-terminal of H 1 receptors, but not by its mutation to alanine. In contrast, the receptor-G-protein coupling, which was evaluated by histamine-induced accumulation of [ 3 H]inositol phosphates, was potentiated by truncation of Ser487, but was lost by its mutation to alanine. These results suggest that the intracellular C-terminal of human H 1 receptors, which only comprises 17 amino acids (Cys471-Ser487), plays crucial roles in both clathrin-dependent internalization of H 1 receptors and G-protein signaling, in which truncation of Ser487 and its mutation to alanine are revealed to result in biased signaling toward activation of G-proteins and clathrin-mediated internalization, respectively. © 2016 International Society for Neurochemistry.

  16. Non-Dioxin-Like Polychlorinated Biphenyls Inhibit G-Protein Coupled Receptor-Mediated Ca2+ Signaling by Blocking Store-Operated Ca2+ Entry.

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    Se-Young Choi

    Full Text Available Polychlorinated biphenyls (PCBs are ubiquitous pollutants which accumulate in the food chain. Recently, several molecular mechanisms by which non-dioxin-like (NDL PCBs mediate neurodevelopmental and neurobehavioral toxicity have been elucidated. However, although the G-protein coupled receptor (GPCR is a significant target for neurobehavioral disturbance, our understanding of the effects of PCBs on GPCR signaling remains unclear. In this study, we investigated the effects of NDL-PCBs on GPCR-mediated Ca2+ signaling in PC12 cells. We found that ortho-substituted 2,2',6-trichlorinated biphenyl (PCB19 caused a rapid decline in the Ca2+ signaling of bradykinin, a typical Gq- and phospholipase Cβ-coupled GPCR, without any effect on its inositol 1,4,5-trisphosphate production. PCB19 reduced thapsigargin-induced sustained cytosolic Ca2+ levels, suggesting that PCB19 inhibits SOCE. The abilities of other NDL-PCBs to inhibit store-operated Ca2+ entry (SOCE were also examined and found to be of similar potencies to that of PCB19. PCB19 also showed a manner equivalent to that of known SOCE inhibitors. PCB19-mediated SOCE inhibition was confirmed by demonstrating the ability of PCB19 to inhibit the SOCE current and thapsigargin-induced Mn2+ influx. These results imply that one of the molecular mechanism by which NDL-PCBs cause neurobehavioral disturbances involves NDL-PCB-mediated inhibition of SOCE, thereby interfering with GPCR-mediated Ca2+ signaling.

  17. Regulator of G protein signaling 6 is a critical mediator of both reward-related behavioral and pathological responses to alcohol.

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    Stewart, Adele; Maity, Biswanath; Anderegg, Simon P; Allamargot, Chantal; Yang, Jianqi; Fisher, Rory A

    2015-02-17

    Alcohol is the most commonly abused drug worldwide, and chronic alcohol consumption is a major etiological factor in the development of multiple pathological sequelae, including alcoholic cardiomyopathy and hepatic cirrhosis. Here, we identify regulator of G protein signaling 6 (RGS6) as a critical regulator of both alcohol-seeking behaviors and the associated cardiac and hepatic morbidities through two mechanistically divergent signaling actions. RGS6(-/-) mice consume less alcohol when given free access and are less susceptible to alcohol-induced reward and withdrawal. Antagonism of GABA(B) receptors or dopamine D2 receptors partially reversed the reduction in alcohol consumption in RGS6(-/-) animals. Strikingly, dopamine transporter inhibition completely restored alcohol seeking in mice lacking RGS6. RGS6 deficiency was associated with alterations in the expression of genes controlling dopamine (DA) homeostasis and a reduction in DA levels in the striatum. Taken together, these data implicate RGS6 as an essential regulator of DA bioavailability. RGS6 deficiency also provided dramatic protection against cardiac hypertrophy and fibrosis, hepatic steatosis, and gastrointestinal barrier dysfunction and endotoxemia when mice were forced to consume alcohol. Although RGS proteins canonically function as G-protein regulators, RGS6-dependent, alcohol-mediated toxicity in the heart, liver, and gastrointestinal tract involves the ability of RGS6 to promote reactive oxygen species-dependent apoptosis, an action independent of its G-protein regulatory capacity. We propose that inhibition of RGS6 might represent a viable means to reduce alcohol cravings and withdrawal in human patients, while simultaneously protecting the heart and liver from further damage upon relapse.

  18. G protein-mediated signaling in the myogenic response – role of Rho-kinase and aging

    DEFF Research Database (Denmark)

    Björling, Karl; Joseph, Philomeena Daphne; Egebjerg, Kristian

    2017-01-01

    . Aim: 1) to investigate the signaling events from Gq/11 and/or G12 activation to MT development; 2) to elucidate the impact of aging. We used pressure myography, calcium imaging, Q-PCR and immunofluorescence to study small (lumen D ...M) suppressed MT development. The Phosholipase C inhibitors U73122 (0.5 µM) and ET18-OCH3 (10 µM) robustly inhibited MT, and the TRPC channel blocker SKF 96365 (10 µM) slightly reduced MT. There was a significant effect of age (P

  19. G protein-mediated signaling in the myogenic response – role of Rho-kinase and aging

    DEFF Research Database (Denmark)

    Björling, Karl; Joseph, Philomeena Daphne; Egebjerg, Kristian

    . Aim: 1) to investigate the signaling events from Gq/11 and/or G12 activation to MT development; 2) to elucidate the impact of aging. We used pressure myography, calcium imaging, Q-PCR and immunofluorescence to study small (lumen D ...M) suppressed MT development. The Phosholipase C inhibitors U73122 (0.5 µM) and ET18-OCH3 (10 µM) robustly inhibited MT, and the TRPC channel blocker SKF 96365 (10 µM) slightly reduced MT. There was a significant effect of age (P

  20. Cell transformation mediated by the Epstein-Barr virus G protein-coupled receptor BILF1 is dependent on constitutive signaling

    DEFF Research Database (Denmark)

    Lyngaa, Rikke Birgitte; Nørregaard, K.; Kristensen, Martin

    2010-01-01

    Epstein-Barr virus (EBV) open reading frame BILF1 encodes a seven trans-membrane (TM) G protein-coupled receptor that signals with high constitutive activity through G alpha(i) (Beisser et al., 2005; Paulsen et al., 2005). In this paper, the transforming potential of BILF1 is investigated in vitro...

  1. Thematic minireview series: cell biology of G protein signaling.

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    Dohlman, Henrik G

    2015-03-13

    This thematic series is on the topic of cell signaling from a cell biology perspective, with a particular focus on G proteins. G protein-coupled receptors (GPCRs, also known as seven-transmembrane receptors) are typically found at the cell surface. Upon agonist binding, these receptors will activate a GTP-binding G protein at the cytoplasmic face of the plasma membrane. Additionally, there is growing evidence that G proteins can also be activated by non-receptor binding partners, and they can signal from non-plasma membrane compartments. The production of second messengers at multiple, spatially distinct locations represents a type of signal encoding that has been largely neglected. The first minireview in the series describes biosensors that are being used to monitor G protein signaling events in live cells. The second describes the implementation of antibody-based biosensors to dissect endosome signaling by G proteins and their receptors. The third describes the function of a non-receptor, cytoplasmic activator of G protein signaling, called GIV (Girdin). Collectively, the advances described in these articles provide a deeper understanding and emerging opportunities for new pharmacology. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  2. Rap G protein signal in normal and disordered lymphohematopoiesis.

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    Minato, Nagahiro

    2013-09-10

    Rap proteins (Rap1, Rap2a, b, c) are small molecular weight GTPases of the Ras family. Rap G proteins mediate diverse cellular events such as cell adhesion, proliferation, and gene activation through various signaling pathways. Activation of Rap signal is regulated tightly by several specific regulatory proteins including guanine nucleotide exchange factors and GTPase-activating proteins. Beyond cell biological studies, increasing attempts have been made in the past decade to define the roles of Rap signal in specific functions of normal tissue systems as well as in cancer. In the immune and hematopoietic systems, Rap signal plays crucial roles in the development and function of essentially all lineages of lymphocytes and hematopoietic cells, and importantly, deregulated Rap signal may lead to unique pathological conditions depending on the affected cell types, including various types of leukemia and autoimmunity. The phenotypical studies have unveiled novel, even unexpected functional aspects of Rap signal in cells from a variety of tissues, providing potentially important clues for controlling human diseases, including malignancy. © 2013 Elsevier Inc. All rights reserved.

  3. Biased and g protein-independent signaling of chemokine receptors

    DEFF Research Database (Denmark)

    Steen, Anne; Larsen, Olav; Thiele, Stefanie

    2014-01-01

    ), different receptors (with the same ligand), or different tissues or cells (for the same ligand-receptor pair). Most often biased signaling is differentiated into G protein-dependent and β-arrestin-dependent signaling. Yet, it may also cover signaling differences within these groups. Moreover, it may...

  4. Regulator of G-protein signaling - 5 (RGS5 is a novel repressor of hedgehog signaling.

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    William M Mahoney

    Full Text Available Hedgehog (Hh signaling plays fundamental roles in morphogenesis, tissue repair, and human disease. Initiation of Hh signaling is controlled by the interaction of two multipass membrane proteins, patched (Ptc and smoothened (Smo. Recent studies identify Smo as a G-protein coupled receptor (GPCR-like protein that signals through large G-protein complexes which contain the Gαi subunit. We hypothesize Regulator of G-Protein Signaling (RGS proteins, and specifically RGS5, are endogenous repressors of Hh signaling via their ability to act as GTPase activating proteins (GAPs for GTP-bound Gαi, downstream of Smo. In support of this hypothesis, we demonstrate that RGS5 over-expression inhibits sonic hedgehog (Shh-mediated signaling and osteogenesis in C3H10T1/2 cells. Conversely, signaling is potentiated by siRNA-mediated knock-down of RGS5 expression, but not RGS4 expression. Furthermore, using immuohistochemical analysis and co-immunoprecipitation (Co-IP, we demonstrate that RGS5 is present with Smo in primary cilia. This organelle is required for canonical Hh signaling in mammalian cells, and RGS5 is found in a physical complex with Smo in these cells. We therefore conclude that RGS5 is an endogenous regulator of Hh-mediated signaling and that RGS proteins are potential targets for novel therapeutics in Hh-mediated diseases.

  5. Hypoxic induction of the regulator of G-protein signalling 4 gene is mediated by the hypoxia-inducible factor pathway.

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    Sam W Z Olechnowicz

    Full Text Available The transcriptional response to hypoxia is largely dependent on the Hypoxia Inducible Factors (HIF-1 and HIF-2 in mammalian cells. Many target genes have been characterised for these heterodimeric transcription factors, yet there is evidence that the full range of HIF-regulated genes has not yet been described. We constructed a TetON overexpression system in the rat pheochromocytoma PC-12 cell line to search for novel HIF and hypoxia responsive genes. The Rgs4 gene encodes the Regulator of G-Protein Signalling 4 (RGS4 protein, an inhibitor of signalling from G-protein coupled receptors, and dysregulation of Rgs4 is linked to disease states such as schizophrenia and cardiomyopathy. Rgs4 was found to be responsive to HIF-2α overexpression, hypoxic treatment, and hypoxia mimetic drugs in PC-12 cells. Similar responses were observed in human neuroblastoma cell lines SK-N-SH and SK-N-BE(2C, but not in endothelial cells, where Rgs4 transcript is readily detected but does not respond to hypoxia. Furthermore, this regulation was found to be dependent on transcription, and occurs in a manner consistent with direct HIF transactivation of Rgs4 transcription. However, no HIF binding site was detectable within 32 kb of the human Rgs4 gene locus, leading to the possibility of regulation by long-distance genomic interactions. Further research into Rgs4 regulation by hypoxia and HIF may result in better understanding of disease states such as schizophrenia, and also shed light on the other roles of HIF yet to be discovered.

  6. Hypoxic Induction of the Regulator of G-Protein Signalling 4 Gene Is Mediated by the Hypoxia-Inducible Factor Pathway

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    Olechnowicz, Sam W. Z.; Fedele, Anthony O.; Peet, Daniel J.

    2012-01-01

    The transcriptional response to hypoxia is largely dependent on the Hypoxia Inducible Factors (HIF-1 and HIF-2) in mammalian cells. Many target genes have been characterised for these heterodimeric transcription factors, yet there is evidence that the full range of HIF-regulated genes has not yet been described. We constructed a TetON overexpression system in the rat pheochromocytoma PC-12 cell line to search for novel HIF and hypoxia responsive genes. The Rgs4 gene encodes the Regulator of G-Protein Signalling 4 (RGS4) protein, an inhibitor of signalling from G-protein coupled receptors, and dysregulation of Rgs4 is linked to disease states such as schizophrenia and cardiomyopathy. Rgs4 was found to be responsive to HIF-2α overexpression, hypoxic treatment, and hypoxia mimetic drugs in PC-12 cells. Similar responses were observed in human neuroblastoma cell lines SK-N-SH and SK-N-BE(2)C, but not in endothelial cells, where Rgs4 transcript is readily detected but does not respond to hypoxia. Furthermore, this regulation was found to be dependent on transcription, and occurs in a manner consistent with direct HIF transactivation of Rgs4 transcription. However, no HIF binding site was detectable within 32 kb of the human Rgs4 gene locus, leading to the possibility of regulation by long-distance genomic interactions. Further research into Rgs4 regulation by hypoxia and HIF may result in better understanding of disease states such as schizophrenia, and also shed light on the other roles of HIF yet to be discovered. PMID:22970249

  7. Comparative expression profiling of AtRAD5B and AtNDL1: Hints towards a role in G protein mediated signaling.

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    Khatri, Nisha; Singh, Swati; Hakim, Nasmeen; Mudgil, Yashwanti

    2017-11-01

    Arabidopsis AtRAD5B encodes for a putative helicase of the class SWItch/Sucrose Non-Fermentable (SWI/SNF) ATPases. We identified AtRAD5B as an interactor of N-MYC DOWNREGULATED-LIKE1 (AtNDL1) in a yeast two-hybrid screen. AtNDL1 is a G protein signaling component which regulates auxin transport and gradients together with GTP binding protein beta 1 (AGB1). Auxin gradients are known to recruit SWI/SNF remodeling complexes to the chromatin and regulate expression of genes involved in flower and leaf formation. In current study, a comparative spatial and temporal co-expression/localization analysis of AtNDL1, AGB1 with AtRAD5B was carried out in order to explore the possibility of their coexistence in a common signaling network. Translational fusion (GUS) of AtNDL1 and AtRAD5B in seedlings and reproductive organs revealed that both shared similar expression patterns with the highest expression observed in male reproductive organs. Moreover, they shared similar domains of localization in roots, suggesting their potential functioning together in reproductive and root development processes. This study predicts the existence of a signaling network involving AtNDL1, AGB1 with AtRAD5B. Copyright © 2017 Elsevier B.V. All rights reserved.

  8. Regulation of heterotrimeric G-protein signaling by NDPK/NME proteins and caveolins: an update.

    Science.gov (United States)

    Abu-Taha, Issam H; Heijman, Jordi; Feng, Yuxi; Vettel, Christiane; Dobrev, Dobromir; Wieland, Thomas

    2018-02-01

    Heterotrimeric G proteins are pivotal mediators of cellular signal transduction in eukaryotic cells and abnormal G-protein signaling plays an important role in numerous diseases. During the last two decades it has become evident that the activation status of heterotrimeric G proteins is both highly localized and strongly regulated by a number of factors, including a receptor-independent activation pathway of heterotrimeric G proteins that does not involve the classical GDP/GTP exchange and relies on nucleoside diphosphate kinases (NDPKs). NDPKs are NTP/NDP transphosphorylases encoded by the nme/nm23 genes that are involved in a variety of cellular events such as proliferation, migration, and apoptosis. They therefore contribute, for example, to tumor metastasis, angiogenesis, retinopathy, and heart failure. Interestingly, NDPKs are translocated and/or upregulated in human heart failure. Here we describe recent advances in the current understanding of NDPK functions and how they have an impact on local regulation of G-protein signaling.

  9. G protein-coupled receptor (GPCR) signaling via heterotrimeric G proteins from endosomes.

    Science.gov (United States)

    Tsvetanova, Nikoleta G; Irannejad, Roshanak; von Zastrow, Mark

    2015-03-13

    Some G protein-coupled receptors (GPCRs), in addition to activating heterotrimeric G proteins in the plasma membrane, appear to elicit a "second wave" of G protein activation after ligand-induced internalization. We briefly summarize evidence supporting this view and then discuss what is presently known about the functional significance of GPCR-G protein activation in endosomes. Endosomal activation can shape the cellular response temporally by prolonging its overall duration, and may shape the response spatially by moving the location of intracellular second messenger production relative to effectors. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  10. Regulation of G protein-coupled receptor signalling: focus on the cardiovascular system and regulator of G protein signalling proteins

    NARCIS (Netherlands)

    Hendriks-Balk, Mariëlle C.; Peters, Stephan L. M.; Michel, Martin C.; Alewijnse, Astrid E.

    2008-01-01

    G protein-coupled receptors (GPCRs) are involved in many biological processes. Therefore, GPCR function is tightly controlled both at receptor level and at the level of signalling components. Well-known mechanisms by which GPCR function can be regulated comprise desensitization/resensitization

  11. Constitutively active signaling by the G protein βγ-subunit mediates intrinsically increased phosphodiesterase-4 activity in human asthmatic airway smooth muscle cells.

    Directory of Open Access Journals (Sweden)

    Aihua Hu

    Full Text Available Signaling by the Gβγ subunit of Gi protein, leading to downstream c-Src-induced activation of the Ras/c-Raf1/MEK-ERK1/2 signaling pathway and its upregulation of phosphodiesterase-4 (PDE4 activity, was recently shown to mediate the heightened contractility in proasthmatic sensitized isolated airway smooth muscle (ASM, as well as allergen-induced airway hyperresponsiveness and inflammation in an in vivo animal model of allergic asthma. This study investigated whether cultured human ASM (HASM cells derived from asthmatic donor lungs exhibit constitutively increased PDE activity that is attributed to intrinsically upregulated Gβγ signaling coupled to c-Src activation of the Ras/MEK/ERK1/2 cascade. We show that, relative to normal cells, asthmatic HASM cells constitutively exhibit markedly increased intrinsic PDE4 activity coupled to heightened Gβγ-regulated phosphorylation of c-Src and ERK1/2, and direct co-localization of the latter with the PDE4D isoform. These signaling events and their induction of heightened PDE activity are acutely suppressed by treating asthmatic HASM cells with a Gβγ inhibitor. Importantly, along with increased Gβγ activation, asthmatic HASM cells also exhibit constitutively increased direct binding of the small Rap1 GTPase-activating protein, Rap1GAP, to the α-subunit of Gi protein, which serves to cooperatively facilitate Ras activation and, thereby, enable enhanced Gβγ-regulated ERK1/2-stimulated PDE activity. Collectively, these data are the first to identify that intrinsically increased signaling via the Gβγ subunit, facilitated by Rap1GAP recruitment to the α-subunit, mediates the constitutively increased PDE4 activity detected in asthmatic HASM cells. These new findings support the notion that interventions targeted at suppressing Gβγ signaling may lead to novel approaches to treat asthma.

  12. Structural Elements in the Gαs and Gαq C Termini That Mediate Selective G Protein-coupled Receptor (GPCR) Signaling.

    Science.gov (United States)

    Semack, Ansley; Sandhu, Manbir; Malik, Rabia U; Vaidehi, Nagarajan; Sivaramakrishnan, Sivaraj

    2016-08-19

    Although the importance of the C terminus of the α subunit of the heterotrimeric G protein in G protein-coupled receptor (GPCR)-G protein pairing is well established, the structural basis of selective interactions remains unknown. Here, we combine live cell FRET-based measurements and molecular dynamics simulations of the interaction between the GPCR and a peptide derived from the C terminus of the Gα subunit (Gα peptide) to dissect the molecular mechanisms of G protein selectivity. We observe a direct link between Gα peptide binding and stabilization of the GPCR conformational ensemble. We find that cognate and non-cognate Gα peptides show deep and shallow binding, respectively, and in distinct orientations within the GPCR. Binding of the cognate Gα peptide stabilizes the agonist-bound GPCR conformational ensemble resulting in favorable binding energy and lower flexibility of the agonist-GPCR pair. We identify three hot spot residues (Gαs/Gαq-Gln-384/Leu-349, Gln-390/Glu-355, and Glu-392/Asn-357) that contribute to selective interactions between the β2-adrenergic receptor (β2-AR)-Gαs and V1A receptor (V1AR)-Gαq The Gαs and Gαq peptides adopt different orientations in β2-AR and V1AR, respectively. The β2-AR/Gαs peptide interface is dominated by electrostatic interactions, whereas the V1AR/Gαq peptide interactions are predominantly hydrophobic. Interestingly, our study reveals a role for both favorable and unfavorable interactions in G protein selection. Residue Glu-355 in Gαq prevents this peptide from interacting strongly with β2-AR. Mutagenesis to the Gαs counterpart (E355Q) imparts a cognate-like interaction. Overall, our study highlights the synergy in molecular dynamics and FRET-based approaches to dissect the structural basis of selective G protein interactions. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  13. Making Sense of G Proteins: Genetic analysis of sensory G protein signaling in the nematode C. elegans

    NARCIS (Netherlands)

    H. Lans (Hannes)

    2005-01-01

    textabstractAmong the key molecules involved in sensory perception are G proteins, which act in every cell to activate a cascade of signaling molecules in response to certain environmental cues. In this thesis, several studies on the role of G proteins in the sensory system of C. elegans are

  14. Integration of G protein α (Gα) signaling by the regulator of G protein signaling 14 (RGS14).

    Science.gov (United States)

    Brown, Nicole E; Goswami, Devrishi; Branch, Mary Rose; Ramineni, Suneela; Ortlund, Eric A; Griffin, Patrick R; Hepler, John R

    2015-04-03

    RGS14 contains distinct binding sites for both active (GTP-bound) and inactive (GDP-bound) forms of Gα subunits. The N-terminal regulator of G protein signaling (RGS) domain binds active Gαi/o-GTP, whereas the C-terminal G protein regulatory (GPR) motif binds inactive Gαi1/3-GDP. The molecular basis for how RGS14 binds different activation states of Gα proteins to integrate G protein signaling is unknown. Here we explored the intramolecular communication between the GPR motif and the RGS domain upon G protein binding and examined whether RGS14 can functionally interact with two distinct forms of Gα subunits simultaneously. Using complementary cellular and biochemical approaches, we demonstrate that RGS14 forms a stable complex with inactive Gαi1-GDP at the plasma membrane and that free cytosolic RGS14 is recruited to the plasma membrane by activated Gαo-AlF4(-). Bioluminescence resonance energy transfer studies showed that RGS14 adopts different conformations in live cells when bound to Gα in different activation states. Hydrogen/deuterium exchange mass spectrometry revealed that RGS14 is a very dynamic protein that undergoes allosteric conformational changes when inactive Gαi1-GDP binds the GPR motif. Pure RGS14 forms a ternary complex with Gαo-AlF4(-) and an AlF4(-)-insensitive mutant (G42R) of Gαi1-GDP, as observed by size exclusion chromatography and differential hydrogen/deuterium exchange. Finally, a preformed RGS14·Gαi1-GDP complex exhibits full capacity to stimulate the GTPase activity of Gαo-GTP, demonstrating that RGS14 can functionally engage two distinct forms of Gα subunits simultaneously. Based on these findings, we propose a working model for how RGS14 integrates multiple G protein signals in host CA2 hippocampal neurons to modulate synaptic plasticity. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  15. Two Chimeric Regulators of G-protein Signaling (RGS) Proteins Differentially Modulate Soybean Heterotrimeric G-protein Cycle*

    Science.gov (United States)

    Roy Choudhury, Swarup; Westfall, Corey S.; Laborde, John P.; Bisht, Naveen C.; Jez, Joseph M.; Pandey, Sona

    2012-01-01

    Heterotrimeric G-proteins and the regulator of G-protein signaling (RGS) proteins, which accelerate the inherent GTPase activity of Gα proteins, are common in animals and encoded by large gene families; however, in plants G-protein signaling is thought to be more limited in scope. For example, Arabidopsis thaliana contains one Gα, one Gβ, three Gγ, and one RGS protein. Recent examination of the Glycine max (soybean) genome reveals a larger set of G-protein-related genes and raises the possibility of more intricate G-protein networks than previously observed in plants. Stopped-flow analysis of GTP-binding and GDP/GTP exchange for the four soybean Gα proteins (GmGα1–4) reveals differences in their kinetic properties. The soybean genome encodes two chimeric RGS proteins with an N-terminal seven transmembrane domain and a C-terminal RGS box. Both GmRGS interact with each of the four GmGα and regulate their GTPase activity. The GTPase-accelerating activities of GmRGS1 and -2 differ for each GmGα, suggesting more than one possible rate of the G-protein cycle initiated by each of the Gα proteins. The differential effects of GmRGS1 and GmRGS2 on GmGα1–4 result from a single valine versus alanine difference. The emerging picture suggests complex regulation of the G-protein cycle in soybean and in other plants with expanded G-protein networks. PMID:22474294

  16. A G Protein-biased Designer G Protein-coupled Receptor Useful for Studying the Physiological Relevance of Gq/11-dependent Signaling Pathways.

    Science.gov (United States)

    Hu, Jianxin; Stern, Matthew; Gimenez, Luis E; Wanka, Lizzy; Zhu, Lu; Rossi, Mario; Meister, Jaroslawna; Inoue, Asuka; Beck-Sickinger, Annette G; Gurevich, Vsevolod V; Wess, Jürgen

    2016-04-08

    Designerreceptorsexclusivelyactivated by adesignerdrug (DREADDs) are clozapine-N-oxide-sensitive designer G protein-coupled receptors (GPCRs) that have emerged as powerful novel chemogenetic tools to study the physiological relevance of GPCR signaling pathways in specific cell types or tissues. Like endogenous GPCRs, clozapine-N-oxide-activated DREADDs do not only activate heterotrimeric G proteins but can also trigger β-arrestin-dependent (G protein-independent) signaling. To dissect the relative physiological relevance of G protein-mediatedversusβ-arrestin-mediated signaling in different cell types or physiological processes, the availability of G protein- and β-arrestin-biased DREADDs would be highly desirable. In this study, we report the development of a mutationally modified version of a non-biased DREADD derived from the M3muscarinic receptor that can activate Gq/11with high efficacy but lacks the ability to interact with β-arrestins. We also demonstrate that this novel DREADD is activein vivoand that cell type-selective expression of this new designer receptor can provide novel insights into the physiological roles of G protein (Gq/11)-dependentversusβ-arrestin-dependent signaling in hepatocytes. Thus, this novel Gq/11-biased DREADD represents a powerful new tool to study the physiological relevance of Gq/11-dependent signaling in distinct tissues and cell types, in the absence of β-arrestin-mediated cellular effects. Such studies should guide the development of novel classes of functionally biased ligands that show high efficacy in various pathophysiological conditions but display a reduced incidence of side effects. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  17. Structure and Function of Vps15 in the Endosomal G Protein Signaling Pathway

    Energy Technology Data Exchange (ETDEWEB)

    Heenan, Erin J.; Vanhooke, Janeen L.; Temple, Brenda R.; Betts, Laurie; Sondek, John E.; Dohlman, Henrik G.; (UNC)

    2009-09-11

    G protein-coupled receptors mediate cellular responses to a wide variety of stimuli, including taste, light, and neurotransmitters. In the yeast Saccharomyces cerevisiae, activation of the pheromone pathway triggers events leading to mating. The view had long been held that the G protein-mediated signal occurs principally at the plasma membrane. Recently, it has been shown that the G protein {alpha} subunit Gpa1 can promote signaling at endosomes and requires two components of the sole phosphatidylinositol-3-kinase in yeast, Vps15 and Vps34. Vps15 contains multiple WD repeats and also binds to Gpa1 preferentially in the GDP-bound state; these observations led us to hypothesize that Vps15 may function as a G protein {beta} subunit at the endosome. Here we show an X-ray crystal structure of the Vps15 WD domain that reveals a seven-bladed propeller resembling that of typical G{beta} subunits. We show further that the WD domain is sufficient to bind Gpa1 as well as to Atg14, a potential G{gamma} protein that exists in a complex with Vps15. The Vps15 kinase domain together with the intermediate domain (linking the kinase and WD domains) also contributes to Gpa1 binding and is necessary for Vps15 to sustain G protein signaling. These findings reveal that the Vps15 G{beta}-like domain serves as a scaffold to assemble Gpa1 and Atg14, whereas the kinase and intermediate domains are required for proper signaling at the endosome.

  18. G-protein coupled receptor 56 promotes myoblast fusion through serum response factor- and nuclear factor of activated T-cell-mediated signalling but is not essential for muscle development in vivo.

    Science.gov (United States)

    Wu, Melissa P; Doyle, Jamie R; Barry, Brenda; Beauvais, Ariane; Rozkalne, Anete; Piao, Xianhua; Lawlor, Michael W; Kopin, Alan S; Walsh, Christopher A; Gussoni, Emanuela

    2013-12-01

    Mammalian muscle cell differentiation is a complex process of multiple steps for which many of the factors involved have not yet been defined. In a screen to identify the regulators of myogenic cell fusion, we found that the gene for G-protein coupled receptor 56 (GPR56) was transiently up-regulated during the early fusion of human myoblasts. Human mutations in the gene for GPR56 cause the disease bilateral frontoparietal polymicrogyria; however, the consequences of receptor dysfunction on muscle development have not been explored. Using knockout mice, we defined the role of GPR56 in skeletal muscle. GPR56(-/-) myoblasts have decreased fusion and smaller myotube sizes in culture. In addition, a loss of GPR56 expression in muscle cells results in decreases or delays in the expression of myogenic differentiation 1, myogenin and nuclear factor of activated T-cell (NFAT)c2. Our data suggest that these abnormalities result from decreased GPR56-mediated serum response element and NFAT signalling. Despite these changes, no overt differences in phenotype were identified in the muscle of GPR56 knockout mice, which presented only a mild but statistically significant elevation of serum creatine kinase compared to wild-type. In agreement with these findings, clinical data from 13 bilateral frontoparietal polymicrogyria patients revealed mild serum creatine kinase increase in only two patients. In summary, targeted disruption of GPR56 in mice results in myoblast abnormalities. The absence of a severe muscle phenotype in GPR56 knockout mice and human patients suggests that other factors may compensate for the lack of this G-protein coupled receptor during muscle development and that the motor delay observed in these patients is likely not a result of primary muscle abnormalities. © 2013 FEBS.

  19. Role of polarized G protein signaling in tracking pheromone gradients

    Science.gov (United States)

    McClure, Allison W.; Minakova, Maria; Dyer, Jayme M.; Zyla, Trevin R.; Elston, Timothy C.; Lew, Daniel J.

    2015-01-01

    Summary Yeast cells track gradients of pheromones to locate mating partners. Intuition suggests that uniform distribution of pheromone receptors over the cell surface would yield optimal gradient sensing. However, yeast cells display polarized receptors. The benefit of such polarization was unknown. During gradient tracking, cell growth is directed by a patch of polarity regulators that wanders around the cortex. Patch movement is sensitive to pheromone dose, with wandering reduced on the up-gradient side of the cell, resulting in net growth in that direction. Mathematical modeling suggests that active receptors and associated G proteins lag behind the polarity patch and act as an effective drag on patch movement. In vivo, the polarity patch is trailed by a G protein-rich domain, and this polarized distribution of G proteins is required to constrain patch wandering. Our findings explain why G protein polarization is beneficial, and illuminate a novel mechanism for gradient tracking. PMID:26609960

  20. G Protein-coupled Receptors and Resistance to Inhibitors of Cholinesterase-8A (Ric-8A) Both Regulate the Regulator of G Protein Signaling 14 (RGS14)·Gαi1 Complex in Live Cells*

    OpenAIRE

    Vellano, Christopher P.; Maher, Ellen M.; Hepler, John R.; Blumer, Joe B.

    2011-01-01

    Background: Regulator of G protein signaling 14 (RGS14) is a G protein regulatory (GPR) protein that participates in unconventional G protein signaling independent of G protein-coupled receptors (GPCRs).

  1. VEGFR-3 signaling is regulated by a G-protein activator, activator of G-protein signaling 8, in lymphatic endothelial cells.

    Science.gov (United States)

    Sakima, Miho; Hayashi, Hisaki; Mamun, Abdullah Al; Sato, Motohiko

    2018-07-01

    Vascular endothelial growth factor C (VEGFC) and its cognate receptor VEGFR-3 play a key role in lymphangiogenesis. We previously reported that an ischemia-inducible Gβγ signal regulator, activator of G-protein signaling 8 (AGS8), regulated the subcellular distribution of vascular endothelial growth factor receptor-2 (VEGFR-2) and influenced VEGFA-induced signaling in vascular endothelial cells. Here, we report that AGS8 regulates VEGFR-3, which is another subtype of the VEGF receptor family, and mediates VEGFC signaling in human dermal lymphatic endothelial cells (HDLECs). VEGFC stimulated the proliferation of HDLECs and tube formation by HDLECs, which were inhibited by knocking down AGS8 by small interfering RNA (siRNA). AGS8 siRNA inhibited VEGFC-mediated phosphorylation of VEGFR-3 and its downstream molecules, including ERK1/2 and AKT. Analysis of fluorescence-activated cell sorting and immunofluorescence staining demonstrated that AGS8 knockdown was associated with a reduction of VEGFR-3 at the cell surface. Endocytosis inhibitors did not rescue the decrease of cell-surface VEGFR-3, suggesting that AGS8 regulated the trafficking of VEGFR-3 to the plasma membrane. An immunoprecipitation assay indicated that VEGFR-3 formed a complex including AGS8 and Gβγ in cells. These data suggest the novel regulation of VEGFC-VEGFR-3 by AGS8 in HDLECs and a potential role for AGS8 in lymphangiogenesis. Copyright © 2018 Elsevier Inc. All rights reserved.

  2. Defective chemokine signal integration in leukocytes lacking activator of G protein signaling 3 (AGS3).

    Science.gov (United States)

    Branham-O'Connor, Melissa; Robichaux, William G; Zhang, Xian-Kui; Cho, Hyeseon; Kehrl, John H; Lanier, Stephen M; Blumer, Joe B

    2014-04-11

    Activator of G-protein signaling 3 (AGS3, gene name G-protein signaling modulator-1, Gpsm1), an accessory protein for G-protein signaling, has functional roles in the kidney and CNS. Here we show that AGS3 is expressed in spleen, thymus, and bone marrow-derived dendritic cells, and is up-regulated upon leukocyte activation. We explored the role of AGS3 in immune cell function by characterizing chemokine receptor signaling in leukocytes from mice lacking AGS3. No obvious differences in lymphocyte subsets were observed. Interestingly, however, AGS3-null B and T lymphocytes and bone marrow-derived dendritic cells exhibited significant chemotactic defects as well as reductions in chemokine-stimulated calcium mobilization and altered ERK and Akt activation. These studies indicate a role for AGS3 in the regulation of G-protein signaling in the immune system, providing unexpected venues for the potential development of therapeutic agents that modulate immune function by targeting these regulatory mechanisms.

  3. G Protein and β-arrestin signaling bias at the ghrelin receptor.

    Science.gov (United States)

    Evron, Tama; Peterson, Sean M; Urs, Nikhil M; Bai, Yushi; Rochelle, Lauren K; Caron, Marc G; Barak, Larry S

    2014-11-28

    The G protein-coupled ghrelin receptor GHSR1a is a potential pharmacological target for treating obesity and addiction because of the critical role ghrelin plays in energy homeostasis and dopamine-dependent reward. GHSR1a enhances growth hormone release, appetite, and dopamine signaling through G(q/11), G(i/o), and G(12/13) as well as β-arrestin-based scaffolds. However, the contribution of individual G protein and β-arrestin pathways to the diverse physiological responses mediated by ghrelin remains unknown. To characterize whether a signaling bias occurs for GHSR1a, we investigated ghrelin signaling in a number of cell-based assays, including Ca(2+) mobilization, serum response factor response element, stress fiber formation, ERK1/2 phosphorylation, and β-arrestin translocation, utilizing intracellular second loop and C-tail mutants of GHSR1a. We observed that GHSR1a and β-arrestin rapidly form metastable plasma membrane complexes following exposure to an agonist, but replacement of the GHSR1a C-tail by the tail of the vasopressin 2 receptor greatly stabilizes them, producing complexes observable on the plasma membrane and also in endocytic vesicles. Mutations of the contiguous conserved amino acids Pro-148 and Leu-149 in the GHSR1a intracellular second loop generate receptors with a strong bias to G protein and β-arrestin, respectively, supporting a role for conformation-dependent signaling bias in the wild-type receptor. Our results demonstrate more balance in GHSR1a-mediated ERK signaling from G proteins and β-arrestin but uncover an important role for β-arrestin in RhoA activation and stress fiber formation. These findings suggest an avenue for modulating drug abuse-associated changes in synaptic plasticity via GHSR1a and indicate the development of GHSR1a-biased ligands as a promising strategy for selectively targeting downstream signaling events. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

  4. G Protein-Coupled Receptor Signaling in Stem Cells and Cancer.

    Science.gov (United States)

    Lynch, Jennifer R; Wang, Jenny Yingzi

    2016-05-11

    G protein-coupled receptors (GPCRs) are a large superfamily of cell-surface signaling proteins that bind extracellular ligands and transduce signals into cells via heterotrimeric G proteins. GPCRs are highly tractable drug targets. Aberrant expression of GPCRs and G proteins has been observed in various cancers and their importance in cancer stem cells has begun to be appreciated. We have recently reported essential roles for G protein-coupled receptor 84 (GPR84) and G protein subunit Gαq in the maintenance of cancer stem cells in acute myeloid leukemia. This review will discuss how GPCRs and G proteins regulate stem cells with a focus on cancer stem cells, as well as their implications for the development of novel targeted cancer therapies.

  5. G Protein-Coupled Receptor Signaling in Stem Cells and Cancer

    Directory of Open Access Journals (Sweden)

    Jennifer R. Lynch

    2016-05-01

    Full Text Available G protein-coupled receptors (GPCRs are a large superfamily of cell-surface signaling proteins that bind extracellular ligands and transduce signals into cells via heterotrimeric G proteins. GPCRs are highly tractable drug targets. Aberrant expression of GPCRs and G proteins has been observed in various cancers and their importance in cancer stem cells has begun to be appreciated. We have recently reported essential roles for G protein-coupled receptor 84 (GPR84 and G protein subunit Gαq in the maintenance of cancer stem cells in acute myeloid leukemia. This review will discuss how GPCRs and G proteins regulate stem cells with a focus on cancer stem cells, as well as their implications for the development of novel targeted cancer therapies.

  6. The essential role of G protein-coupled receptor (GPCR) signaling in regulating T cell immunity.

    Science.gov (United States)

    Wang, Dashan

    2018-06-01

    The aim of this paper is to clarify the critical role of GPCR signaling in T cell immunity. The G protein-coupled receptors (GPCRs) are the most common targets in current pharmaceutical industry, and represent the largest and most versatile family of cell surface communicating molecules. GPCRs can be activated by a diverse array of ligands including neurotransmitters, chemokines as well as sensory stimuli. Therefore, GPCRs are involved in many key cellular and physiological processes, such as sense of light, taste and smell, neurotransmission, metabolism, endocrine and exocrine secretion. In recent years, GPCRs have been found to play an important role in immune system. T cell is an important type of immune cell, which plays a central role in cell-mediated immunity. A variety of GPCRs and their signaling mediators (RGS proteins, GRKs and β-arrestin) have been found to express in T cells and involved T cell-mediated immunity. We will summarize the role of GPCR signaling and their regulatory molecules in T cell activation, homeostasis and function in this article. GPCR signaling plays an important role in T cell activation, homeostasis and function. GPCR signaling is critical in regulating T cell immunity.

  7. Extracellular Ca2+ is a danger signal activating the NLRP3 inflammasome through G protein-coupled calcium sensing receptors

    DEFF Research Database (Denmark)

    Rossol, Manuela; Pierer, Matthias; Raulien, Nora

    2012-01-01

    calcium activates the NLRP3 inflammasome via stimulation of G protein-coupled calcium sensing receptors. Activation is mediated by signalling through the calcium-sensing receptor and GPRC6A via the phosphatidyl inositol/Ca(2+) pathway. The resulting increase in the intracellular calcium concentration......, and this effect was inhibited in GPRC6A(-/-) mice. Our results demonstrate that G-protein-coupled receptors can activate the inflammasome, and indicate that increased extracellular calcium has a role as a danger signal and amplifier of inflammation....

  8. How a mycoparasite employs g-protein signaling: using the example of trichoderma.

    Science.gov (United States)

    Omann, Markus; Zeilinger, Susanne

    2010-01-01

    Mycoparasitic Trichoderma spp. act as potent biocontrol agents against a number of plant pathogenic fungi, whereupon the mycoparasitic attack includes host recognition followed by infection structure formation and secretion of lytic enzymes and antifungal metabolites leading to the host's death. Host-derived signals are suggested to be recognized by receptors located on the mycoparasite's cell surface eliciting an internal signal transduction cascade which results in the transcription of mycoparasitism-relevant genes. Heterotrimeric G proteins of fungi transmit signals originating from G-protein-coupled receptors mainly to the cAMP and the MAP kinase pathways resulting in regulation of downstream effectors. Components of the G-protein signaling machinery such as Gα subunits and G-protein-coupled receptors were recently shown to play crucial roles in Trichoderma mycoparasitism as they govern processes such as the production of extracellular cell wall lytic enzymes, the secretion of antifungal metabolites, and the formation of infection structures.

  9. Imaging of persistent cAMP signaling by internalized G protein-coupled receptors.

    Science.gov (United States)

    Calebiro, Davide; Nikolaev, Viacheslav O; Lohse, Martin J

    2010-07-01

    G protein-coupled receptors (GPCRs) are the largest family of plasma membrane receptors. They mediate the effects of several endogenous cues and serve as important pharmacological targets. Although many biochemical events involved in GPCR signaling have been characterized in great detail, little is known about their spatiotemporal dynamics in living cells. The recent advent of optical methods based on fluorescent resonance energy transfer allows, for the first time, to directly monitor GPCR signaling in living cells. Utilizing these methods, it has been recently possible to show that the receptors for two protein/peptide hormones, the TSH and the parathyroid hormone, continue signaling to cAMP after their internalization into endosomes. This type of intracellular signaling is persistent and apparently triggers specific cellular outcomes. Here, we review these recent data and explain the optical methods used for such studies. Based on these findings, we propose a revision of the current model of the GPCR-cAMP signaling pathway to accommodate receptor signaling at endosomes.

  10. Chemotaxis to cyclic AMP and folic acid is mediated by different G proteins in Dictyostelium discoideum

    NARCIS (Netherlands)

    Kesbeke, Fanja; Haastert, Peter J.M. van; Wit, René J.W. de; Snaar-Jagalska, B. Ewa

    1990-01-01

    Mutant Frigid A (fgdA) of Dictyostelium discoideum is defective in a functional Gα2 subunit of a G protein and is characterized by a complete blockade of the cyclic AMP-mediated sensory transduction steps, including cyclic AMP relay, chemotaxis and the cyclic GMP response. Folic acid-mediated

  11. Regulator of G-Protein Signaling 7 Regulates Reward Behavior by Controlling Opioid Signaling in the Striatum.

    Science.gov (United States)

    Sutton, Laurie P; Ostrovskaya, Olga; Dao, Maria; Xie, Keqiang; Orlandi, Cesare; Smith, Roy; Wee, Sunmee; Martemyanov, Kirill A

    2016-08-01

    Morphine mediates its euphoric and analgesic effects by acting on the μ-opioid receptor (MOR). MOR belongs to the family of G-protein coupled receptors whose signaling efficiency is controlled by the regulator of G-protein signaling (RGS) proteins. Our understanding of the molecular diversity of RGS proteins that control MOR signaling, their circuit specific actions, and underlying cellular mechanisms is very limited. We used genetic approaches to ablate regulator of G-protein signaling 7 (RGS7) both globally and in specific neuronal populations. We used conditioned place preference and self-administration paradigms to examine reward-related behavior and a battery of tests to assess analgesia, tolerance, and physical dependence to morphine. Electrophysiology approaches were applied to investigate the impact of RGS7 on morphine-induced alterations in neuronal excitability and plasticity of glutamatergic synapses. At least three animals were used for each assessment. Elimination of RGS7 enhanced reward, increased analgesia, delayed tolerance, and heightened withdrawal in response to morphine administration. RGS7 in striatal neurons was selectively responsible for determining the sensitivity of rewarding and reinforcing behaviors to morphine without affecting analgesia, tolerance, and withdrawal. In contrast, deletion of RGS7 in dopaminergic neurons did not influence morphine reward. RGS7 exerted its effects by controlling morphine-induced changes in excitability of medium spiny neurons in nucleus accumbens and gating the compositional plasticity of α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid and N-methyl-D-aspartate receptors. This study identifies RGS7 as a novel regulator of MOR signaling by dissecting its circuit specific actions and pinpointing its role in regulating morphine reward by controlling the activity of nucleus accumbens neurons. Copyright © 2016 Society of Biological Psychiatry. Published by Elsevier Inc. All rights reserved.

  12. The heterotrimeric G protein Gβ1 interacts with the catalytic subunit of protein phosphatase 1 and modulates G protein-coupled receptor signaling in platelets.

    Science.gov (United States)

    Pradhan, Subhashree; Khatlani, Tanvir; Nairn, Angus C; Vijayan, K Vinod

    2017-08-11

    Thrombosis is caused by the activation of platelets at the site of ruptured atherosclerotic plaques. This activation involves engagement of G protein-coupled receptors (GPCR) on platelets that promote their aggregation. Although it is known that protein kinases and phosphatases modulate GPCR signaling, how serine/threonine phosphatases integrate with G protein signaling pathways is less understood. Because the subcellular localization and substrate specificity of the catalytic subunit of protein phosphatase 1 (PP1c) is dictated by PP1c-interacting proteins, here we sought to identify new PP1c interactors. GPCRs signal via the canonical heterotrimeric Gα and Gβγ subunits. Using a yeast two-hybrid screen, we discovered an interaction between PP1cα and the heterotrimeric G protein Gβ 1 subunit. Co-immunoprecipitation studies with epitope-tagged PP1c and Gβ 1 revealed that Gβ 1 interacts with the PP1c α, β, and γ1 isoforms. Purified PP1c bound to recombinant Gβ 1 -GST protein, and PP1c co-immunoprecipitated with Gβ 1 in unstimulated platelets. Thrombin stimulation of platelets induced the dissociation of the PP1c-Gβ 1 complex, which correlated with an association of PP1c with phospholipase C β3 (PLCβ3), along with a concomitant dephosphorylation of the inhibitory Ser 1105 residue in PLCβ3. siRNA-mediated depletion of GNB1 (encoding Gβ 1 ) in murine megakaryocytes reduced protease-activated receptor 4, activating peptide-induced soluble fibrinogen binding. Thrombin-induced aggregation was decreased in PP1cα -/- murine platelets and in human platelets treated with a small-molecule inhibitor of Gβγ. Finally, disruption of PP1c-Gβ 1 complexes with myristoylated Gβ 1 peptides containing the PP1c binding site moderately decreased thrombin-induced human platelet aggregation. These findings suggest that Gβ 1 protein enlists PP1c to modulate GPCR signaling in platelets.

  13. Cholesterol activates the G-protein coupled receptor Smoothened to promote Hedgehog signaling

    Science.gov (United States)

    Luchetti, Giovanni; Sircar, Ria; Kong, Jennifer H; Nachtergaele, Sigrid; Sagner, Andreas; Byrne, Eamon FX; Covey, Douglas F; Siebold, Christian; Rohatgi, Rajat

    2016-01-01

    Cholesterol is necessary for the function of many G-protein coupled receptors (GPCRs). We find that cholesterol is not just necessary but also sufficient to activate signaling by the Hedgehog (Hh) pathway, a prominent cell-cell communication system in development. Cholesterol influences Hh signaling by directly activating Smoothened (SMO), an orphan GPCR that transmits the Hh signal across the membrane in all animals. Unlike many GPCRs, which are regulated by cholesterol through their heptahelical transmembrane domains, SMO is activated by cholesterol through its extracellular cysteine-rich domain (CRD). Residues shown to mediate cholesterol binding to the CRD in a recent structural analysis also dictate SMO activation, both in response to cholesterol and to native Hh ligands. Our results show that cholesterol can initiate signaling from the cell surface by engaging the extracellular domain of a GPCR and suggest that SMO activity may be regulated by local changes in cholesterol abundance or accessibility. DOI: http://dx.doi.org/10.7554/eLife.20304.001 PMID:27705744

  14. DMPD: G-protein-coupled receptor expression, function, and signaling in macrophages. [Dynamic Macrophage Pathway CSML Database

    Lifescience Database Archive (English)

    Full Text Available 17456803 G-protein-coupled receptor expression, function, and signaling in macropha...2007 Apr 24. (.png) (.svg) (.html) (.csml) Show G-protein-coupled receptor expression, function, and signali...ng in macrophages. PubmedID 17456803 Title G-protein-coupled receptor expression, function

  15. G protein-coupled receptor 91 signaling in diabetic retinopathy and hypoxic retinal diseases.

    Science.gov (United States)

    Hu, Jianyan; Li, Tingting; Du, Xinhua; Wu, Qiang; Le, Yun-Zheng

    2017-10-01

    G protein-coupled receptor 91 (GPR91) is a succinate-specific receptor and activation of GPR91 could initiate a complex signal transduction cascade and upregulate inflammatory and pro-angiogenic cytokines. In the retina, GPR91 is predominately expressed in ganglion cells, a major cellular entity involved in the pathogenesis of diabetic retinopathy (DR) and other hypoxic retinal diseases. During the development of DR and retinopathy of prematurity (ROP), chronic hypoxia causes an increase in the levels of local succinate. Succinate-mediated GPR91 activation upregulates vascular endothelial growth factor (VEGF) through ERK1/2-C/EBP β (c-Fos) and/or ERK1/2-COX-2/PGE2 signaling pathways, which in turn, leads to the breakdown of blood-retina barriers in these disorders. In this review, we will have a brief introduction of GPR91 and its biological functions and a more detailed discussion about the role and mechanisms of GPR91 in DR and ROP. A better understanding of GPR91 regulation may be of great significance in identifying new biomarkers and drug targets for the prediction and treatment of DR, ROP, and hypoxic retinal diseases. Copyright © 2017 Elsevier Ltd. All rights reserved.

  16. Cyclic nucleotide dependent dephosphorylation of regulator of G-protein signaling 18 in human platelets.

    LENUS (Irish Health Repository)

    Gegenbauer, Kristina

    2013-11-01

    Regulator of G-protein signaling 18 (RGS18) is a GTPase-activating protein that turns off Gq signaling in platelets. RGS18 is regulated by binding to the adaptor protein 14-3-3 via phosphorylated serine residues S49 and S218 on RGS18. In this study we confirm that thrombin, thromboxane A2, or ADP stimulate the interaction of RGS18 and 14-3-3 by increasing the phosphorylation of S49. Cyclic AMP- and cyclic GMP-dependent kinases (PKA, PKG) inhibit the interaction of RGS18 and 14-3-3 by phosphorylating S216. To understand the effect of S216 phosphorylation we studied the phosphorylation kinetics of S49, S216, and S218 using Phos-tag gels and phosphorylation site-specific antibodies in transfected cells and in platelets. Cyclic nucleotide-induced detachment of 14-3-3 from RGS18 coincides initially with double phosphorylation of S216 and S218. This is followed by dephosphorylation of S49 and S218. Dephosphorylation of S49 and S218 might be mediated by protein phosphatase 1 (PP1) which is linked to RGS18 by the regulatory subunit PPP1R9B (spinophilin). We conclude that PKA and PKG induced S216 phosphorylation triggers the dephosphorylation of the 14-3-3 binding sites of RGS18 in platelets.

  17. PDZ domain-mediated interactions of G protein-coupled receptors with postsynaptic density protein 95

    DEFF Research Database (Denmark)

    Møller, Thor C; Wirth, Volker F; Roberts, Nina Ingerslev

    2013-01-01

    G protein-coupled receptors (GPCRs) constitute the largest family of membrane proteins in the human genome. Their signaling is regulated by scaffold proteins containing PDZ domains, but although these interactions are important for GPCR function, they are still poorly understood. We here present...

  18. Spatiotemporal aspects of G protein signaling : Where GPCRs and Rho GTPases meet

    NARCIS (Netherlands)

    van Unen, J.

    2017-01-01

    The experimental work conducted for this thesis is aimed towards a better understanding of the fundamental aspects of G protein signaling at the plasma membrane and beyond. The use of advanced microscopy techniques in living cells allows the collection of quantitative information on reaction

  19. Time-dependent, glucose-regulated Arabidopsis Regulator of G-protein Signaling 1 network

    Directory of Open Access Journals (Sweden)

    Dinesh Kumar Jaiswal

    2016-04-01

    Full Text Available Plants lack 7-transmembrane, G-protein coupled receptors (GPCRs because the G alpha subunit of the heterotrimeric G protein complex is “self-activating”—meaning that it spontaneously exchanges bound GDP for GTP without the need of a GPCR. In lieu of GPCRs, most plants have a seven transmembrane receptor-like regulator of G-protein signaling (RGS protein, a component of the complex that keeps G-protein signaling in its non-activated state. The addition of glucose physically uncouples AtRGS1 from the complex through specific endocytosis leaving the activated G protein at the plasma membrane. The complement of proteins in the AtRGS1/G-protein complex over time from glucose-induced endocytosis was profiled by immunoprecipitation coupled to mass spectrometry (IP-MS. A total of 119 proteins in the AtRGS1 complex were identified. Several known interactors of the complex were identified, thus validating the approach, but the vast majority (93/119 were not known previously. AtRGS1 protein interactions were dynamically modulated by d-glucose. At low glucose levels, the AtRGS1 complex is comprised of proteins involved in transport, stress and metabolism. After glucose application, the AtRGS1 complex rapidly sheds many of these proteins and recruits other proteins involved in vesicular trafficking and signal transduction. The profile of the AtRGS1 components answers several questions about the type of coat protein and vesicular trafficking GTPases used in AtRGS1 endocytosis and the function of endocytic AtRGS1.

  20. G protein betagamma-subunits activated by serotonin mediate presynaptic inhibition by regulating vesicle fusion properties.

    Science.gov (United States)

    Photowala, Huzefa; Blackmer, Trillium; Schwartz, Eric; Hamm, Heidi E; Alford, Simon

    2006-03-14

    Neurotransmitters are thought to be released as quanta, where synaptic vesicles deliver packets of neurotransmitter to the synaptic cleft by fusion with the plasma membrane. However, synaptic vesicles may undergo incomplete fusion. We provide evidence that G protein-coupled receptors inhibit release by causing such incomplete fusion. 5-hydroxytryptamine (5-HT) receptor signaling potently inhibits excitatory postsynaptic currents (EPSCs) between lamprey reticulospinal axons and their postsynaptic targets by a direct action on the vesicle fusion machinery. We show that 5-HT receptor-mediated presynaptic inhibition, at this synapse, involves a reduction in EPSC quantal size. Quantal size was measured directly by comparing unitary quantal amplitudes of paired EPSCs before and during 5-HT application and indirectly by determining the effect of 5-HT on the relationship between mean-evoked EPSC amplitude and variance. Results from FM dye-labeling experiments indicate that 5-HT prevents full fusion of vesicles. 5-HT reduces FM1-43 staining of vesicles with a similar efficacy to its effect on the EPSC. However, destaining of FM1-43-labeled vesicles is abolished by lower concentrations of 5-HT that leave a substantial EPSC. The use of a water-soluble membrane impermeant quenching agent in the extracellular space reduced FM1-43 fluorescence during stimulation in 5-HT. Thus vesicles contact the extracellular space during inhibition of synaptic transmission by 5-HT. We conclude that 5-HT, via free Gbetagamma, prevents the collapse of synaptic vesicles into the presynaptic membrane.

  1. Mitogen-Activated Protein Kinase 8 (MAP3K8) Mediates the Signaling Pathway of Estradiol Stimulating Progesterone Production Through G Protein-Coupled Receptor 30 (GPR30) in Mouse Corpus Luteum.

    Science.gov (United States)

    Liu, Ying; Li, Yueqin; Zhang, Di; Liu, Jiali; Gou, Kemian; Cui, Sheng

    2015-05-01

    The corpus luteum (CL) is a transient endocrine gland developed from the ovulated follicles, and the most important function is to synthesize and secrete progesterone (P(4)), a key hormone to maintain normal pregnancy and estrous cycle in most mammals. It is known that estrogen has a vital role in stimulating P(4) synthesis in CL, but it still remains unclear about the mechanism of estradiol (E(2)) regulating P(4) production in CL. Our results here first show that all of the CL cells express MAPK 8 (MAP3K8), and the MAP3K8 level is much higher at the midstage than at the early and late stages during CL development. The further functional studies show that the forced inhibition of endogenous MAP3K8 by using MAP3K8 small interfering RNA and MAP3K8 signaling inhibitor (MAP3K8i) in the luteal cells significantly block the P(4) synthesis and neutralize the enhancing effect of E(2) on P(4) production in the CL. In addition, our results here demonstrate that the stimulating effect of E(2) on P(4) synthesis relies on the estrogen no-classical protein-coupled receptor 30, and MAP3K8 is involved in mediating the protein-coupled receptor 30signaling of E(2) affecting P(4) synthesis via stimulating ERK phosphorylation. These novel findings are critical for our understanding the ovary physiology and pathological mechanism.

  2. G-protein signaling leverages subunit-dependent membrane affinity to differentially control βγ translocation to intracellular membranes.

    Science.gov (United States)

    O'Neill, Patrick R; Karunarathne, W K Ajith; Kalyanaraman, Vani; Silvius, John R; Gautam, N

    2012-12-18

    Activation of G-protein heterotrimers by receptors at the plasma membrane stimulates βγ-complex dissociation from the α-subunit and translocation to internal membranes. This intermembrane movement of lipid-modified proteins is a fundamental but poorly understood feature of cell signaling. The differential translocation of G-protein βγ-subunit types provides a valuable experimental model to examine the movement of signaling proteins between membranes in a living cell. We used live cell imaging, mathematical modeling, and in vitro measurements of lipidated fluorescent peptide dissociation from vesicles to determine the mechanistic basis of the intermembrane movement and identify the interactions responsible for differential translocation kinetics in this family of evolutionarily conserved proteins. We found that the reversible translocation is mediated by the limited affinity of the βγ-subunits for membranes. The differential kinetics of the βγ-subunit types are determined by variations among a set of basic and hydrophobic residues in the γ-subunit types. G-protein signaling thus leverages the wide variation in membrane dissociation rates among different γ-subunit types to differentially control βγ-translocation kinetics in response to receptor activation. The conservation of primary structures of γ-subunits across mammalian species suggests that there can be evolutionary selection for primary structures that confer specific membrane-binding affinities and consequent rates of intermembrane movement.

  3. Antiatherosclerotic Effect of Korean Red Ginseng Extract Involves Regulator of G-Protein Signaling 5

    Directory of Open Access Journals (Sweden)

    Eun Ju Im

    2014-01-01

    Full Text Available Regulator of G-protein signaling 5 (RGS5, an inhibitor of Gα(q and Gα(i activation, has been reported to have antiatherosclerosis. Previous studies showed antiatherosclerotic effect of Korean red ginseng water extract (KRGE via multiple signaling pathways. However, potential protective effect of KRGE through RGS5 expression has not been elucidated. Here, we investigated the antiatherosclerotic effect of KRGE in vivo and in vitro and its role on RGS5 mRNA expression. Elevated levels of total cholesterol, lactate dehydrogenase (LDH, and triglyceride (TG in western diet groups of low-density lipoprotein receptor deficient LDLr−/− mice were reversed by oral administration of KRGE. KRGE suppressed transcriptional activity of tumor necrotic factor alpha (TNF-α, interleukin-6 (IL-6, and leptin in adipose tissue. It also potently repressed western diet-induced atheroma formation in aortic sinus. While KRGE showed reduced mRNA expression of inducible nitric oxide synthase (iNOS, cyclooxygenase-2 (COX-2, IL-1β, IL-6, and TNF-α in LPS-stimulated RAW264.7 cells, it enhanced mRNA expression of RGS5. Moreover, RGS5 siRNA transfection of microglia cells pretreated with KRGE reversed its inhibitory effect on the expression of iNOS, COX-2, and IL-1β mRNA. In conclusion, KRGE showed antiatherosclerotic and anti-inflammatory effects in western diet fed LDLr−/− mice and this effect could partly be mediated by RGS5 expression.

  4. Natural products induce a G protein-mediated calcium pathway activating p53 in cancer cells

    Energy Technology Data Exchange (ETDEWEB)

    Ginkel, Paul R. van; Yan, Michael B. [UW Carbone Cancer Center, University of Wisconsin, Madison, WI 53792 (United States); Department of Ophthalmology and Visual Sciences, University of Wisconsin, Madison, WI 53792 (United States); Bhattacharya, Saswati [UW Carbone Cancer Center, University of Wisconsin, Madison, WI 53792 (United States); Department of Ophthalmology and Visual Sciences, University of Wisconsin, Madison, WI 53792 (United States); Department of Pediatrics, University of Wisconsin, Madison, WI 53792 (United States); Polans, Arthur S., E-mail: aspolans@wisc.edu [UW Carbone Cancer Center, University of Wisconsin, Madison, WI 53792 (United States); Department of Ophthalmology and Visual Sciences, University of Wisconsin, Madison, WI 53792 (United States); Kenealey, Jason D. [UW Carbone Cancer Center, University of Wisconsin, Madison, WI 53792 (United States); Department of Ophthalmology and Visual Sciences, University of Wisconsin, Madison, WI 53792 (United States); Department of Nutrition, Dietetics and Food Science, Brigham Young University, Provo, UT 84602 (United States)

    2015-11-01

    Paclitaxel, etoposide, vincristine and doxorubicin are examples of natural products being used as chemotherapeutics but with adverse side effects that limit their therapeutic window. Natural products derived from plants and having low toxicity, such as quercetin, resveratrol, epigallocatechin gallate and piceatannol, have been shown to inhibit tumor cell growth both in vitro and in pre-clinical models of cancer, but their mechanisms of action have not been fully elucidated, thus restricting their use as prototypes for developing synthetic analogs with improved anti-cancer properties. We and others have demonstrated that one of the earliest and consistent events upon exposure of tumor cells to these less toxic natural products is a rise in cytoplasmic calcium, activating several pro-apoptotic pathways. We describe here a G protein/inositol 1,4,5-trisphosphate pathway (InsP3) in MDA-MB-231 human breast cancer cells that mediates between these less toxic natural products and the release of calcium from the endoplasmic reticulum. Further, we demonstrate that this elevation of intracellular calcium modulates p53 activity and the subsequent transcription of several pro-apoptotic genes encoding PIG8, CD95, PIDD, TP53INP, RRM2B, Noxa, p21 and PUMA. We conclude from our findings that less toxic natural products likely bind to a G protein coupled receptor that activates a G protein-mediated and calcium-dependent pathway resulting selectively in tumor cell death. - Highlights: • Natural products having low toxicity increase cytoplasmic calcium in cancer cells. • A G-protein/IP{sub 3} pathway mediates the release of calcium from the ER. • The elevation of intracellular calcium modulates p53 activity. • p53 and other Ca{sup 2+}-dependent pro-apoptotic pathways inhibit cancer cell growth.

  5. Natural products induce a G protein-mediated calcium pathway activating p53 in cancer cells

    International Nuclear Information System (INIS)

    Ginkel, Paul R. van; Yan, Michael B.; Bhattacharya, Saswati; Polans, Arthur S.; Kenealey, Jason D.

    2015-01-01

    Paclitaxel, etoposide, vincristine and doxorubicin are examples of natural products being used as chemotherapeutics but with adverse side effects that limit their therapeutic window. Natural products derived from plants and having low toxicity, such as quercetin, resveratrol, epigallocatechin gallate and piceatannol, have been shown to inhibit tumor cell growth both in vitro and in pre-clinical models of cancer, but their mechanisms of action have not been fully elucidated, thus restricting their use as prototypes for developing synthetic analogs with improved anti-cancer properties. We and others have demonstrated that one of the earliest and consistent events upon exposure of tumor cells to these less toxic natural products is a rise in cytoplasmic calcium, activating several pro-apoptotic pathways. We describe here a G protein/inositol 1,4,5-trisphosphate pathway (InsP3) in MDA-MB-231 human breast cancer cells that mediates between these less toxic natural products and the release of calcium from the endoplasmic reticulum. Further, we demonstrate that this elevation of intracellular calcium modulates p53 activity and the subsequent transcription of several pro-apoptotic genes encoding PIG8, CD95, PIDD, TP53INP, RRM2B, Noxa, p21 and PUMA. We conclude from our findings that less toxic natural products likely bind to a G protein coupled receptor that activates a G protein-mediated and calcium-dependent pathway resulting selectively in tumor cell death. - Highlights: • Natural products having low toxicity increase cytoplasmic calcium in cancer cells. • A G-protein/IP 3 pathway mediates the release of calcium from the ER. • The elevation of intracellular calcium modulates p53 activity. • p53 and other Ca 2+ -dependent pro-apoptotic pathways inhibit cancer cell growth.

  6. G Protein-Linked Signaling Pathways in Bipolar and Major Depressive Disorders

    Directory of Open Access Journals (Sweden)

    Hiroaki eTomita

    2013-12-01

    Full Text Available The G-protein linked signaling system (GPLS comprises a large number of G-proteins, G protein-coupled receptors (GPCRs, GPCR ligands, and downstream effector molecules. G-proteins interact with both GPCRs and downstream effectors such as cyclic adenosine monophosphate (cAMP, phosphatidylinositols, and ion channels. The GPLS is implicated in the pathophysiology and pharmacology of both major depressive disorder (MDD and bipolar disorder (BPD. This study evaluated whether GPLS is altered at the transcript level. The gene expression in the dorsolateral prefrontal (DLPFC and anterior cingulate (ACC were compared from MDD, BPD, and control subjects using Affymetrix Gene Chips and real time quantitative PCR. High quality brain tissue was used in the study to control for confounding effects of agonal events, tissue pH, RNA integrity, gender, and age. GPLS signaling transcripts were altered especially in the ACC of BPD and MDD subjects. Transcript levels of molecules which repress cAMP activity were increased in BPD and decreased in MDD. Two orphan GPCRs, GPRC5B and GPR37, showed significantly decreased expression levels in MDD, and significantly increased expression levels in BPD. Our results suggest opposite changes in BPD and MDD in the GPLS, ‘activated’ cAMP signaling activity in BPD and ‘blunted’ cAMP signaling activity in MDD. GPRC5B and GPR37 both appear to have behavioral effects, and are also candidate genes for neurodegenerative disorders. In the context of the opposite changes observed in BPD and MDD, these GPCRs warrant further study of their brain effects.

  7. Development of full sweet, umami, and bitter taste responsiveness requires Regulator of G protein Signaling-21 (RGS21).

    Science.gov (United States)

    Schroer, Adam B; Gross, Joshua D; Kaski, Shane W; Wix, Kim; Siderovski, David P; Vandenbeuch, Aurelie; Setola, Vincent

    2018-04-26

    The mammalian tastes of sweet, umami, and bitter are initiated by activation of G protein-coupled receptors (GPCRs) of the T1R and T2R families on taste receptor cells. GPCRs signal via nucleotide exchange and hydrolysis, the latter hastened by GTPase-accelerating proteins (GAPs) that include the Regulators of G protein Signaling (RGS) protein family. We previously reported that RGS21, uniquely expressed in Type II taste receptor cells, decreases the potency of bitter-stimulated T2R signaling in cultured cells, consistent with its in vitro GAP activity. However, the role of RGS21 in organismal responses to GPCR-mediated tastants was not established. Here, we characterized mice lacking the Rgs21 fifth exon. Eliminating Rgs21 expression had no effect on body mass accumulation (a measure of alimentation), fungiform papillae number and morphology, circumvallate papillae morphology, and taste bud number. Two-bottle preference tests, however, revealed that Rgs21-null mice have blunted aversion to quinine and denatonium, and blunted preference for monosodium glutamate, the sweeteners sucrose and SC45647, and (surprisingly) NaCl. Observed reductions in GPCR-mediated tastant responses upon Rgs21 loss are opposite to original expectations, given that loss of RGS21 -- a GPCR signaling negative regulator -- should lead to increased responsiveness to tastant-mediated GPCR signaling (all else being equal). Yet, reduced organismal tastant responses are consistent with observations of reduced chorda tympani nerve recordings in Rgs21-null mice. Reduced tastant-mediated responses and behaviors exhibited by adult mice lacking Rgs21 expression since birth have thus revealed an underappreciated requirement for a GPCR GAP to establish the full character of tastant signaling.

  8. Regulator of G protein signaling 5 (RGS5) inhibits sonic hedgehog function in mouse cortical neurons.

    Science.gov (United States)

    Liu, Chuanliang; Hu, Qiongqiong; Jing, Jia; Zhang, Yun; Jin, Jing; Zhang, Liulei; Mu, Lili; Liu, Yumei; Sun, Bo; Zhang, Tongshuai; Kong, Qingfei; Wang, Guangyou; Wang, Dandan; Zhang, Yao; Liu, Xijun; Zhao, Wei; Wang, Jinghua; Feng, Tao; Li, Hulun

    2017-09-01

    Regulator of G protein signaling 5 (RGS5) acts as a GTPase-activating protein (GAP) for the Gαi subunit and negatively regulates G protein-coupled receptor signaling. However, its presence and function in postmitotic differentiated primary neurons remains largely uncharacterized. During neural development, sonic hedgehog (Shh) signaling is involved in cell signaling pathways via Gαi activity. In particular, Shh signaling is essential for embryonic neural tube patterning, which has been implicated in neuronal polarization involving neurite outgrowth. Here, we examined whether RGS5 regulates Shh signaling in neurons. RGS5 transcripts were found to be expressed in cortical neurons and their expression gradually declined in a time-dependent manner in culture system. When an adenovirus expressing RGS5 was introduced into an in vitro cell culture model of cortical neurons, RGS5 overexpression significantly reduced neurite outgrowth and FM4-64 uptake, while cAMP-PKA signaling was also affected. These findings suggest that RGS5 inhibits Shh function during neurite outgrowth and the presynaptic terminals of primary cortical neurons mature via modulation of cAMP. Copyright © 2017 Elsevier Inc. All rights reserved.

  9. Conformational analysis of g protein-coupled receptor signaling by hydrogen/deuterium exchange mass spectrometry.

    Science.gov (United States)

    Li, Sheng; Lee, Su Youn; Chung, Ka Young

    2015-01-01

    Conformational change and protein-protein interactions are two major mechanisms of membrane protein signal transduction, including G protein-coupled receptors (GPCRs). Upon agonist binding, GPCRs change conformation, resulting in interaction with downstream signaling molecules such as G proteins. To understand the precise signaling mechanism, studies have investigated the structural mechanism of GPCR signaling using X-ray crystallography, nuclear magnetic resonance (NMR), or electron paramagnetic resonance. In addition to these techniques, hydrogen/deuterium exchange mass spectrometry (HDX-MS) has recently been used in GPCR studies. HDX-MS measures the rate at which peptide amide hydrogens exchange with deuterium in the solvent. Exposed or flexible regions have higher exchange rates and excluded or ordered regions have lower exchange rates. Therefore, HDX-MS is a useful tool for studying protein-protein interfaces and conformational changes after protein activation or protein-protein interactions. Although HDX-MS does not give high-resolution structures, it analyzes protein conformations that are difficult to study with X-ray crystallography or NMR. Furthermore, conformational information from HDX-MS can help in the crystallization of X-ray crystallography by suggesting highly flexible regions. Interactions between GPCRs and downstream signaling molecules are not easily analyzed by X-ray crystallography or NMR because of the large size of the GPCR-signaling molecule complexes, hydrophobicity, and flexibility of GPCRs. HDX-MS could be useful for analyzing the conformational mechanism of GPCR signaling. In this chapter, we discuss details of HDX-MS for analyzing GPCRs using the β2AR-G protein complex as a model system. © 2015 Elsevier Inc. All rights reserved.

  10. Regulation of protease-activated receptor 1 signaling by the adaptor protein complex 2 and R4 subfamily of regulator of G protein signaling proteins.

    Science.gov (United States)

    Chen, Buxin; Siderovski, David P; Neubig, Richard R; Lawson, Mark A; Trejo, Joann

    2014-01-17

    The G protein-coupled protease-activated receptor 1 (PAR1) is irreversibly proteolytically activated by thrombin. Hence, the precise regulation of PAR1 signaling is important for proper cellular responses. In addition to desensitization, internalization and lysosomal sorting of activated PAR1 are critical for the termination of signaling. Unlike most G protein-coupled receptors, PAR1 internalization is mediated by the clathrin adaptor protein complex 2 (AP-2) and epsin-1, rather than β-arrestins. However, the function of AP-2 and epsin-1 in the regulation of PAR1 signaling is not known. Here, we report that AP-2, and not epsin-1, regulates activated PAR1-stimulated phosphoinositide hydrolysis via two different mechanisms that involve, in part, a subset of R4 subfamily of "regulator of G protein signaling" (RGS) proteins. A significantly greater increase in activated PAR1 signaling was observed in cells depleted of AP-2 using siRNA or in cells expressing a PAR1 (420)AKKAA(424) mutant with defective AP-2 binding. This effect was attributed to AP-2 modulation of PAR1 surface expression and efficiency of G protein coupling. We further found that ectopic expression of R4 subfamily members RGS2, RGS3, RGS4, and RGS5 reduced activated PAR1 wild-type signaling, whereas signaling by the PAR1 AKKAA mutant was minimally affected. Intriguingly, siRNA-mediated depletion analysis revealed a function for RGS5 in the regulation of signaling by the PAR1 wild type but not the AKKAA mutant. Moreover, activation of the PAR1 wild type, and not the AKKAA mutant, induced Gαq association with RGS3 via an AP-2-dependent mechanism. Thus, AP-2 regulates activated PAR1 signaling by altering receptor surface expression and through recruitment of RGS proteins.

  11. Identification of serine 348 on the apelin receptor as a novel regulatory phosphorylation site in apelin-13-induced G protein-independent biased signaling.

    Science.gov (United States)

    Chen, Xiaoyu; Bai, Bo; Tian, Yanjun; Du, Hui; Chen, Jing

    2014-11-07

    Phosphorylation plays vital roles in the regulation of G protein-coupled receptor (GPCR) functions. The apelin and apelin receptor (APJ) system is involved in the regulation of cardiovascular function and central control of body homeostasis. Here, using tandem mass spectrometry, we first identified phosphorylated serine residues in the C terminus of APJ. To determine the role of phosphorylation sites in APJ-mediated G protein-dependent and -independent signaling and function, we induced a mutation in the C-terminal serine residues and examined their effects on the interaction between APJ with G protein or GRK/β-arrestin and their downstream signaling. Mutation of serine 348 led to an elimination of both GRK and β-arrestin recruitment to APJ induced by apelin-13. Moreover, APJ internalization and G protein-independent ERK signaling were also abolished by point mutation at serine 348. In contrast, this mutant at serine residues had no demonstrable impact on apelin-13-induced G protein activation and its intracellular signaling. These findings suggest that mutation of serine 348 resulted in inactive GRK/β-arrestin. However, there was no change in the active G protein thus, APJ conformation was biased. These results provide important information on the molecular interplay and impact of the APJ function, which may be extrapolated to design novel drugs for cardiac hypertrophy based on this biased signal pathway. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

  12. Regulator of G protein signaling 2 (RGS2 and RGS4 form distinct G protein-dependent complexes with protease activated-receptor 1 (PAR1 in live cells.

    Directory of Open Access Journals (Sweden)

    Sungho Ghil

    Full Text Available Protease-activated receptor 1 (PAR1 is a G-protein coupled receptor (GPCR that is activated by natural proteases to regulate many physiological actions. We previously reported that PAR1 couples to Gi, Gq and G12 to activate linked signaling pathways. Regulators of G protein signaling (RGS proteins serve as GTPase activating proteins to inhibit GPCR/G protein signaling. Some RGS proteins interact directly with certain GPCRs to modulate their signals, though cellular mechanisms dictating selective RGS/GPCR coupling are poorly understood. Here, using bioluminescence resonance energy transfer (BRET, we tested whether RGS2 and RGS4 bind to PAR1 in live COS-7 cells to regulate PAR1/Gα-mediated signaling. We report that PAR1 selectively interacts with either RGS2 or RGS4 in a G protein-dependent manner. Very little BRET activity is observed between PAR1-Venus (PAR1-Ven and either RGS2-Luciferase (RGS2-Luc or RGS4-Luc in the absence of Gα. However, in the presence of specific Gα subunits, BRET activity was markedly enhanced between PAR1-RGS2 by Gαq/11, and PAR1-RGS4 by Gαo, but not by other Gα subunits. Gαq/11-YFP/RGS2-Luc BRET activity is promoted by PAR1 and is markedly enhanced by agonist (TFLLR stimulation. However, PAR1-Ven/RGS-Luc BRET activity was blocked by a PAR1 mutant (R205A that eliminates PAR1-Gq/11 coupling. The purified intracellular third loop of PAR1 binds directly to purified His-RGS2 or His-RGS4. In cells, RGS2 and RGS4 inhibited PAR1/Gα-mediated calcium and MAPK/ERK signaling, respectively, but not RhoA signaling. Our findings indicate that RGS2 and RGS4 interact directly with PAR1 in Gα-dependent manner to modulate PAR1/Gα-mediated signaling, and highlight a cellular mechanism for selective GPCR/G protein/RGS coupling.

  13. New functions and signaling mechanisms for the class of adhesion G protein-coupled receptors

    DEFF Research Database (Denmark)

    Liebscher, Ines; Ackley, Brian; Araç, Demet

    2014-01-01

    The class of adhesion G protein-coupled receptors (aGPCRs), with 33 human homologs, is the second largest family of GPCRs. In addition to a seven-transmembrane α-helix-a structural feature of all GPCRs-the class of aGPCRs is characterized by the presence of a large N-terminal extracellular region....... In addition, all aGPCRs but one (GPR123) contain a GPCR autoproteolysis-inducing (GAIN) domain that mediates autoproteolytic cleavage at the GPCR autoproteolysis site motif to generate N- and a C-terminal fragments (NTF and CTF, respectively) during protein maturation. Subsequently, the NTF and CTF...

  14. PhotosynthateRegulation of the Root System Architecture Mediated bythe Heterotrimeric G Protein Complex in Arabidopsis

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    Yashwanti Mudgil

    2016-08-01

    Full Text Available Assimilate partitioning to the root system is a desirable developmental trait to control but little is known of the signaling pathway underlying partitioning. A null mutation in the gene encoding the Gβ subunit of the heterotrimeric G protein complex, a nexus for a variety of signaling pathways, confers altered sugar partitioning in roots. While fixed carbon rapidly reached the roots of wild type and agb1-2 mutant seedlings, agb1 roots had more of this fixed carbon in the form of glucose, fructose, and sucrose which manifested as a higher lateral root density. Upon glucose treatment, the agb1-2 mutant had abnormal gene expression in the root tip validated by transcriptome analysis. In addition, PIN2 membrane localization and level was altered in the agb1-2 mutant. The heterotrimeric G protein complex integrates photosynthesis-derived sugar signaling incorporating both membrane-and transcriptional-based mechanisms. The time constants for these signaling mechanisms are in the same range as photosynthate delivery to the root, raising the possibility that root cells are able to use changes in carbon fixation in real time to adjust growth behavior.

  15. Photosynthate Regulation of the Root System Architecture Mediated by the Heterotrimeric G Protein Complex in Arabidopsis.

    Science.gov (United States)

    Mudgil, Yashwanti; Karve, Abhijit; Teixeira, Paulo J P L; Jiang, Kun; Tunc-Ozdemir, Meral; Jones, Alan M

    2016-01-01

    Assimilate partitioning to the root system is a desirable developmental trait to control but little is known of the signaling pathway underlying partitioning. A null mutation in the gene encoding the Gβ subunit of the heterotrimeric G protein complex, a nexus for a variety of signaling pathways, confers altered sugar partitioning in roots. While fixed carbon rapidly reached the roots of wild type and agb1-2 mutant seedlings, agb1 roots had more of this fixed carbon in the form of glucose, fructose, and sucrose which manifested as a higher lateral root density. Upon glucose treatment, the agb1-2 mutant had abnormal gene expression in the root tip validated by transcriptome analysis. In addition, PIN2 membrane localization was altered in the agb1-2 mutant. The heterotrimeric G protein complex integrates photosynthesis-derived sugar signaling incorporating both membrane-and transcriptional-based mechanisms. The time constants for these signaling mechanisms are in the same range as photosynthate delivery to the root, raising the possibility that root cells are able to use changes in carbon fixation in real time to adjust growth behavior.

  16. The Multiple Faces of Prostaglandin E2 G-Protein Coupled Receptor Signaling during the Dendritic Cell Life Cycle

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    Alessandra Cambi

    2013-03-01

    Full Text Available Many processes regulating immune responses are initiated by G-protein coupled receptors (GPCRs and report biochemical changes in the microenvironment. Dendritic cells (DCs are the most potent antigen-presenting cells and crucial for the regulation of innate and adaptive immune responses. The lipid mediator Prostaglandin E2 (PGE2 via four GPCR subtypes (EP1-4 critically regulates DC generation, maturation and migration. The role of PGE2 signaling in DC biology was unraveled by the characterization of EP receptor subtype expression in DC progenitor cells and DCs, the identification of the signaling pathways initiated by these GPCR subtypes and the classification of DC responses to PGE2 at different stages of differentiation. Here, we review the advances in PGE2 signaling in DCs and describe the efforts still to be made to understand the spatio-temporal fine-tuning of PGE2 responses by DCs.

  17. Diverse Regulation of Temperature Sensation by Trimeric G-Protein Signaling in Caenorhabditis elegans.

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    Tomoyo Ujisawa

    Full Text Available Temperature sensation by the nervous system is essential for life and proliferation of animals. The molecular-physiological mechanisms underlying temperature signaling have not been fully elucidated. We show here that diverse regulatory machinery underlies temperature sensation through trimeric G-protein signaling in the nematode Caenorhabditis elegans. Molecular-genetic studies demonstrated that cold tolerance is regulated by additive functions of three Gα proteins in a temperature-sensing neuron, ASJ, which is also known to be a light-sensing neuron. Optical recording of calcium concentration in ASJ upon temperature-changes demonstrated that three Gα proteins act in different aspects of temperature signaling. Calcium concentration changes in ASJ upon temperature change were unexpectedly decreased in a mutant defective in phosphodiesterase, which is well known as a negative regulator of calcium increase. Together, these data demonstrate commonalities and differences in the molecular components concerned with light and temperature signaling in a single sensory neuron.

  18. Diverse Regulation of Temperature Sensation by Trimeric G-Protein Signaling in Caenorhabditis elegans

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    Ujisawa, Tomoyo; Ohta, Akane; Uda-Yagi, Misato

    2016-01-01

    Temperature sensation by the nervous system is essential for life and proliferation of animals. The molecular-physiological mechanisms underlying temperature signaling have not been fully elucidated. We show here that diverse regulatory machinery underlies temperature sensation through trimeric G-protein signaling in the nematode Caenorhabditis elegans. Molecular-genetic studies demonstrated that cold tolerance is regulated by additive functions of three Gα proteins in a temperature-sensing neuron, ASJ, which is also known to be a light-sensing neuron. Optical recording of calcium concentration in ASJ upon temperature-changes demonstrated that three Gα proteins act in different aspects of temperature signaling. Calcium concentration changes in ASJ upon temperature change were unexpectedly decreased in a mutant defective in phosphodiesterase, which is well known as a negative regulator of calcium increase. Together, these data demonstrate commonalities and differences in the molecular components concerned with light and temperature signaling in a single sensory neuron. PMID:27788246

  19. Isolation and structure-function characterization of a signaling-active rhodopsin-G protein complex.

    Science.gov (United States)

    Gao, Yang; Westfield, Gerwin; Erickson, Jon W; Cerione, Richard A; Skiniotis, Georgios; Ramachandran, Sekar

    2017-08-25

    The visual photo-transduction cascade is a prototypical G protein-coupled receptor (GPCR) signaling system, in which light-activated rhodopsin (Rho*) is the GPCR catalyzing the exchange of GDP for GTP on the heterotrimeric G protein transducin (G T ). This results in the dissociation of G T into its component α T -GTP and β 1 γ 1 subunit complex. Structural information for the Rho*-G T complex will be essential for understanding the molecular mechanism of visual photo-transduction. Moreover, it will shed light on how GPCRs selectively couple to and activate their G protein signaling partners. Here, we report on the preparation of a stable detergent-solubilized complex between Rho* and a heterotrimer (G T *) comprising a Gα T /Gα i1 chimera (α T *) and β 1 γ 1 The complex was formed on native rod outer segment membranes upon light activation, solubilized in lauryl maltose neopentyl glycol, and purified with a combination of affinity and size-exclusion chromatography. We found that the complex is fully functional and that the stoichiometry of Rho* to Gα T * is 1:1. The molecular weight of the complex was calculated from small-angle X-ray scattering data and was in good agreement with a model consisting of one Rho* and one G T *. The complex was visualized by negative-stain electron microscopy, which revealed an architecture similar to that of the β 2 -adrenergic receptor-G S complex, including a flexible α T * helical domain. The stability and high yield of the purified complex should allow for further efforts toward obtaining a high-resolution structure of this important signaling complex. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  20. Heterotrimeric G Protein-coupled Receptor Signaling in Yeast Mating Pheromone Response.

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    Alvaro, Christopher G; Thorner, Jeremy

    2016-04-08

    The DNAs encoding the receptors that respond to the peptide mating pheromones of the budding yeastSaccharomyces cerevisiaewere isolated in 1985, and were the very first genes for agonist-binding heterotrimeric G protein-coupled receptors (GPCRs) to be cloned in any organism. Now, over 30 years later, this yeast and its receptors continue to provide a pathfinding experimental paradigm for investigating GPCR-initiated signaling and its regulation, as described in this retrospective overview. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  1. Induction of the unfolded protein response by constitutive G-protein signaling in rod photoreceptor cells.

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    Wang, Tian; Chen, Jeannie

    2014-10-17

    Phototransduction is a G-protein signal transduction cascade that converts photon absorption to a change in current at the plasma membrane. Certain genetic mutations affecting the proteins in the phototransduction cascade cause blinding disorders in humans. Some of these mutations serve as a genetic source of "equivalent light" that activates the cascade, whereas other mutations lead to amplification of the light response. How constitutive phototransduction causes photoreceptor cell death is poorly understood. We showed that persistent G-protein signaling, which occurs in rod arrestin and rhodopsin kinase knock-out mice, caused a rapid and specific induction of the PERK pathway of the unfolded protein response. These changes were not observed in the cGMP-gated channel knock-out rods, an equivalent light condition that mimics light-stimulated channel closure. Thus transducin signaling, but not channel closure, triggers rapid cell death in light damage caused by constitutive phototransduction. Additionally, we show that in the albino light damage model cell death was not associated with increase in global protein ubiquitination or unfolded protein response induction. Taken together, these observations provide novel mechanistic insights into the cell death pathway caused by constitutive phototransduction and identify the unfolded protein response as a potential target for therapeutic intervention. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

  2. Switch I-dependent allosteric signaling in a G-protein chaperone-B12 enzyme complex.

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    Campanello, Gregory C; Lofgren, Michael; Yokom, Adam L; Southworth, Daniel R; Banerjee, Ruma

    2017-10-27

    G-proteins regulate various processes ranging from DNA replication and protein synthesis to cytoskeletal dynamics and cofactor assimilation and serve as models for uncovering strategies deployed for allosteric signal transduction. MeaB is a multifunctional G-protein chaperone, which gates loading of the active 5'-deoxyadenosylcobalamin cofactor onto methylmalonyl-CoA mutase (MCM) and precludes loading of inactive cofactor forms. MeaB also safeguards MCM, which uses radical chemistry, against inactivation and rescues MCM inactivated during catalytic turnover by using the GTP-binding energy to offload inactive cofactor. The conserved switch I and II signaling motifs used by G-proteins are predicted to mediate allosteric regulation in response to nucleotide binding and hydrolysis in MeaB. Herein, we targeted conserved residues in the MeaB switch I motif to interrogate the function of this loop. Unexpectedly, the switch I mutations had only modest effects on GTP binding and on GTPase activity and did not perturb stability of the MCM-MeaB complex. However, these mutations disrupted multiple MeaB chaperone functions, including cofactor editing, loading, and offloading. Hence, although residues in the switch I motif are not essential for catalysis, they are important for allosteric regulation. Furthermore, single-particle EM analysis revealed, for the first time, the overall architecture of the MCM-MeaB complex, which exhibits a 2:1 stoichiometry. These EM studies also demonstrate that the complex exhibits considerable conformational flexibility. In conclusion, the switch I element does not significantly stabilize the MCM-MeaB complex or influence the affinity of MeaB for GTP but is required for transducing signals between MeaB and MCM. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  3. R7-binding protein targets the G protein β5/R7-regulator of G protein signaling complex to lipid rafts in neuronal cells and brain

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    Zhang Jian-Hua

    2007-09-01

    Full Text Available Abstract Background Heterotrimeric guanine nucleotide-binding regulatory proteins (G proteins, composed of Gα, Gβ, and Gγ subunits, are positioned at the inner face of the plasma membrane and relay signals from activated G protein-coupled cell surface receptors to various signaling pathways. Gβ5 is the most structurally divergent Gβ isoform and forms tight heterodimers with regulator of G protein signalling (RGS proteins of the R7 subfamily (R7-RGS. The subcellular localization of Gβ 5/R7-RGS protein complexes is regulated by the palmitoylation status of the associated R7-binding protein (R7BP, a recently discovered SNARE-like protein. We investigate here whether R7BP controls the targeting of Gβ5/R7-RGS complexes to lipid rafts, cholesterol-rich membrane microdomains where conventional heterotrimeric G proteins and some effector proteins are concentrated in neurons and brain. Results We show that endogenous Gβ5/R7-RGS/R7BP protein complexes are present in native neuron-like PC12 cells and that a fraction is targeted to low-density, detergent-resistant membrane lipid rafts. The buoyant density of endogenous raft-associated Gβ5/R7-RGS protein complexes in PC12 cells was similar to that of lipid rafts containing the palmitoylated marker proteins PSD-95 and LAT, but distinct from that of the membrane microdomain where flotillin was localized. Overexpression of wild-type R7BP, but not its palmitoylation-deficient mutant, greatly enriched the fraction of endogenous Gβ5/R7-RGS protein complexes in the lipid rafts. In HEK-293 cells the palmitoylation status of R7BP also regulated the lipid raft targeting of co-expressed Gβ5/R7-RGS/R7BP proteins. A fraction of endogenous Gβ5/R7-RGS/R7BP complexes was also present in lipid rafts in mouse brain. Conclusion A fraction of Gβ5/R7-RGS/R7BP protein complexes is targeted to low-density, detergent-resistant membrane lipid rafts in PC12 cells and brain. In cultured cells, the palmitoylation status of

  4. Membrane-mediated oligomerization of G protein coupled receptors and its implications for GPCR function

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    Stefan Gahbauer

    2016-10-01

    Full Text Available The dimerization or even oligomerization of G protein coupled receptors (GPCRs causes ongoing, controversial debates about its functional role and the coupled biophysical, biochemical or biomedical implications. A continously growing number of studies hints to a relation between oligomerization and function of GPCRs and strengthens the assumption that receptor assembly plays a key role in the regulation of protein function. Additionally, progress in the structural analysis of GPCR-G protein and GPCR-ligand interactions allows to distinguish between actively functional and non-signalling complexes. Recent findings further suggest that the surrounding membrane, i.e. its lipid composition may modulate the preferred dimerization interface and as a result the abundance of distinct dimeric conformations. In this review, the association of GPCRs and the role of the membrane in oligomerization will be discussed. An overview of the different reported oligomeric interfaces is provided and their capability for signaling discussed. The currently available data is summarized with regard to the formation of GPCR oligomers, their structures and dependency on the membrane microenvironment as well as the coupling of oligomerization to receptor function.

  5. Chemogenetic Modulation of G Protein-Coupled Receptor Signalling in Visual Attention Research

    DEFF Research Database (Denmark)

    Jørgensen, Søren H; Fitzpatrick, Ciarán Martin; Gether, Ulrik

    2017-01-01

    Exclusively Activated by Designer Drugs (DREADDs). The DREADD technology is an emerging and transformative method that allows selective manipulation of G protein-coupled receptor (GPCR) signalling, and its broad-ranging usefulness in attention research is now beginning to emerge. We first describe......Attention is a fundamental cognitive process involved in nearly all aspects of life. Abnormal attentional control is a symptom of many neurological disorders, most notably recognized in ADHD (attention deficit hyperactivity disorder). Although attentional performance and its malfunction has been...... the different DREADDs available and explain how unprecedented specificity of neuronal signalling can be achieved using DREADDs. We next discuss various studies performed in animal models of visual attention, where different brain regions and neuronal populations have been probed by DREADDs. We highlight...

  6. Polycystin 1 loss of function is directly linked to an imbalance in G-protein signaling in the kidney.

    Science.gov (United States)

    Zhang, Bo; Tran, Uyen; Wessely, Oliver

    2018-03-22

    The development of the kidney relies on the establishment and maintenance of a precise tubular diameter of its functional units, the nephrons. This process is disrupted in polycystic kidney disease (PKD), resulting in dilations of the nephron and renal cyst formation. In the course of exploring G-protein-coupled signaling in the Xenopus pronephric kidney, we discovered that loss of the G-protein α subunit, Gnas, results in a PKD phenotype. Polycystin 1, one of the genes mutated in human PKD, encodes a protein resembling a G-protein-coupled receptor. Furthermore, deletion of the G-protein-binding domain present in the intracellular C terminus of polycystin 1 impacts functionality. A comprehensive analysis of all the G-protein α subunits expressed in the Xenopus pronephric kidney demonstrates that polycystin 1 recruits a select subset of G-protein α subunits and that their knockdown - as in the case of Gnas - results in a PKD phenotype. Mechanistically, the phenotype is caused by increased endogenous G-protein β/γ signaling and can be reversed by pharmacological inhibitors as well as knocking down Gnb1. Together, our data support the hypothesis that G proteins are recruited to the intracellular domain of PKD1 and that this interaction is crucial for its function in the kidney. © 2018. Published by The Company of Biologists Ltd.

  7. Isolation and structure–function characterization of a signaling-active rhodopsin–G protein complex

    Science.gov (United States)

    Gao, Yang; Westfield, Gerwin; Erickson, Jon W.; Cerione, Richard A.; Skiniotis, Georgios; Ramachandran, Sekar

    2017-01-01

    The visual photo-transduction cascade is a prototypical G protein–coupled receptor (GPCR) signaling system, in which light-activated rhodopsin (Rho*) is the GPCR catalyzing the exchange of GDP for GTP on the heterotrimeric G protein transducin (GT). This results in the dissociation of GT into its component αT–GTP and β1γ1 subunit complex. Structural information for the Rho*–GT complex will be essential for understanding the molecular mechanism of visual photo-transduction. Moreover, it will shed light on how GPCRs selectively couple to and activate their G protein signaling partners. Here, we report on the preparation of a stable detergent-solubilized complex between Rho* and a heterotrimer (GT*) comprising a GαT/Gαi1 chimera (αT*) and β1γ1. The complex was formed on native rod outer segment membranes upon light activation, solubilized in lauryl maltose neopentyl glycol, and purified with a combination of affinity and size-exclusion chromatography. We found that the complex is fully functional and that the stoichiometry of Rho* to GαT* is 1:1. The molecular weight of the complex was calculated from small-angle X-ray scattering data and was in good agreement with a model consisting of one Rho* and one GT*. The complex was visualized by negative-stain electron microscopy, which revealed an architecture similar to that of the β2-adrenergic receptor–GS complex, including a flexible αT* helical domain. The stability and high yield of the purified complex should allow for further efforts toward obtaining a high-resolution structure of this important signaling complex. PMID:28655769

  8. The late endosomal HOPS complex anchors active G-protein signaling essential for pathogenesis in magnaporthe oryzae.

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    Ravikrishna Ramanujam

    Full Text Available In Magnaporthe oryzae, the causal ascomycete of the devastating rice blast disease, the conidial germ tube tip must sense and respond to a wide array of requisite cues from the host in order to switch from polarized to isotropic growth, ultimately forming the dome-shaped infection cell known as the appressorium. Although the role for G-protein mediated Cyclic AMP signaling in appressorium formation was first identified almost two decades ago, little is known about the spatio-temporal dynamics of the cascade and how the signal is transmitted through the intracellular network during cell growth and morphogenesis. In this study, we demonstrate that the late endosomal compartments, comprising of a PI3P-rich (Phosphatidylinositol 3-phosphate highly dynamic tubulo-vesicular network, scaffold active MagA/GαS, Rgs1 (a GAP for MagA, Adenylate cyclase and Pth11 (a non-canonical GPCR in the likely absence of AKAP-like anchors during early pathogenic development in M. oryzae. Loss of HOPS component Vps39 and consequently the late endosomal function caused a disruption of adenylate cyclase localization, cAMP signaling and appressorium formation. Remarkably, exogenous cAMP rescued the appressorium formation defects associated with VPS39 deletion in M. oryzae. We propose that sequestration of key G-protein signaling components on dynamic late endosomes and/or endolysosomes, provides an effective molecular means to compartmentalize and control the spatio-temporal activation and rapid downregulation (likely via vacuolar degradation of cAMP signaling amidst changing cellular geometry during pathogenic development in M. oryzae.

  9. Targeting G-Protein Signaling for the Therapeutics of Prostate Tumor Bone Metastases and the Associated Chronic Bone Pain

    Science.gov (United States)

    2015-09-01

    Cancer Bone Metastasis, heterotrimeric G protein  subunits, G protein-coupled receptors, signal transduction 16. SECURITY CLASSIFICATION OF: 17...TRPV1 expression/function in cultured mouse DRG sensory neurons. Accomplishments: we initially attempted to manipulate Gsignaling in isolated DRG ...increase in AKT activation. Since AKT activation will activate TRPV1 channel in DRG neurons, we cannot further assess the effect of G1 and Gt

  10. Regulation of G-protein coupled receptor traffic by an evolutionary conserved hydrophobic signal.

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    Angelotti, Tim; Daunt, David; Shcherbakova, Olga G; Kobilka, Brian; Hurt, Carl M

    2010-04-01

    Plasma membrane (PM) expression of G-protein coupled receptors (GPCRs) is required for activation by extracellular ligands; however, mechanisms that regulate PM expression of GPCRs are poorly understood. For some GPCRs, such as alpha2c-adrenergic receptors (alpha(2c)-ARs), heterologous expression in non-native cells results in limited PM expression and extensive endoplasmic reticulum (ER) retention. Recently, ER export/retentions signals have been proposed to regulate cellular trafficking of several GPCRs. By utilizing a chimeric alpha(2a)/alpha(2c)-AR strategy, we identified an evolutionary conserved hydrophobic sequence (ALAAALAAAAA) in the extracellular amino terminal region that is responsible in part for alpha(2c)-AR subtype-specific trafficking. To our knowledge, this is the first luminal ER retention signal reported for a GPCR. Removal or disruption of the ER retention signal dramatically increased PM expression and decreased ER retention. Conversely, transplantation of this hydrophobic sequence into alpha(2a)-ARs reduced their PM expression and increased ER retention. This evolutionary conserved hydrophobic trafficking signal within alpha(2c)-ARs serves as a regulator of GPCR trafficking.

  11. Transmembrane signal transduction by peptide hormones via family B G protein-coupled receptors

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    Kelly J Culhane

    2015-11-01

    Full Text Available Although family B G protein-coupled receptors (GPCRs contain only 15 members, they play key roles in transmembrane signal transduction of hormones. Family B GPCRs are drug targets for developing therapeutics for diseases ranging from metabolic to neurological disorders. Despite their importance, the molecular mechanism of activation of family B GPCRs remains largely unexplored due to the challenges in expression and purification of functional receptors to the quantity for biophysical characterization. Currently, there is no crystal structure available of a full-length family B GPCR. However, structures of key domains, including the extracellular ligand binding regions and seven-helical transmembrane regions, have been solved by X-ray crystallography and NMR, providing insights into the mechanisms of ligand recognition and selectivity, and helical arrangements within the cell membrane. Moreover, biophysical and biochemical methods have been used to explore functions, key residues for signaling, and the kinetics and dynamics of signaling processes. This review summarizes the current knowledge of the signal transduction mechanism of family B GPCRs at the molecular level and comments on the challenges and outlook for mechanistic studies of family B GPCRs.

  12. Role of G protein-regulated inducer of neurite outgrowth 3 (GRIN3) in β-arrestin 2-Akt signaling and dopaminergic behaviors.

    Science.gov (United States)

    Mototani, Yasumasa; Okamura, Tadashi; Goto, Motohito; Shimizu, Yukiko; Yanobu-Takanashi, Rieko; Ito, Aiko; Kawamura, Naoya; Yagisawa, Yuka; Umeki, Daisuke; Nariyama, Megumi; Suita, Kenji; Ohnuki, Yoshiki; Shiozawa, Kouichi; Sahara, Yoshinori; Kozasa, Tohru; Saeki, Yasutake; Okumura, Satoshi

    2018-06-01

    The G protein-regulated inducer of neurite growth (GRIN) family has three isoforms (GRIN1-3), which bind to the Gαi/o subfamily of G protein that mediate signal processing via G protein-coupled receptors (GPCRs). Here, we show that GRIN3 is involved in regulation of dopamine-dependent behaviors and is essential for activation of the dopamine receptors (DAR)-β-arrestin signaling cascade. Analysis of functional regions of GRIN3 showed that a di-cysteine motif (Cys751/752) is required for plasma membrane localization. GRIN3 was co-immunoprecipitated with GPCR kinases 2/6 and β-arrestins 1/2. Among GRINs, only GRIN3, which is highly expressed in striatum, strongly interacted with β-arrestin 2. We also generated GRIN3-knockout mice (GRIN3KO). GRIN3KO exhibited reduced locomotor activity and increased anxiety-like behavior in the elevated maze test, as well as a reduced locomoter response to dopamine stimulation. We also examined the phosphorylation of Akt at threonine 308 (phospho308-Akt), which is dephosphorylated via a β-arrestin 2-mediated pathway. Dephosphorylation of phospho308-Akt via the D2R-β-arrestin 2 signaling pathway was completely abolished in striatum of GRIN3KO. Our results suggest that GRIN3 has a role in recruitment and assembly of proteins involved in β-arrestin-dependent, G protein-independent signaling.

  13. Proteolytic degradation of regulator of G protein signaling 2 facilitates temporal regulation of Gq/11 signaling and vascular contraction.

    Science.gov (United States)

    Kanai, Stanley M; Edwards, Alethia J; Rurik, Joel G; Osei-Owusu, Patrick; Blumer, Kendall J

    2017-11-24

    Regulator of G protein signaling 2 (RGS2) controls signaling by receptors coupled to the G q/11 class heterotrimeric G proteins. RGS2 deficiency causes several phenotypes in mice and occurs in several diseases, including hypertension in which a proteolytically unstable RGS2 mutant has been reported. However, the mechanisms and functions of RGS2 proteolysis remain poorly understood. Here we addressed these questions by identifying degradation signals in RGS2, and studying dynamic regulation of G q/11 -evoked Ca 2+ signaling and vascular contraction. We identified a novel bipartite degradation signal in the N-terminal domain of RGS2. Mutations disrupting this signal blunted proteolytic degradation downstream of E3 ubiquitin ligase binding to RGS2. Analysis of RGS2 mutants proteolyzed at various rates and the effects of proteasome inhibition indicated that proteolytic degradation controls agonist efficacy by setting RGS2 protein expression levels, and affecting the rate at which cells regain agonist responsiveness as synthesis of RGS2 stops. Analyzing contraction of mesenteric resistance arteries supported the biological relevance of this mechanism. Because RGS2 mRNA expression often is strikingly and transiently up-regulated and then down-regulated upon cell stimulation, our findings indicate that proteolytic degradation tightly couples RGS2 transcription, protein levels, and function. Together these mechanisms provide tight temporal control of G q/11 -coupled receptor signaling in the cardiovascular, immune, and nervous systems. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  14. Regulation of neurite morphogenesis by interaction between R7 regulator of G protein signaling complexes and G protein subunit Gα13.

    Science.gov (United States)

    Scherer, Stephanie L; Cain, Matthew D; Kanai, Stanley M; Kaltenbronn, Kevin M; Blumer, Kendall J

    2017-06-16

    The R7 regulator of G protein signaling family (R7-RGS) critically regulates nervous system development and function. Mice lacking all R7-RGS subtypes exhibit diverse neurological phenotypes, and humans bearing mutations in the retinal R7-RGS isoform RGS9-1 have vision deficits. Although each R7-RGS subtype forms heterotrimeric complexes with Gβ 5 and R7-RGS-binding protein (R7BP) that regulate G protein-coupled receptor signaling by accelerating deactivation of G i/o α-subunits, several neurological phenotypes of R7-RGS knock-out mice are not readily explained by dysregulated G i/o signaling. Accordingly, we used tandem affinity purification and LC-MS/MS to search for novel proteins that interact with R7-RGS heterotrimers in the mouse brain. Among several proteins detected, we focused on Gα 13 because it had not been linked to R7-RGS complexes before. Split-luciferase complementation assays indicated that Gα 13 in its active or inactive state interacts with R7-RGS heterotrimers containing any R7-RGS isoform. LARG (leukemia-associated Rho guanine nucleotide exchange factor (GEF)), PDZ-RhoGEF, and p115RhoGEF augmented interaction between activated Gα 13 and R7-RGS heterotrimers, indicating that these effector RhoGEFs can engage Gα 13 ·R7-RGS complexes. Because Gα 13 /R7-RGS interaction required R7BP, we analyzed phenotypes of neuronal cell lines expressing RGS7 and Gβ 5 with or without R7BP. We found that neurite retraction evoked by Gα 12/13 -dependent lysophosphatidic acid receptors was augmented in R7BP-expressing cells. R7BP expression blunted neurite formation evoked by serum starvation by signaling mechanisms involving Gα 12/13 but not Gα i/o These findings provide the first evidence that R7-RGS heterotrimers interact with Gα 13 to augment signaling pathways that regulate neurite morphogenesis. This mechanism expands the diversity of functions whereby R7-RGS complexes regulate critical aspects of nervous system development and function. © 2017 by

  15. PDZ Protein Regulation of G Protein-Coupled Receptor Trafficking and Signaling Pathways.

    Science.gov (United States)

    Dunn, Henry A; Ferguson, Stephen S G

    2015-10-01

    G protein-coupled receptors (GPCRs) contribute to the regulation of every aspect of human physiology and are therapeutic targets for the treatment of numerous diseases. As a consequence, understanding the myriad of mechanisms controlling GPCR signaling and trafficking is essential for the development of new pharmacological strategies for the treatment of human pathologies. Of the many GPCR-interacting proteins, postsynaptic density protein of 95 kilodaltons, disc large, zona occludens-1 (PDZ) domain-containing proteins appear most abundant and have similarly been implicated in disease mechanisms. PDZ proteins play an important role in regulating receptor and channel protein localization within synapses and tight junctions and function to scaffold intracellular signaling protein complexes. In the current study, we review the known functional interactions between PDZ domain-containing proteins and GPCRs and provide insight into the potential mechanisms of action. These PDZ domain-containing proteins include the membrane-associated guanylate-like kinases [postsynaptic density protein of 95 kilodaltons; synapse-associated protein of 97 kilodaltons; postsynaptic density protein of 93 kilodaltons; synapse-associated protein of 102 kilodaltons; discs, large homolog 5; caspase activation and recruitment domain and membrane-associated guanylate-like kinase domain-containing protein 3; membrane protein, palmitoylated 3; calcium/calmodulin-dependent serine protein kinase; membrane-associated guanylate kinase protein (MAGI)-1, MAGI-2, and MAGI-3], Na(+)/H(+) exchanger regulatory factor proteins (NHERFs) (NHERF1, NHERF2, PDZ domain-containing kidney protein 1, and PDZ domain-containing kidney protein 2), Golgi-associated PDZ proteins (Gα-binding protein interacting protein, C-terminus and CFTR-associated ligand), PDZ domain-containing guanine nucleotide exchange factors (GEFs) 1 and 2, regulator of G protein signaling (RGS)-homology-RhoGEFs (PDZ domain-containing RhoGEF and

  16. Nicotinic Acid Increases Adiponectin Secretion from Differentiated Bovine Preadipocytes through G-Protein Coupled Receptor Signaling

    Directory of Open Access Journals (Sweden)

    Christina Kopp

    2014-11-01

    Full Text Available The transition period in dairy cows (3 weeks prepartum until 3 weeks postpartum is associated with substantial mobilization of energy stores, which is often associated with metabolic diseases. Nicotinic acid (NA is an antilipolytic and lipid-lowering compound used to treat dyslipidaemia in humans, and it also reduces non-esterified fatty acids in cattle. In mice the G-protein coupled receptor 109A (GPR109A ligand NA positively affects the secretion of adiponectin, an important modulator of glucose and fat metabolism. In cattle, the corresponding data linking NA to adiponectin are missing. Our objective was to examine the effects of NA on adiponectin and AMPK protein abundance and the expression of mRNAs of related genes such as chemerin, an adipokine that enhances adiponectin secretion in vitro. Differentiated bovine adipocytes were incubated with pertussis toxin (PTX to verify the involvement of GPR signaling, and treated with 10 or 15 µM NA for 12 or 24 h. NA increased adiponectin concentrations (p ≤ 0.001 and the mRNA abundances of GPR109A (p ≤ 0.05 and chemerin (p ≤ 0.01. Pre-incubation with PTX reduced the adiponectin response to NA (p ≤ 0.001. The NA-stimulated secretion of adiponectin and the mRNA expression of chemerin in the bovine adipocytes were suggestive of GPR signaling-dependent improved insulin sensitivity and/or adipocyte metabolism in dairy cows.

  17. The PDZ and band 4.1 containing protein Frmpd1 regulates the subcellular location of activator of G-protein signaling 3 and its interaction with G-proteins.

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    An, Ningfei; Blumer, Joe B; Bernard, Michael L; Lanier, Stephen M

    2008-09-05

    Activator of G-protein signaling 3 (AGS3) is one of nine mammalian proteins containing one or more G-protein regulatory (GPR) motifs that stabilize the GDP-bound conformation of Galphai. Such proteins have revealed unexpected functional diversity for the "G-switch" in the control of events within the cell independent of the role of heterotrimeric G-proteins as transducers for G-protein-coupled receptors at the cell surface. A key question regarding this class of proteins is what controls their subcellular positioning and interaction with G-proteins. We conducted a series of yeast two-hybrid screens to identify proteins interacting with the tetratricopeptide repeat (TPR) of AGS3, which plays an important role in subcellular positioning of the protein. We report the identification of Frmpd1 (FERM and PDZ domain containing 1) as a regulatory binding partner of AGS3. Frmpd1 binds to the TPR domain of AGS3 and coimmunoprecipitates with AGS3 from cell lysates. Cell fractionation indicated that Frmpd1 stabilizes AGS3 in a membrane fraction. Upon cotransfection of COS7 cells with Frmpd1-GFP and AGS3-mRFP, AGS3-mRFP is observed in regions of the cell cortex and also in membrane extensions or processes where it appears to be colocalized with Frmpd1-GFP based upon the merged fluorescent signals. Frmpd1 knockdown (siRNA) in Cath.a-differentiated neuronal cells decreased the level of endogenous AGS3 in membrane fractions by approximately 50% and enhanced the alpha2-adrenergic receptor-mediated inhibition of forskolin-induced increases in cAMP. The coimmunoprecipitation of Frmpd1 with AGS3 is lost as the amount of Galphai3 in the cell is increased and AGS3 apparently switches its binding partner from Frmpd1 to Galphai3 indicating that the interaction of AGS3 with Frmpd1 and Galphai3 is mutually exclusive. Mechanistically, Frmpd1 may position AGS3 in a membrane environment where it then interacts with Galphai in a regulated manner.

  18. Regulator of G-protein signaling 18 controls both platelet generation and function.

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    Nathalie Delesque-Touchard

    Full Text Available RGS18 is a myeloerythroid lineage-specific regulator of G-protein signaling, highly expressed in megakaryocytes (MKs and platelets. In the present study, we describe the first generation of a RGS18 knockout mouse model (RGS18-/-. Interesting phenotypic differences between RGS18-/- and wild-type (WT mice were identified, and show that RGS18 plays a significant role in both platelet generation and function. RGS18 deficiency produced a gain of function phenotype in platelets. In resting platelets, the level of CD62P expression was increased in RGS18-/- mice. This increase correlated with a higher level of plasmatic serotonin concentration. RGS18-/- platelets displayed a higher sensitivity to activation in vitro. RGS18 deficiency markedly increased thrombus formation in vivo. In addition, RGS18-/- mice presented a mild thrombocytopenia, accompanied with a marked deficit in MK number in the bone marrow. Analysis of MK maturation in vitro and in vivo revealed a defective megakaryopoiesis in RGS18-/- mice, with a lower bone marrow content of only the most committed MK precursors. Finally, RGS18 deficiency was correlated to a defect of platelet recovery in vivo under acute conditions of thrombocytopenia. Thus, we highlight a role for RGS18 in platelet generation and function, and provide additional insights into the physiology of RGS18.

  19. Loss of regulator of G protein signaling 5 exacerbates obesity, hepatic steatosis, inflammation and insulin resistance.

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    Wei Deng

    Full Text Available BACKGROUND: The effect of regulator of G protein signaling 5 (RGS5 on cardiac hypertrophy, atherosclerosis and angiogenesis has been well demonstrated, but the role in the development of obesity and insulin resistance remains completely unknown. We determined the effect of RGS5 deficiency on obesity, hepatic steatosis, inflammation and insulin resistance in mice fed either a normal-chow diet (NC or a high-fat diet (HF. METHODOLOGY/PRINCIPAL FINDINGS: Male, 8-week-old RGS5 knockout (KO and littermate control mice were fed an NC or an HF for 24 weeks and were phenotyped accordingly. RGS5 KO mice exhibited increased obesity, fat mass and ectopic lipid deposition in the liver compared with littermate control mice, regardless of diet. When fed an HF, RGS5 KO mice had a markedly exacerbated metabolic dysfunction and inflammatory state in the blood serum. Meanwhile, macrophage recruitment and inflammation were increased and these increases were associated with the significant activation of JNK, IκBα and NF-κBp65 in the adipose tissue, liver and skeletal muscle of RGS5 KO mice fed an HF relative to control mice. These exacerbated metabolic dysfunction and inflammation are accompanied with decreased systemic insulin sensitivity in the adipose tissue, liver and skeletal muscle of RGS5 KO mice, reflected by weakened Akt/GSK3β phosphorylation. CONCLUSIONS/SIGNIFICANCE: Our data suggest that loss of RGS5 exacerbates HF-induced obesity, hepatic steatosis, inflammation and insulin resistance.

  20. G-protein mediates voltage regulation of agonist binding to muscarinic receptors: effects on receptor-Na+ channel interaction

    International Nuclear Information System (INIS)

    Cohen-Armon, M.; Garty, H.; Sokolovsky, M.

    1988-01-01

    The authors previous experiments in membranes prepared from rat heart and brain led them to suggest that the binding of agonist to the muscarinic receptors and to the Na + channels is a coupled event mediated by guanine nucleotide binding protein(s) [G-protein(s)]. These in vitro findings prompted us to employ synaptoneurosomes from brain stem tissue to examine (i) the binding properties of [ 3 H] acetylcholine at resting potential and under depolarization conditions in the absence and presence of pertussis toxin; (ii) the binding of [ 3 H]batrachotoxin to Na + channel(s) in the presence of the muscarinic agonists; and (iii) muscarinically induced 22 Na + uptake in the presence and absence of tetrodotoxin, which blocks Na + channels. The findings indicate that agonist binding to muscarinic receptors is voltage dependent, that this process is mediated by G-protein(s), and that muscarinic agonists induce opening of Na + channels. The latter process persists even after pertussis toxin treatment, indicating that it is not likely to be mediated by pertussis toxin sensitive G-protein(s). The system with its three interacting components-receptor, G-protein, and Na + channel-is such that at resting potential the muscarinic receptor induces opening of Na + channels; this property may provide a possible physiological mechanism for the depolarization stimulus necessary for autoexcitation or repetitive firing in heart or brain tissues

  1. Estrogen-mediated inactivation of FOXO3a by the G protein-coupled estrogen receptor GPER

    International Nuclear Information System (INIS)

    Zekas, Erin; Prossnitz, Eric R.

    2015-01-01

    Estrogen (17β-estradiol) promotes the survival and proliferation of breast cancer cells and its receptors represent important therapeutic targets. The cellular actions of estrogen are mediated by the nuclear estrogen receptors ERα and ERβ as well as the 7-transmembrane spanning G protein-coupled estrogen receptor (GPER). We previously reported that estrogen activates the phosphoinositide 3-kinase (PI3Kinase) pathway via GPER, resulting in phosphatidylinositol (3,4,5)-trisphosphate (PIP3) production within the nucleus of breast cancer cells; however, the mechanisms and consequences of this activity remained unclear. MCF7 breast cancer cells were transfected with GFP-fused Forkhead box O3 (FOXO3) as a reporter to assess localization in response to estrogen stimulation. Inhibitors of PI3Kinases and EGFR were employed to determine the mechanisms of estrogen-mediated FOXO3a inactivation. Receptor knockdown with siRNA and the selective GPER agonist G-1 elucidated the estrogen receptor(s) responsible for estrogen-mediated FOXO3a inactivation. The effects of selective estrogen receptor modulators and downregulators (SERMs and SERDs) on FOXO3a in MCF7 cells were also determined. Cell survival (inhibition of apoptosis) was assessed by caspase activation. In the estrogen-responsive breast cancer cell line MCF7, FOXO3a inactivation occurs on a rapid time scale as a result of GPER, but not ERα, stimulation by estrogen, established by the GPER-selective agonist G-1 and knockdown of GPER and ERα. GPER-mediated inactivation of FOXO3a is effected by the p110α catalytic subunit of PI3Kinase as a result of transactivation of the EGFR. The SERMs tamoxifen and raloxifene, as well as the SERD ICI182,780, were active in mediating FOXO3a inactivation in a GPER-dependent manner. Additionally, estrogen-and G-1-mediated stimulation of MCF7 cells results in a decrease in caspase activation under proapoptotic conditions. Our results suggest that non-genomic signaling by GPER contributes

  2. PKD1 mediates negative feedback of PI3K/Akt activation in response to G protein-coupled receptors.

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    Yang Ni

    Full Text Available We examined whether protein kinase D1 (PKD1 mediates negative feeback of PI3K/Akt signaling in intestinal epithelial cells stimulated with G protein-coupled receptor (GPCR agonists. Exposure of intestinal epithelial IEC-18 cells to increasing concentrations of the PKD family inhibitor kb NB 142-70, at concentrations that inhibited PKD1 activation, strikingly potentiated Akt phosphorylation at Thr(308 and Ser(473 in response to the mitogenic GPCR agonist angiotensin II (ANG II. Enhancement of Akt activation by kb NB 142-70 was also evident in cells with other GPCR agonists, including vasopressin and lysophosphatidic acid. Cell treatment with the structurally unrelated PKD family inhibitor CRT0066101 increased Akt phosphorylation as potently as kb NB 142-70 [corrected]. Knockdown of PKD1 with two different siRNAs strikingly enhanced Akt phosphorylation in response to ANG II stimulation in IEC-18 cells. To determine whether treatment with kb NB 142-70 enhances accumulation of phosphatidylinositol (3,4,5-trisphosphate (PIP3 in the plasma membrane, we monitored the redistribution of Akt-pleckstrin homology domain-green fluorescent protein (Akt-PH-GFP in single IEC-18 cells. Exposure to kb NB 142-70 strikingly increased membrane accumulation of Akt-PH-GFP in response to ANG II. The translocation of the PIP3 sensor to the plasma membrane and the phosphorylation of Akt was completed prevented by prior exposure to the class I p110α specific inhibitor A66. ANG II markedly increased the phosphorylation of p85α detected by a PKD motif-specific antibody and enhanced the association of p85α with PTEN. Transgenic mice overexpressing PKD1 showed a reduced phosphorylation of Akt at Ser(473 in intestinal epithelial cells compared to wild type littermates. Collectively these results indicate that PKD1 activation mediates feedback inhibition of PI3K/Akt signaling in intestinal epithelial cells in vitro and in vivo.

  3. Disease-associated extracellular loop mutations in the adhesion G protein-coupled receptor G1 (ADGRG1; GPR56) differentially regulate downstream signaling.

    Science.gov (United States)

    Kishore, Ayush; Hall, Randy A

    2017-06-09

    Mutations to the adhesion G protein-coupled receptor ADGRG1 (G1; also known as GPR56) underlie the neurological disorder bilateral frontoparietal polymicrogyria. Disease-associated mutations in G1 studied to date are believed to induce complete loss of receptor function through disruption of either receptor trafficking or signaling activity. Given that N-terminal truncation of G1 and other adhesion G protein-coupled receptors has been shown to significantly increase the receptors' constitutive signaling, we examined two different bilateral frontoparietal polymicrogyria-inducing extracellular loop mutations (R565W and L640R) in the context of both full-length and N-terminally truncated (ΔNT) G1. Interestingly, we found that these mutations reduced surface expression of full-length G1 but not G1-ΔNT in HEK-293 cells. Moreover, the mutations ablated receptor-mediated activation of serum response factor luciferase, a classic measure of Gα 12/13 -mediated signaling, but had no effect on G1-mediated signaling to nuclear factor of activated T cells (NFAT) luciferase. Given these differential signaling results, we sought to further elucidate the pathway by which G1 can activate NFAT luciferase. We found no evidence that ΔNT activation of NFAT is dependent on Gα q/11 -mediated or β-arrestin-mediated signaling but rather involves liberation of Gβγ subunits and activation of calcium channels. These findings reveal that disease-associated mutations to the extracellular loops of G1 differentially alter receptor trafficking, depending on the presence of the N terminus, and differentially alter signaling to distinct downstream pathways. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  4. Dissecting signaling and functions of adhesion G protein-coupled receptors

    DEFF Research Database (Denmark)

    Araç, Demet; Aust, Gabriela; Calebiro, Davide

    2012-01-01

    G protein-coupled receptors (GPCRs) comprise an expanded superfamily of receptors in the human genome. Adhesion class G protein-coupled receptors (adhesion-GPCRs) form the second largest class of GPCRs. Despite the abundance, size, molecular structure, and functions in facilitating cell and matrix...... contacts in a variety of organ systems, adhesion-GPCRs are by far the most poorly understood GPCR class. Adhesion-GPCRs possess a unique molecular structure, with extended N-termini containing various adhesion domains. In addition, many adhesion-GPCRs are autoproteolytically cleaved into an N......-terminal fragment (NTF, NT, α-subunit) and C-terminal fragment (CTF, CT, β-subunit) at a conserved GPCR autoproteolysis-inducing (GAIN) domain that contains a GPCR proteolysis site (GPS). These two features distinguish adhesion-GPCRs from other GPCR classes. Though active research on adhesion-GPCRs in diverse areas...

  5. Mechanisms of G Protein-Coupled Estrogen Receptor-Mediated Spinal Nociception

    DEFF Research Database (Denmark)

    Deliu, Elena; Brailoiu, G. Cristina; Arterburn, Jeffrey B.

    2012-01-01

    . Cytosolic calcium concentration elevates faster and with higher amplitude following G-1 intracellular microinjections compared to extracellular exposure, suggesting subcellular GPER functionality. Thus, GPER activation results in spinal nociception, and the downstream mechanisms involve cytosolic calcium......Human and animal studies suggest that estrogens are involved in the processing of nociceptive sensory information and analgesic responses in the central nervous system. Rapid pronociceptive estrogenic effects have been reported, some of which likely involve G protein-coupled estrogen receptor (GPER......) activation. Membrane depolarization and increases in cytosolic calcium and reactive oxygen species (ROS) levels are markers of neuronal activation, underlying pain sensitization in the spinal cord. Using behavioral, electrophysiological, and fluorescent imaging studies, we evaluated GPER involvement...

  6. The Orphan G Protein-coupled Receptor Gpr175 (Tpra40) Enhances Hedgehog Signaling by Modulating cAMP Levels.

    Science.gov (United States)

    Singh, Jaskirat; Wen, Xiaohui; Scales, Suzie J

    2015-12-04

    The Hedgehog (Hh) signaling pathway plays an essential role in vertebrate embryonic tissue patterning of many developing organs. Signaling occurs predominantly in primary cilia and is initiated by the entry of the G protein-coupled receptor (GPCR)-like protein Smoothened into cilia and culminates in gene transcription via the Gli family of transcription factors upon their nuclear entry. Here we identify an orphan GPCR, Gpr175 (also known as Tpra1 or Tpra40: transmembrane protein, adipocyte associated 1 or of 40 kDa), which also localizes to primary cilia upon Hh stimulation and positively regulates Hh signaling. Interaction experiments place Gpr175 at the level of PKA and upstream of the Gαi component of heterotrimeric G proteins, which itself localizes to cilia and can modulate Hh signaling. Gpr175 or Gαi1 depletion leads to increases in cellular cAMP levels and in Gli3 processing into its repressor form. Thus we propose that Gpr175 coupled to Gαi1 normally functions to inhibit the production of cAMP by adenylyl cyclase upon Hh stimulation, thus maximizing signaling by turning off PKA activity and hence Gli3 repressor formation. Taken together our data suggest that Gpr175 is a novel positive regulator of the Hh signaling pathway. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  7. Cytosolic phospholipase A2: a member of the signalling pathway of a new G protein α subunit in Sporothrix schenckii

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    González-Méndez Ricardo

    2009-05-01

    Full Text Available Abstract Background Sporothrix schenckii is a pathogenic dimorphic fungus, the etiological agent of sporotrichosis, a lymphocutaneous disease that can remain localized or can disseminate, involving joints, lungs, and the central nervous system. Pathogenic fungi use signal transduction pathways to rapidly adapt to changing environmental conditions and S. schenckii is no exception. S. schenckii yeast cells, either proliferate (yeast cell cycle or engage in a developmental program that includes proliferation accompanied by morphogenesis (yeast to mycelium transition depending on the environmental conditions. The principal intracellular receptors of environmental signals are the heterotrimeric G proteins, suggesting their involvement in fungal dimorphism and pathogenicity. Identifying these G proteins in fungi and their involvement in protein-protein interactions will help determine their role in signal transduction pathways. Results In this work we describe a new G protein α subunit gene in S. schenckii, ssg-2. The cDNA sequence of ssg-2 revealed a predicted open reading frame of 1,065 nucleotides encoding a 355 amino acids protein with a molecular weight of 40.9 kDa. When used as bait in a yeast two-hybrid assay, a cytoplasmic phospholipase A2 catalytic subunit was identified as interacting with SSG-2. The sspla2 gene, revealed an open reading frame of 2538 bp and encoded an 846 amino acid protein with a calculated molecular weight of 92.62 kDa. The principal features that characterize cPLA2 were identified in this enzyme such as a phospholipase catalytic domain and the characteristic invariable arginine and serine residues. A role for SSPLA2 in the control of dimorphism in S. schenckii is suggested by observing the effects of inhibitors of the enzyme on the yeast cell cycle and the yeast to mycelium transition in this fungus. Phospholipase A2 inhibitors such as AACOCF3 (an analogue of archidonic acid and isotetrandrine (an inhibitor of G protein

  8. Membrane Recruitment of the Non-receptor Protein GIV/Girdin (Gα-interacting, Vesicle-associated Protein/Girdin) Is Sufficient for Activating Heterotrimeric G Protein Signaling.

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    Parag-Sharma, Kshitij; Leyme, Anthony; DiGiacomo, Vincent; Marivin, Arthur; Broselid, Stefan; Garcia-Marcos, Mikel

    2016-12-30

    GIV (aka Girdin) is a guanine nucleotide exchange factor that activates heterotrimeric G protein signaling downstream of RTKs and integrins, thereby serving as a platform for signaling cascade cross-talk. GIV is recruited to the cytoplasmic tail of receptors upon stimulation, but the mechanism of activation of its G protein regulatory function is not well understood. Here we used assays in humanized yeast models and G protein activity biosensors in mammalian cells to investigate the role of GIV subcellular compartmentalization in regulating its ability to promote G protein signaling. We found that in unstimulated cells GIV does not co-fractionate with its substrate G protein Gα i3 on cell membranes and that constitutive membrane anchoring of GIV in yeast cells or rapid membrane translocation in mammalian cells via chemically induced dimerization leads to robust G protein activation. We show that membrane recruitment of the GIV "Gα binding and activating" motif alone is sufficient for G protein activation and that it does not require phosphomodification. Furthermore, we engineered a synthetic protein to show that recruitment of the GIV "Gα binding and activating" motif to membranes via association with active RTKs, instead of via chemically induced dimerization, is also sufficient for G protein activation. These results reveal that recruitment of GIV to membranes in close proximity to its substrate G protein is a major mechanism responsible for the activation of its G protein regulatory function. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  9. Pertussis toxin-sensitive G-protein mediates the alpha 2-adrenergic receptor inhibition of melatonin release in photoreceptive chick pineal cell cultures

    International Nuclear Information System (INIS)

    Pratt, B.L.; Takahashi, J.S.

    1988-01-01

    The avian pineal gland is a photoreceptive organ that has been shown to contain postjunctional alpha 2-adrenoceptors that inhibit melatonin synthesis and/or release upon receptor activation. Physiological response and [32P]ADP ribosylation experiments were performed to investigate whether pertussis toxin-sensitive guanine nucleotide-binding proteins (G-proteins) were involved in the transduction of the alpha 2-adrenergic signal. For physiological response studies, the effects of pertussis toxin on melatonin release in dissociated cell cultures exposed to norepinephrine were assessed. Pertussis toxin blocked alpha 2-adrenergic receptor-mediated inhibition in a dose-dependent manner. Pertussis toxin-induced blockade appeared to be noncompetitive. One and 10 ng/ml doses of pertussis toxin partially blocked and a 100 ng/ml dose completely blocked norepinephrine-induced inhibition. Pertussis toxin-catalyzed [32P]ADP ribosylation of G-proteins in chick pineal cell membranes was assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. Membranes were prepared from cells that had been pretreated with 0, 1, 10, or 100 ng/ml pertussis toxin. In the absence of pertussis toxin pretreatment, two major proteins of 40K and 41K mol wt (Mr) were labeled by [32P]NAD. Pertussis toxin pretreatment of pineal cells abolished [32P] radiolabeling of the 40K Mr G-protein in a dose-dependent manner. The norepinephrine-induced inhibition of both cAMP efflux and melatonin release, as assessed by RIA of medium samples collected before membrane preparation, was also blocked in a dose-dependent manner by pertussis toxin. Collectively, these results suggest that a pertussis toxin-sensitive 40K Mr G-protein labeled by [32P]NAD may be functionally associated with alpha 2-adrenergic signal transduction in chick pineal cells

  10. Chemogenomic analysis of G-protein coupled receptors and their ligands deciphers locks and keys governing diverse aspects of signalling.

    Directory of Open Access Journals (Sweden)

    Jörg D Wichard

    Full Text Available Understanding the molecular mechanism of signalling in the important super-family of G-protein-coupled receptors (GPCRs is causally related to questions of how and where these receptors can be activated or inhibited. In this context, it is of great interest to unravel the common molecular features of GPCRs as well as those related to an active or inactive state or to subtype specific G-protein coupling. In our underlying chemogenomics study, we analyse for the first time the statistical link between the properties of G-protein-coupled receptors and GPCR ligands. The technique of mutual information (MI is able to reveal statistical inter-dependence between variations in amino acid residues on the one hand and variations in ligand molecular descriptors on the other. Although this MI analysis uses novel information that differs from the results of known site-directed mutagenesis studies or published GPCR crystal structures, the method is capable of identifying the well-known common ligand binding region of GPCRs between the upper part of the seven transmembrane helices and the second extracellular loop. The analysis shows amino acid positions that are sensitive to either stimulating (agonistic or inhibitory (antagonistic ligand effects or both. It appears that amino acid positions for antagonistic and agonistic effects are both concentrated around the extracellular region, but selective agonistic effects are cumulated between transmembrane helices (TMHs 2, 3, and ECL2, while selective residues for antagonistic effects are located at the top of helices 5 and 6. Above all, the MI analysis provides detailed indications about amino acids located in the transmembrane region of these receptors that determine G-protein signalling pathway preferences.

  11. Association with the Plasma Membrane Is Sufficient for Potentiating Catalytic Activity of Regulators of G Protein Signaling (RGS) Proteins of the R7 Subfamily.

    Science.gov (United States)

    Muntean, Brian S; Martemyanov, Kirill A

    2016-03-25

    Regulators of G protein Signaling (RGS) promote deactivation of heterotrimeric G proteins thus controlling the magnitude and kinetics of responses mediated by G protein-coupled receptors (GPCR). In the nervous system, RGS7 and RGS9-2 play essential role in vision, reward processing, and movement control. Both RGS7 and RGS9-2 belong to the R7 subfamily of RGS proteins that form macromolecular complexes with R7-binding protein (R7BP). R7BP targets RGS proteins to the plasma membrane and augments their GTPase-accelerating protein (GAP) activity, ultimately accelerating deactivation of G protein signaling. However, it remains unclear if R7BP serves exclusively as a membrane anchoring subunit or further modulates RGS proteins to increase their GAP activity. To directly answer this question, we utilized a rapidly reversible chemically induced protein dimerization system that enabled us to control RGS localization independent from R7BP in living cells. To monitor kinetics of Gα deactivation, we coupled this strategy with measuring changes in the GAP activity by bioluminescence resonance energy transfer-based assay in a cellular system containing μ-opioid receptor. This approach was used to correlate changes in RGS localization and activity in the presence or absence of R7BP. Strikingly, we observed that RGS activity is augmented by membrane recruitment, in an orientation independent manner with no additional contributions provided by R7BP. These findings argue that the association of R7 RGS proteins with the membrane environment provides a major direct contribution to modulation of their GAP activity. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  12. Angiotensin II Type 1 Receptor Mechanoactivation Involves RGS5 (Regulator of G Protein Signaling 5) in Skeletal Muscle Arteries: Impaired Trafficking of RGS5 in Hypertension.

    Science.gov (United States)

    Hong, Kwangseok; Li, Min; Nourian, Zahra; Meininger, Gerald A; Hill, Michael A

    2017-12-01

    Studies suggest that arteriolar pressure-induced vasoconstriction can be initiated by GPCRs (G protein-coupled receptors), including the AT 1 R (angiotensin II type 1 receptor). This raises the question, are such mechanisms regulated by negative feedback? The present studies examined whether RGS (regulators of G protein signaling) proteins in vascular smooth muscle cells are colocalized with the AT 1 R when activated by mechanical stress or angiotensin II and whether this modulates AT 1 R-mediated vasoconstriction. To determine whether activation of the AT 1 R recruits RGS5, an in situ proximity ligation assay was performed in primary cultures of cremaster muscle arteriolar vascular smooth muscle cells treated with angiotensin II or hypotonic solution in the absence or presence of candesartan (an AT 1 R blocker). Proximity ligation assay results revealed a concentration-dependent increase in trafficking/translocation of RGS5 toward the activated AT 1 R, which was attenuated by candesartan. In intact arterioles, knockdown of RGS5 enhanced constriction to angiotensin II and augmented myogenic responses to increased intraluminal pressure. Myogenic constriction was attenuated to a higher degree by candesartan in RGS5 siRNA-transfected arterioles, consistent with RGS5 contributing to downregulation of AT 1 R-mediated signaling. Further, translocation of RGS5 was impaired in vascular smooth muscle cells of spontaneously hypertensive rats. This is consistent with dysregulated (RGS5-mediated) AT 1 R signaling that could contribute to excessive vasoconstriction in hypertension. In intact vessels, candesartan reduced myogenic vasoconstriction to a greater extent in spontaneously hypertensive rats compared with controls. Collectively, these findings suggest that AT 1 R activation results in translocation of RGS5 toward the plasma membrane, limiting AT 1 R-mediated vasoconstriction through its role in G q/11 protein-dependent signaling. © 2017 American Heart Association, Inc.

  13. Structural basis for different phosphoinositide specificities of the PX domains of sorting nexins regulating G-protein signaling.

    Science.gov (United States)

    Mas, Caroline; Norwood, Suzanne J; Bugarcic, Andrea; Kinna, Genevieve; Leneva, Natalya; Kovtun, Oleksiy; Ghai, Rajesh; Ona Yanez, Lorena E; Davis, Jasmine L; Teasdale, Rohan D; Collins, Brett M

    2014-10-10

    Sorting nexins (SNXs) or phox homology (PX) domain containing proteins are central regulators of cell trafficking and signaling. A subfamily of PX domain proteins possesses two unique PX-associated domains, as well as a regulator of G protein-coupled receptor signaling (RGS) domain that attenuates Gαs-coupled G protein-coupled receptor signaling. Here we delineate the structural organization of these RGS-PX proteins, revealing a protein family with a modular architecture that is conserved in all eukaryotes. The one exception to this is mammalian SNX19, which lacks the typical RGS structure but preserves all other domains. The PX domain is a sensor of membrane phosphoinositide lipids and we find that specific sequence alterations in the PX domains of the mammalian RGS-PX proteins, SNX13, SNX14, SNX19, and SNX25, confer differential phosphoinositide binding preferences. Although SNX13 and SNX19 PX domains bind the early endosomal lipid phosphatidylinositol 3-phosphate, SNX14 shows no membrane binding at all. Crystal structures of the SNX19 and SNX14 PX domains reveal key differences, with alterations in SNX14 leading to closure of the binding pocket to prevent phosphoinositide association. Our findings suggest a role for alternative membrane interactions in spatial control of RGS-PX proteins in cell signaling and trafficking. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

  14. Serotonergic Chemosensory Neurons Modify the C. elegans Immune Response by Regulating G-Protein Signaling in Epithelial Cells

    Science.gov (United States)

    Anderson, Alexandra; Laurenson-Schafer, Henry; Partridge, Frederick A.; Hodgkin, Jonathan; McMullan, Rachel

    2013-01-01

    The nervous and immune systems influence each other, allowing animals to rapidly protect themselves from changes in their internal and external environment. However, the complex nature of these systems in mammals makes it difficult to determine how neuronal signaling influences the immune response. Here we show that serotonin, synthesized in Caenorhabditis elegans chemosensory neurons, modulates the immune response. Serotonin released from these cells acts, directly or indirectly, to regulate G-protein signaling in epithelial cells. Signaling in these cells is required for the immune response to infection by the natural pathogen Microbacterium nematophilum. Here we show that serotonin signaling suppresses the innate immune response and limits the rate of pathogen clearance. We show that C. elegans uses classical neurotransmitters to alter the immune response. Serotonin released from sensory neurons may function to modify the immune system in response to changes in the animal's external environment such as the availability, or quality, of food. PMID:24348250

  15. Three regulators of G protein signaling differentially affect mating, morphology and virulence in the smut fungus Ustilago maydis.

    Science.gov (United States)

    Moretti, Marino; Wang, Lei; Grognet, Pierre; Lanver, Daniel; Link, Hannes; Kahmann, Regine

    2017-09-01

    Regulators of G protein signaling (RGS) proteins modulate heterotrimeric G protein signaling negatively. To broaden an understanding of the roles of RGS proteins in fungal pathogens, we functionally characterized the three RGS protein-encoding genes (rgs1, rgs2 and rgs3) in the phytopathogenic fungus Ustilago maydis. It was found that RGS proteins played distinct roles in the regulation of development and virulence. rgs1 had a minor role in virulence when deleted in a solopathogenic strain. In crosses, rgs1 was dispensable for mating and filamentation, but was required for teliospore production. Haploid rgs2 mutants were affected in cell morphology, growth, mating and were unable to cause disease symptoms in crosses. However, virulence was unaffected when rgs2 was deleted in a solopathogenic strain, suggesting an exclusive involvement in pre-fusion events. These rgs2 phenotypes are likely connected to elevated intracellular cAMP levels. rgs3 mutants were severely attenuated in mating, in their response to pheromone, virulence and formation of mature teliospores. The mating defect could be traced back to reduced expression of the transcription factor rop1. It was speculated that the distinct roles of the three U. maydis RGS proteins were achieved by direct modulation of the Gα subunit-activated signaling pathways as well as through Gα-independent functions. © 2017 John Wiley & Sons Ltd.

  16. G-Protein Gαs controls medulloblastoma initiation by suppressing sonic hedgehog signaling.

    Science.gov (United States)

    He, Xuelian; Lu, Q Richard

    2015-01-01

    We identify Gαs as a novel tumor suppressor in medulloblastoma that functions principally by inhibition of sonic hedgehog signaling. Gαs not only stimulates cyclic adenosine monophosphate (cAMP)-dependent signaling but also inhibits ciliary trafficking of hedgehog components. Elevation of cAMP inhibits medulloblastoma growth and augments inhibition of smoothened to decrease tumor cell proliferation, thus highlighting Gαs as a potential therapeutic target.

  17. Regulator of G protein signaling-12 modulates the dopamine transporter in ventral striatum and locomotor responses to psychostimulants.

    Science.gov (United States)

    Gross, Joshua D; Kaski, Shane W; Schroer, Adam B; Wix, Kimberley A; Siderovski, David P; Setola, Vincent

    2018-02-01

    Regulators of G protein signaling are proteins that accelerate the termination of effector stimulation after G protein-coupled receptor activation. Many regulators of G protein signaling proteins are highly expressed in the brain and therefore considered potential drug discovery targets for central nervous system pathologies; for example, here we show that RGS12 is highly expressed in microdissected mouse ventral striatum. Given a role for the ventral striatum in psychostimulant-induced locomotor activity, we tested whether Rgs12 genetic ablation affected behavioral responses to amphetamine and cocaine. RGS12 loss significantly decreased hyperlocomotion to lower doses of both amphetamine and cocaine; however, other outcomes of administration (sensitization and conditioned place preference) were unaffected, suggesting that RGS12 does not function in support of the rewarding properties of these psychostimulants. To test whether observed response changes upon RGS12 loss were caused by changes to dopamine transporter expression and/or function, we prepared crude membranes from the brains of wild-type and RGS12-null mice and measured dopamine transporter-selective [ 3 H]WIN 35428 binding, revealing an increase in dopamine transporter levels in the ventral-but not dorsal-striatum of RGS12-null mice. To address dopamine transporter function, we prepared striatal synaptosomes and measured [ 3 H]dopamine uptake. Consistent with increased [ 3 H]WIN 35428 binding, dopamine transporter-specific [ 3 H]dopamine uptake in RGS12-null ventral striatal synaptosomes was found to be increased. Decreased amphetamine-induced locomotor activity and increased [ 3 H]WIN 35428 binding were recapitulated with an independent RGS12-null mouse strain. Thus, we propose that RGS12 regulates dopamine transporter expression and function in the ventral striatum, affecting amphetamine- and cocaine-induced increases in dopamine levels that specifically elicit acute hyperlocomotor responses.

  18. Phosphorylation of the Usher syndrome 1G protein SANS controls Magi2-mediated endocytosis.

    Science.gov (United States)

    Bauß, Katharina; Knapp, Barbara; Jores, Pia; Roepman, Ronald; Kremer, Hannie; Wijk, Erwin V; Märker, Tina; Wolfrum, Uwe

    2014-08-01

    The human Usher syndrome (USH) is a complex ciliopathy with at least 12 chromosomal loci assigned to three clinical subtypes, USH1-3. The heterogeneous USH proteins are organized into protein networks. Here, we identified Magi2 (membrane-associated guanylate kinase inverted-2) as a new component of the USH protein interactome, binding to the multifunctional scaffold protein SANS (USH1G). We showed that the SANS-Magi2 complex assembly is regulated by the phosphorylation of an internal PDZ-binding motif in the sterile alpha motif domain of SANS by the protein kinase CK2. We affirmed Magi2's role in receptor-mediated, clathrin-dependent endocytosis and showed that phosphorylated SANS tightly regulates Magi2-mediated endocytosis. Specific depletions by RNAi revealed that SANS and Magi2-mediated endocytosis regulates aspects of ciliogenesis. Furthermore, we demonstrated the localization of the SANS-Magi2 complex in the periciliary membrane complex facing the ciliary pocket of retinal photoreceptor cells in situ. Our data suggest that endocytotic processes may not only contribute to photoreceptor cell homeostasis but also counterbalance the periciliary membrane delivery accompanying the exocytosis processes for the cargo vesicle delivery. In USH1G patients, mutations in SANS eliminate Magi2 binding and thereby deregulate endocytosis, lead to defective ciliary transport modules and ultimately disrupt photoreceptor cell function inducing retinal degeneration. © The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

  19. Adenosine Receptors Differentially Regulate the Expression of Regulators of G-Protein Signalling (RGS 2, 3 and 4 in Astrocyte-Like Cells.

    Directory of Open Access Journals (Sweden)

    Till Nicolas Eusemann

    Full Text Available The "regulators of g-protein signalling" (RGS comprise a large family of proteins that limit by virtue of their GTPase accelerating protein domain the signal transduction of G-protein coupled receptors. RGS proteins have been implicated in various neuropsychiatric diseases such as schizophrenia, drug abuse, depression and anxiety and aggressive behaviour. Since conditions associated with a large increase of adenosine in the brain such as seizures or ischemia were reported to modify the expression of some RGS proteins we hypothesized that adenosine might regulate RGS expression in neural cells. We measured the expression of RGS-2,-3, and -4 in both transformed glia cells (human U373 MG astrocytoma cells and in primary rat astrocyte cultures stimulated with adenosine agonists. Expression of RGS-2 mRNA as well as RGS2 protein was increased up to 30-fold by adenosine agonists in astrocytes. The order of potency of agonists and the blockade by the adenosine A2B-antagonist MRS1706 indicated that this effect was largely mediated by adenosine A2B receptors. However, a smaller effect was observed due to activation of adenosine A2A receptors. In astrocytoma cells adenosine agonists elicited an increase in RGS-2 expression solely mediated by A2B receptors. Expression of RGS-3 was inhibited by adenosine agonists in both astrocytoma cells and astrocytes. However while this effect was mediated by A2B receptors in astrocytoma cells it was mediated by A2A receptors in astrocytes as assessed by the order of potency of agonists and selective blockade by the specific antagonists MRS1706 and ZM241385 respectively. RGS-4 expression was inhibited in astrocytoma cells but enhanced in astrocytes by adenosine agonists.

  20. Insights into Basal Signaling Regulation, Oligomerization, and Structural Organization of the Human G-Protein Coupled Receptor 83.

    Directory of Open Access Journals (Sweden)

    Anne Müller

    Full Text Available The murine G-protein coupled receptor 83 (mGPR83 is expressed in the hypothalamus and was previously suggested to be involved in the regulation of metabolism. The neuropeptide PEN has been recently identified as a potent GPR83 ligand. Moreover, GPR83 constitutes functionally relevant hetero-oligomers with other G-protein coupled receptors (GPCR such as the ghrelin receptor (GHSR or GPR171. Previous deletion studies also revealed that the long N-terminal extracellular receptor domain (eNDo of mGPR83 may act as an intra-molecular ligand, which participates in the regulation of basal signaling activity, which is a key feature of GPCR function. Here, we investigated particular amino acids at the eNDo of human GPR83 (hGPR83 by side-directed mutagenesis to identify determinants of the internal ligand. These studies were accompanied by structure homology modeling to combine functional insights with structural information. The capacity for hetero-oligomer formation of hGPR83 with diverse family A GPCRs such as the melanocortin-4 receptor (MC4R was also investigated, with a specific emphasis on the impact of the eNDo on oligomerization and basal signaling properties. Finally, we demonstrate that hGPR83 exhibits an unusual basal signaling for different effectors, which also supports signaling promiscuity. hGPR83 interacts with a variety of hypothalamic GPCRs such as the MC4R or GHSR. These interactions are not dependent on the ectodomain and most likely occur at interfaces constituted in the transmembrane regions. Moreover, several amino acids at the transition between the eNDo and transmembrane helix 1 were identified, where mutations lead also to biased basal signaling modulation.

  1. Assessing the osteoblast transcriptome in a model of enhanced bone formation due to constitutive G{sub s}–G protein signaling in osteoblasts

    Energy Technology Data Exchange (ETDEWEB)

    Wattanachanya, Lalita, E-mail: lalita_md@yahoo.com [Endocrine Research Unit, Veterans Affairs Medical Center and Departments of Medicine and Physiology, University of California, San Francisco, CA (United States); Division of Endocrinology and Metabolism, Department of Medicine, Faculty of Medicine, Chulalongkorn University and King Chulalongkorn Memorial Hospital, Thai Red Cross Society, Bangkok (Thailand); Wang, Liping, E-mail: lipingwang05@yahoo.com [Endocrine Research Unit, Veterans Affairs Medical Center and Departments of Medicine and Physiology, University of California, San Francisco, CA (United States); Millard, Susan M., E-mail: susan.millard@mater.uq.edu.au [Endocrine Research Unit, Veterans Affairs Medical Center and Departments of Medicine and Physiology, University of California, San Francisco, CA (United States); Lu, Wei-Dar, E-mail: weidar_lu@yahoo.com [Endocrine Research Unit, Veterans Affairs Medical Center and Departments of Medicine and Physiology, University of California, San Francisco, CA (United States); O’Carroll, Dylan, E-mail: dylancocarroll@gmail.com [Endocrine Research Unit, Veterans Affairs Medical Center and Departments of Medicine and Physiology, University of California, San Francisco, CA (United States); Hsiao, Edward C., E-mail: Edward.Hsiao@ucsf.edu [Division of Endocrinology and Metabolism, Department of Medicine, University of California, San Francisco, CA (United States); Conklin, Bruce R., E-mail: bconklin@gladstone.ucsf.edu [Gladstone Institute of Cardiovascular Disease, San Francisco, CA (United States); Department of Medicine, University of California, San Francisco, CA (United States); Department of Cellular and Molecular Pharmacology, University of California, San Francisco, CA (United States); Nissenson, Robert A., E-mail: Robert.Nissenson@ucsf.edu [Endocrine Research Unit, Veterans Affairs Medical Center and Departments of Medicine and Physiology, University of California, San Francisco, CA (United States)

    2015-05-01

    G protein-coupled receptor (GPCR) signaling in osteoblasts (OBs) is an important regulator of bone formation. We previously described a mouse model expressing Rs1, an engineered constitutively active G{sub s}-coupled GPCR, under the control of the 2.3 kb Col I promoter. These mice showed a dramatic age-dependent increase in trabecular bone of femurs. Here, we further evaluated the effects of enhanced G{sub s} signaling in OBs on intramembranous bone formation by examining calvariae of 1- and 9-week-old Col1(2.3)/Rs1 mice and characterized the in vivo gene expression specifically occurring in osteoblasts with activated G{sub s} G protein-coupled receptor signaling, at the cellular level rather than in a whole bone. Rs1 calvariae displayed a dramatic increase in bone volume with partial loss of cortical structure. By immunohistochemistry, Osterix was detected in cells throughout the inter-trabecular space while Osteocalcin was expressed predominantly in cells along bone surfaces, suggesting the role of paracrine mediators secreted from OBs driven by 2.3 kb Col I promoter could influence early OB commitment, differentiation, and/or proliferation. Gene expression analysis of calvarial OBs revealed that genes affected by Rs1 signaling include those encoding proteins important for cell differentiation, cytokines and growth factors, angiogenesis, coagulation, and energy metabolism. The set of G{sub s}-GPCRs and other GPCRs that may contribute to the observed skeletal phenotype and candidate paracrine mediators of the effect of G{sub s} signaling in OBs were also determined. Our results identify novel detailed in vivo cellular changes of the anabolic response of the skeleton to G{sub s} signaling in mature OBs. - Highlights: • OB expression of an engineered G{sub s}-coupled receptor dramatically increases bone mass. • We investigated the changes in gene expression in vivo in enhanced OB G{sub s} signaling. • Genes in cell cycle and transcription were increased in

  2. Biased signaling of G protein-coupled receptors - From a chemokine receptor CCR7 perspective

    DEFF Research Database (Denmark)

    Jørgensen, Astrid Sissel; Rosenkilde, Mette M; Hjortø, Gertrud M

    2018-01-01

    of CCL21 displays an extraordinarily strong glycosaminoglycan (GAG) binding, CCR7 plays a central role in coordinating the meeting between mature antigen presenting DCs and naïve T-cells which normally takes place in the lymph nodes (LNs). This process is a prerequisite for the initiation of an antigen...... the cell-based immune system is controlled. Bias comes in three forms; ligand-, receptor- and tissue-bias. Biased signaling is increasingly being recognized as playing an important role in contributing to the fine-tuned coordination of immune cell chemotaxis. In the current review we discuss the recent...

  3. Microvesicle and tunneling nanotube mediated intercellular transfer of g-protein coupled receptors in cell cultures

    International Nuclear Information System (INIS)

    Guescini, M.; Leo, G.; Genedani, S.; Carone, C.; Pederzoli, F.; Ciruela, F.; Guidolin, D.; Stocchi, V.; Mantuano, M.; Borroto-Escuela, D.O.; Fuxe, K.; Agnati, L.F.

    2012-01-01

    Recent evidence shows that cells exchange collections of signals via microvesicles (MVs) and tunneling nano-tubes (TNTs). In this paper we have investigated whether in cell cultures GPCRs can be transferred by means of MVs and TNTs from a source cell to target cells. Western blot, transmission electron microscopy and gene expression analyses demonstrate that A 2A and D 2 receptors are present in released MVs. In order to further demonstrate the involvement of MVs in cell-to-cell communication we created two populations of cells (HEK293T and COS-7) transiently transfected with D 2 R-CFP or A 2A R-YFP. These two types of cells were co-cultured, and FRET analysis demonstrated simultaneously positive cells to the D 2 R-CFP and A 2A R-YFP. Fluorescence microscopy analysis also showed that GPCRs can move from one cell to another also by means of TNTs. Finally, recipient cells pre-incubated for 24 h with A 2A R positive MVs were treated with the adenosine A 2A receptor agonist CGS-21680. The significant increase in cAMP accumulation clearly demonstrated that A 2A Rs were functionally competent in target cells. These findings demonstrate that A 2A receptors capable of recognizing and decoding extracellular signals can be safely transferred via MVs from source to target cells.

  4. Microvesicle and tunneling nanotube mediated intercellular transfer of g-protein coupled receptors in cell cultures

    Energy Technology Data Exchange (ETDEWEB)

    Guescini, M. [Department of Biomolecular Sciences, University of Urbino ' Carlo Bo' , 61029 Urbino (Italy); Leo, G.; Genedani, S. [Department Biomedical Sciences, University of Modena and Reggio Emilia (Italy); Carone, C. [Department Biomedical Sciences, University of Modena and Reggio Emilia (Italy); IRCCS San Camillo Lido, Venezia (Italy); Pederzoli, F. [Department Biomedical Sciences, University of Modena and Reggio Emilia (Italy); Ciruela, F. [Departament Patologia i Terapeutica Experimental, Universitat de Barcelona (Spain); Guidolin, D. [Department of Human Anatomy and Physiology, University of Padua (Italy); Stocchi, V.; Mantuano, M. [Department of Biomolecular Sciences, University of Urbino ' Carlo Bo' , 61029 Urbino (Italy); Borroto-Escuela, D.O.; Fuxe, K. [Department of Neuroscience, Karolinska Institutet, Stockholm (Sweden); Agnati, L.F., E-mail: luigiagnati@tin.it [IRCCS San Camillo Lido, Venezia (Italy)

    2012-03-10

    Recent evidence shows that cells exchange collections of signals via microvesicles (MVs) and tunneling nano-tubes (TNTs). In this paper we have investigated whether in cell cultures GPCRs can be transferred by means of MVs and TNTs from a source cell to target cells. Western blot, transmission electron microscopy and gene expression analyses demonstrate that A{sub 2A} and D{sub 2} receptors are present in released MVs. In order to further demonstrate the involvement of MVs in cell-to-cell communication we created two populations of cells (HEK293T and COS-7) transiently transfected with D{sub 2}R-CFP or A{sub 2A}R-YFP. These two types of cells were co-cultured, and FRET analysis demonstrated simultaneously positive cells to the D{sub 2}R-CFP and A{sub 2A}R-YFP. Fluorescence microscopy analysis also showed that GPCRs can move from one cell to another also by means of TNTs. Finally, recipient cells pre-incubated for 24 h with A{sub 2A}R positive MVs were treated with the adenosine A{sub 2A} receptor agonist CGS-21680. The significant increase in cAMP accumulation clearly demonstrated that A{sub 2A}Rs were functionally competent in target cells. These findings demonstrate that A{sub 2A} receptors capable of recognizing and decoding extracellular signals can be safely transferred via MVs from source to target cells.

  5. Receptor Activity-modifying Protein-directed G Protein Signaling Specificity for the Calcitonin Gene-related Peptide Family of Receptors.

    Science.gov (United States)

    Weston, Cathryn; Winfield, Ian; Harris, Matthew; Hodgson, Rose; Shah, Archna; Dowell, Simon J; Mobarec, Juan Carlos; Woodlock, David A; Reynolds, Christopher A; Poyner, David R; Watkins, Harriet A; Ladds, Graham

    2016-10-14

    The calcitonin gene-related peptide (CGRP) family of G protein-coupled receptors (GPCRs) is formed through the association of the calcitonin receptor-like receptor (CLR) and one of three receptor activity-modifying proteins (RAMPs). Binding of one of the three peptide ligands, CGRP, adrenomedullin (AM), and intermedin/adrenomedullin 2 (AM2), is well known to result in a Gα s -mediated increase in cAMP. Here we used modified yeast strains that couple receptor activation to cell growth, via chimeric yeast/Gα subunits, and HEK-293 cells to characterize the effect of different RAMP and ligand combinations on this pathway. We not only demonstrate functional couplings to both Gα s and Gα q but also identify a Gα i component to CLR signaling in both yeast and HEK-293 cells, which is absent in HEK-293S cells. We show that the CGRP family of receptors displays both ligand- and RAMP-dependent signaling bias among the Gα s , Gα i , and Gα q/11 pathways. The results are discussed in the context of RAMP interactions probed through molecular modeling and molecular dynamics simulations of the RAMP-GPCR-G protein complexes. This study further highlights the importance of RAMPs to CLR pharmacology and to bias in general, as well as identifying the importance of choosing an appropriate model system for the study of GPCR pharmacology. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  6. Different cAMP sources are critically involved in G protein-coupled receptor CRHR1 signaling.

    Science.gov (United States)

    Inda, Carolina; Dos Santos Claro, Paula A; Bonfiglio, Juan J; Senin, Sergio A; Maccarrone, Giuseppina; Turck, Christoph W; Silberstein, Susana

    2016-07-18

    Corticotropin-releasing hormone receptor 1 (CRHR1) activates G protein-dependent and internalization-dependent signaling mechanisms. Here, we report that the cyclic AMP (cAMP) response of CRHR1 in physiologically relevant scenarios engages separate cAMP sources, involving the atypical soluble adenylyl cyclase (sAC) in addition to transmembrane adenylyl cyclases (tmACs). cAMP produced by tmACs and sAC is required for the acute phase of extracellular signal regulated kinase 1/2 activation triggered by CRH-stimulated CRHR1, but only sAC activity is essential for the sustained internalization-dependent phase. Thus, different cAMP sources are involved in different signaling mechanisms. Examination of the cAMP response revealed that CRH-activated CRHR1 generates cAMP after endocytosis. Characterizing CRHR1 signaling uncovered a specific link between CRH-activated CRHR1, sAC, and endosome-based signaling. We provide evidence of sAC being involved in an endocytosis-dependent cAMP response, strengthening the emerging model of GPCR signaling in which the cAMP response does not occur exclusively at the plasma membrane and introducing the notion of sAC as an alternative source of cAMP. © 2016 Inda et al.

  7. Extracellular signal-regulated kinases control expression of G protein-coupled receptor kinase 2 (GRK2)

    DEFF Research Database (Denmark)

    Theilade, Juliane; Lerche Hansen, Jakob; Haunsø, Stig

    2002-01-01

    G protein-coupled receptor kinase 2 (GRK2) phosphorylates G protein-coupled receptors resulting in uncoupling from G proteins. Receptors modulate GRK2 expression, however the mechanistic basis for this effect is largely unknown. Here we report a novel mechanism by which receptors use...

  8. Epigenetic repression of regulator of G-protein signaling 2 promotes androgen-independent prostate cancer cell growth.

    Science.gov (United States)

    Wolff, Dennis W; Xie, Yan; Deng, Caishu; Gatalica, Zoran; Yang, Mingjie; Wang, Bo; Wang, Jincheng; Lin, Ming-Fong; Abel, Peter W; Tu, Yaping

    2012-04-01

    G-protein-coupled receptor (GPCR)-stimulated androgen-independent activation of androgen receptor (AR) contributes to acquisition of a hormone-refractory phenotype by prostate cancer. We previously reported that regulator of G-protein signaling (RGS) 2, an inhibitor of GPCRs, inhibits androgen-independent AR activation (Cao et al., Oncogene 2006;25:3719-34). Here, we show reduced RGS2 protein expression in human prostate cancer specimens compared to adjacent normal or hyperplastic tissue. Methylation-specific PCR analysis and bisulfite sequencing indicated that methylation of the CpG island in the RGS2 gene promoter correlated with RGS2 downregulation in prostate cancer. In vitro methylation of this promoter suppressed reporter gene expression in transient transfection studies, whereas reversal of this promoter methylation with 5-aza-2'-deoxycytidine (5-Aza-dC) induced RGS2 reexpression in androgen-independent prostate cancer cells and inhibited their growth under androgen-deficient conditions. Interestingly, the inhibitory effect of 5-Aza-dC was significantly reduced by an RGS2-targeted short hairpin RNA, indicating that reexpressed RGS2 contributed to this growth inhibition. Restoration of RGS2 levels by ectopic expression in androgen-independent prostate cancer cells suppressed growth of xenografts in castrated mice. Thus, RGS2 promoter hypermethylation represses its expression and unmasks a latent pathway for AR transactivation in prostate cancer cells. Targeting this reversible process may provide a new strategy for suppressing prostate cancer progression by reestablishing its androgen sensitivity. Copyright © 2011 UICC.

  9. G protein-coupled receptor 84 controls osteoclastogenesis through inhibition of NF-κB and MAPK signaling pathways.

    Science.gov (United States)

    Park, Ji-Wan; Yoon, Hye-Jin; Kang, Woo Youl; Cho, Seungil; Seong, Sook Jin; Lee, Hae Won; Yoon, Young-Ran; Kim, Hyun-Ju

    2018-02-01

    GPR84, a member of the G protein-coupled receptor family, is found predominantly in immune cells, such as macrophages, and functions as a pivotal modulator of inflammatory responses. In this study, we investigated the role of GPR84 in receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclast differentiation. Our microarray data showed that GPR84 was significantly downregulated in osteoclasts compared to in their precursors, macrophages. The overexpression of GPR84 in bone marrow-derived macrophages suppressed the formation of multinucleated osteoclasts without affecting precursor proliferation. In addition, GPR84 overexpression attenuated the induction of c-Fos and nuclear factor of activated T cells, cytoplasmic 1 (NFATc1), which are transcription factors that are critical for osteoclastogenesis. Furthermore, knockdown of GPR84 using a small hairpin RNA promoted RANKL-mediated osteoclast differentiation and gene expression of osteoclastogenic markers. Mechanistically, GPR84 overexpression blocked RANKL-stimulated phosphorylation of IκBα and three MAPKs, JNK, ERK, and p38. GPR84 also suppressed NF-κB transcriptional activity mediated by RANKL. Conversely, GPR84 knockdown enhanced RANKL-induced activation of IκBα and the three MAPKs. Collectively, our results revealed that GPR84 functions as a negative regulator of osteoclastogenesis, suggesting that it may be a potential therapeutic target for osteoclast-mediated bone-destructive diseases. © 2017 Wiley Periodicals, Inc.

  10. Wide range of interacting partners of pea Gβ subunit of G-proteins suggests its multiple functions in cell signalling.

    Science.gov (United States)

    Bhardwaj, Deepak; Lakhanpaul, Suman; Tuteja, Narendra

    2012-09-01

    Climate change is a major concern especially in view of the increasing global population and food security. Plant scientists need to look for genetic tools whose appropriate usage can contribute to sustainable food availability. G-proteins have been identified as some of the potential genetic tools that could be useful for protecting plants from various stresses. Heterotrimeric G-proteins consisting of three subunits Gα, Gβ and Gγ are important components of a number of signalling pathways. Their structure and functions are already well studied in animals but their potential in plants is now gaining attention for their role in stress tolerance. Earlier we have reported that over expressing pea Gβ conferred heat tolerance in tobacco plants. Here we report the interacting partners (proteins) of Gβ subunit of Pisum sativum and their putative role in stress and development. Out of 90 transformants isolated from the yeast-two-hybrid (Y2H) screening, seven were chosen for further investigation due to their recurrence in multiple experiments. These interacting partners were confirmed using β-galactosidase colony filter lift and ONPG (O-nitrophenyl-β-D-galactopyranoside) assays. These partners include thioredoxin H, histidine-containing phosphotransfer protein 5-like, pathogenesis-related protein, glucan endo-beta-1, 3-glucosidase (acidic isoform), glycine rich RNA binding protein, cold and drought-regulated protein (corA gene) and soluble inorganic pyrophosphatase 1. This study suggests the role of pea Gβ subunit in stress signal transduction and development pathways owing to its capability to interact with a wide range of proteins of multiple functions. Copyright © 2012 Elsevier Masson SAS. All rights reserved.

  11. Autoinhibition and signaling by the switch II motif in the G-protein chaperone of a radical B12 enzyme.

    Science.gov (United States)

    Lofgren, Michael; Koutmos, Markos; Banerjee, Ruma

    2013-10-25

    MeaB is an accessory GTPase protein involved in the assembly, protection, and reactivation of 5'-deoxyadenosyl cobalamin-dependent methylmalonyl-CoA mutase (MCM). Mutations in the human ortholog of MeaB result in methylmalonic aciduria, an inborn error of metabolism. G-proteins typically utilize conserved switch I and II motifs for signaling to effector proteins via conformational changes elicited by nucleotide binding and hydrolysis. Our recent discovery that MeaB utilizes an unusual switch III region for bidirectional signaling with MCM raised questions about the roles of the switch I and II motifs in MeaB. In this study, we addressed the functions of conserved switch II residues by performing alanine-scanning mutagenesis. Our results demonstrate that the GTPase activity of MeaB is autoinhibited by switch II and that this loop is important for coupling nucleotide-sensitive conformational changes in switch III to elicit the multiple chaperone functions of MeaB. Furthermore, we report the structure of MeaB·GDP crystallized in the presence of AlFx(-) to form the putative transition state analog, GDP·AlF4(-). The resulting crystal structure and its comparison with related G-proteins support the conclusion that the catalytic site of MeaB is incomplete in the absence of the GTPase-activating protein MCM and therefore unable to stabilize the transition state analog. Favoring an inactive conformation in the absence of the client MCM protein might represent a strategy for suppressing the intrinsic GTPase activity of MeaB in which the switch II loop plays an important role.

  12. Phosphatidic acid regulates signal output by G protein coupled receptors through direct interaction with phospholipase C-beta(1).

    Science.gov (United States)

    Litosch, Irene; Pujari, Rajeshree; Lee, Shawn J

    2009-09-01

    Phosphatidic acid (PA), generated downstream of monomeric Rho GTPases via phospholipase D (PLD) and additionally by diacylglycerol kinases (DGK), both stimulates phospholipase C-beta(1) (PLC-beta(1)) and potentiates stimulation of PLC-beta(1) activity by Galpha(q) in vitro. PA is a potential candidate for integrating signaling by monomeric and heterotrimeric G proteins to regulate signal output by G protein coupled receptors (GPCR), and we have sought to understand the mechanisms involved. We previously identified the region spanning residues 944-957, lying within the PLC-beta(1) C-terminus alphaA helix and flexible loop of the Galpha(q) binding domain, as required for stimulation of lipase activity by PA in vitro. Regulation by PA does not require residues essential for stimulation by Galpha(q) or GTPase activating activity. The present studies evaluated shorter alanine/glycine replacement mutants and finally point mutations to identify Tyr(952) and Ile(955) as key determinants for regulation by PA, assessed by both in vitro enzymatic and cell-based co-transfection assays. Replacement of Tyr(952) and Ile(955), PLC-beta(1) (Y952G/I955G), results in an 85% loss in stimulation by PA relative to WT-PLC-beta(1) in vitro. COS 7 cells co-transfected with PLC-beta(1) (Y952G/I955G) demonstrate a 10-fold increase in the EC(50) for stimulation and a 60% decrease in maximum stimulation by carbachol via Galpha(q) linked m1 muscarinic receptors, relative to cells co-transfected with WT-PLC-beta(1) but otherwise similar conditions. Residues required for regulation by PA are not essential for stimulation by G protein subunits. WT-PLC-beta(1) and PLC-beta(1) (Y952G/I955G) activity is increased comparably by co-transfection with Galpha(q) and neither is markedly affected by co-transfection with Gbeta(1)gamma(2). Inhibiting PLD-generated PA production by 1-butanol has little effect on maximum stimulation, but shifts the EC(50) for agonist stimulation of WT-PLC-beta(1) by 10-fold

  13. Variation in regulator of G-protein signaling 17 gene (RGS17 is associated with multiple substance dependence diagnoses

    Directory of Open Access Journals (Sweden)

    Zhang Huiping

    2012-05-01

    Full Text Available Abstract Background RGS17 and RGS20 encode two members of the regulator of G-protein signaling RGS-Rz subfamily. Variation in these genes may alter their transcription and thereby influence the function of G protein-coupled receptors, including opioid receptors, and modify risk for substance dependence. Methods The association of 13 RGS17 and eight RGS20 tag single nucleotide polymorphisms (SNPs was examined with four substance dependence diagnoses (alcohol (AD, cocaine (CD, opioid (OD or marijuana (MjD] in 1,905 African Americans (AAs: 1,562 cases and 343 controls and 1,332 European Americans (EAs: 981 cases and 351 controls. Analyses were performed using both χ2 tests and logistic regression analyses that covaried sex, age, and ancestry proportion. Correlation of genotypes and mRNA expression levels was assessed by linear regression analyses. Results Seven RGS17 SNPs showed a significant association with at least one of the four dependence traits after a permutation-based correction for multiple testing (0.003≤Pempirical≤0.037. The G allele of SNP rs596359, in the RGS17 promoter region, was associated with AD, CD, OD, or MjD in both populations (0.005≤Pempirical≤0.019. This allele was also associated with significantly lower mRNA expression levels of RGS17 in YRI subjects (P = 0.002 and non-significantly lower mRNA expression levels of RGS17 in CEU subjects (P = 0.185. No RGS20 SNPs were associated with any of the four dependence traits in either population. Conclusions This study demonstrated that variation in RGS17 was associated with risk for substance dependence diagnoses in both AA and EA populations.

  14. Distinct Phosphorylation Clusters Determine the Signaling Outcome of Free Fatty Acid Receptor 4/G Protein-Coupled Receptor 120

    DEFF Research Database (Denmark)

    Prihandoko, Rudi; Alvarez-Curto, Elisa; Hudson, Brian D

    2016-01-01

    of these phosphoacceptor sites to alanine completely prevented phosphorylation of mFFA4 but did not limit receptor coupling to extracellular signal regulated protein kinase 1 and 2 (ERK1/2) activation. Rather, an inhibitor of Gq/11proteins completely prevented receptor signaling to ERK1/2. By contrast, the recruitment...... activation. These unique observations define differential effects on signaling mediated by phosphorylation at distinct locations. This hallmark feature supports the possibility that the signaling outcome of mFFA4 activation can be determined by the pattern of phosphorylation (phosphorylation barcode...

  15. Pro-aging effects of glucose signaling through a G protein-coupled glucose receptor in fission yeast.

    Directory of Open Access Journals (Sweden)

    Antoine E Roux

    2009-03-01

    Full Text Available Glucose is the preferred carbon and energy source in prokaryotes, unicellular eukaryotes, and metazoans. However, excess of glucose has been associated with several diseases, including diabetes and the less understood process of aging. On the contrary, limiting glucose (i.e., calorie restriction slows aging and age-related diseases in most species. Understanding the mechanism by which glucose limits life span is therefore important for any attempt to control aging and age-related diseases. Here, we use the yeast Schizosaccharomyces pombe as a model to study the regulation of chronological life span by glucose. Growth of S. pombe at a reduced concentration of glucose increased life span and oxidative stress resistance as reported before for many other organisms. Surprisingly, loss of the Git3 glucose receptor, a G protein-coupled receptor, also increased life span in conditions where glucose consumption was not affected. These results suggest a role for glucose-signaling pathways in life span regulation. In agreement, constitutive activation of the Galpha subunit acting downstream of Git3 accelerated aging in S. pombe and inhibited the effects of calorie restriction. A similar pro-aging effect of glucose was documented in mutants of hexokinase, which cannot metabolize glucose and, therefore, are exposed to constitutive glucose signaling. The pro-aging effect of glucose signaling on life span correlated with an increase in reactive oxygen species and a decrease in oxidative stress resistance and respiration rate. Likewise, the anti-aging effect of both calorie restriction and the Deltagit3 mutation was accompanied by increased respiration and lower reactive oxygen species production. Altogether, our data suggest an important role for glucose signaling through the Git3/PKA pathway to regulate S. pombe life span.

  16. The role of G-protein-coupled receptors in mediating the effect of fatty acids on inflammation and insulin sensitivity.

    Science.gov (United States)

    Oh, Da Young; Lagakos, William S

    2011-07-01

    Chronic activation of inflammatory pathways mediates the pathogenesis of insulin resistance, and the macrophage/adipocyte nexus provides a key mechanism underlying decreased insulin sensitivity. Free fatty acids are important in the pathogenesis of insulin resistance, although their precise mechanisms of action have yet to be fully elucidated. Recently, a family of G-protein-coupled receptors has been identified that exhibits high affinity for fatty acids. This review summarizes recent findings on six of these receptors, their ligands, and their potential physiological functions in vivo. Upon activation, the free fatty acid receptors affect inflammation, glucose metabolism, and insulin sensitivity. Genetic deletion of GPR40 and GPR41, receptors for long-chain and short-chain fatty acids, respectively, results in resistance to diet-induced obesity. Deletion of GPR43 and GPR84 exacerbates inflammation, and deletion of the long-chain fatty acid receptors GPR119 and GPR120 reduces or is predicted to reduce glucose tolerance. These studies provide a new understanding of the general biology of gastric motility and also shed valuable insight into some potentially beneficial therapeutic targets. Furthermore, highly selective agonists or antagonists for the free fatty acid receptors have been developed and look promising for treating various metabolic diseases.

  17. Serotonin Signaling in Schistosoma mansoni: A Serotonin–Activated G Protein-Coupled Receptor Controls Parasite Movement

    Science.gov (United States)

    Rashid, Mohammed; Ribeiro, Paula

    2014-01-01

    Serotonin is an important neuroactive substance in all the parasitic helminths. In Schistosoma mansoni, serotonin is strongly myoexcitatory; it potentiates contraction of the body wall muscles and stimulates motor activity. This is considered to be a critical mechanism of motor control in the parasite, but the mode of action of serotonin is poorly understood. Here we provide the first molecular evidence of a functional serotonin receptor (Sm5HTR) in S. mansoni. The schistosome receptor belongs to the G protein-coupled receptor (GPCR) superfamily and is distantly related to serotonergic type 7 (5HT7) receptors from other species. Functional expression studies in transfected HEK 293 cells showed that Sm5HTR is a specific serotonin receptor and it signals through an increase in intracellular cAMP, consistent with a 5HT7 signaling mechanism. Immunolocalization studies with a specific anti-Sm5HTR antibody revealed that the receptor is abundantly distributed in the worm's nervous system, including the cerebral ganglia and main nerve cords of the central nervous system and the peripheral innervation of the body wall muscles and tegument. RNA interference (RNAi) was performed both in schistosomulae and adult worms to test whether the receptor is required for parasite motility. The RNAi-suppressed adults and larvae were markedly hypoactive compared to the corresponding controls and they were also resistant to exogenous serotonin treatment. These results show that Sm5HTR is at least one of the receptors responsible for the motor effects of serotonin in S. mansoni. The fact that Sm5HTR is expressed in nerve tissue further suggests that serotonin stimulates movement via this receptor by modulating neuronal output to the musculature. Together, the evidence identifies Sm5HTR as an important neuronal protein and a key component of the motor control apparatus in S. mansoni. PMID:24453972

  18. When Heterotrimeric G Proteins Are Not Activated by G Protein-Coupled Receptors: Structural Insights and Evolutionary Conservation.

    Science.gov (United States)

    DiGiacomo, Vincent; Marivin, Arthur; Garcia-Marcos, Mikel

    2018-01-23

    Heterotrimeric G proteins are signal-transducing switches conserved across eukaryotes. In humans, they work as critical mediators of intercellular communication in the context of virtually any physiological process. While G protein regulation by G protein-coupled receptors (GPCRs) is well-established and has received much attention, it has become recently evident that heterotrimeric G proteins can also be activated by cytoplasmic proteins. However, this alternative mechanism of G protein regulation remains far less studied than GPCR-mediated signaling. This Viewpoint focuses on recent advances in the characterization of a group of nonreceptor proteins that contain a sequence dubbed the "Gα-binding and -activating (GBA) motif". So far, four proteins present in mammals [GIV (also known as Girdin), DAPLE, CALNUC, and NUCB2] and one protein in Caenorhabditis elegans (GBAS-1) have been described as possessing a functional GBA motif. The GBA motif confers guanine nucleotide exchange factor activity on Gαi subunits in vitro and activates G protein signaling in cells. The importance of this mechanism of signal transduction is highlighted by the fact that its dysregulation underlies human diseases, such as cancer, which has made the proteins attractive new candidates for therapeutic intervention. Here we discuss recent discoveries on the structural basis of GBA-mediated activation of G proteins and its evolutionary conservation and compare them with the better-studied mechanism mediated by GPCRs.

  19. The G protein-coupled receptor GPR30 mediates the proliferative and invasive effects induced by hydroxytamoxifen in endometrial cancer cells

    Energy Technology Data Exchange (ETDEWEB)

    Du, Gui-Qiang; Zhou, Long; Chen, Xiao-Yue [Department of Obstetrics and Gynecology, The International Peace Maternity and Child Health Hospital of the China Welfare Institute Affiliated to Shanghai Jiao Tong University, 910, Hengshan Road, Shanghai (China); Wan, Xiao-Ping, E-mail: wanxiaoping61@126.com [Department of Obstetrics and Gynecology, The International Peace Maternity and Child Health Hospital of the China Welfare Institute Affiliated to Shanghai Jiao Tong University, 910, Hengshan Road, Shanghai (China); He, Yin-Yan [Department of Obstetrics and Gynecology, Shanghai First People' s Hospital, Shanghai Jiao Tong University, Shanghai (China)

    2012-04-06

    Highlights: Black-Right-Pointing-Pointer We assessed hydroxytamoxifen (OHT) effects in two endometrial cancer cell lines. Black-Right-Pointing-Pointer GPR30 mediates the proliferative effects induced by OHT. Black-Right-Pointing-Pointer GPR30 mediates the invasive effects induced by OHT. Black-Right-Pointing-Pointer GPR30 expression was up-regulated by OHT in endometrial cancer cell line. -- Abstract: The selective ER modulator tamoxifen (TAM) is the most widely used ER antagonist for treatment of women with hormone-dependent breast tumor. However, long-term treatment is associated with an increased risk of endometrial cancer. The aim of the present study was to demonstrate new insight into the role of G-protein coupled receptor 30 (GPR30) in the activity of TAM, which promoted endometrial cancer. In endometrial cancer cell lines ISHIKAWA and KLE, the potential of 4-hydroxytamoxifen (OHT), the active metabolite of TAM, 17{beta}-estradiol (E2) and G1, a non-steroidal GPR30-specific agonist to promote cell proliferation and invasion was evaluated. All agents above induced high proliferative and invasive effects, while the down-regulation of GPR30 or the interruption of MAPK signal pathway partly or completely prevented the action of the regent. Moreover, the RNA and protein expression of GPR30 was up-regulated by G1, E2 or OHT in both cell lines. The present study provided a new insight into the mechanism involved in the agonistic activity exerted by TAM in the uterus.

  20. The G protein-coupled receptor GPR30 mediates the proliferative and invasive effects induced by hydroxytamoxifen in endometrial cancer cells

    International Nuclear Information System (INIS)

    Du, Gui-Qiang; Zhou, Long; Chen, Xiao-Yue; Wan, Xiao-Ping; He, Yin-Yan

    2012-01-01

    Highlights: ► We assessed hydroxytamoxifen (OHT) effects in two endometrial cancer cell lines. ► GPR30 mediates the proliferative effects induced by OHT. ► GPR30 mediates the invasive effects induced by OHT. ► GPR30 expression was up-regulated by OHT in endometrial cancer cell line. -- Abstract: The selective ER modulator tamoxifen (TAM) is the most widely used ER antagonist for treatment of women with hormone-dependent breast tumor. However, long-term treatment is associated with an increased risk of endometrial cancer. The aim of the present study was to demonstrate new insight into the role of G-protein coupled receptor 30 (GPR30) in the activity of TAM, which promoted endometrial cancer. In endometrial cancer cell lines ISHIKAWA and KLE, the potential of 4-hydroxytamoxifen (OHT), the active metabolite of TAM, 17β-estradiol (E2) and G1, a non-steroidal GPR30-specific agonist to promote cell proliferation and invasion was evaluated. All agents above induced high proliferative and invasive effects, while the down-regulation of GPR30 or the interruption of MAPK signal pathway partly or completely prevented the action of the regent. Moreover, the RNA and protein expression of GPR30 was up-regulated by G1, E2 or OHT in both cell lines. The present study provided a new insight into the mechanism involved in the agonistic activity exerted by TAM in the uterus.

  1. A regulator of G Protein signaling, RGS3, inhibits gonadotropin-releasing hormone (GnRH-stimulated luteinizing hormone (LH secretion

    Directory of Open Access Journals (Sweden)

    Musgrove Lois C

    2001-11-01

    Full Text Available Abstract Background Luteinizing hormone secreted by the anterior pituitary gland regulates gonadal function. Luteinizing hormone secretion is regulated both by alterations in gonadotrope responsiveness to hypothalamic gonadotropin releasing hormone and by alterations in gonadotropin releasing hormone secretion. The mechanisms that determine gonadotrope responsiveness are unknown but may involve regulators of G protein signaling (RGSs. These proteins act by antagonizing or abbreviating interaction of Gα proteins with effectors such as phospholipase Cβ. Previously, we reported that gonadotropin releasing hormone-stimulated second messenger inositol trisphosphate production was inhibited when RGS3 and gonadotropin releasing hormone receptor cDNAs were co-transfected into the COS cell line. Here, we present evidence for RGS3 inhibition of gonadotropin releasing hormone-induced luteinizing hormone secretion from cultured rat pituitary cells. Results A truncated version of RGS3 (RGS3T = RGS3 314–519 inhibited gonadotropin releasing hormone-stimulated inositol trisphosphate production more potently than did RSG3 in gonadotropin releasing hormone receptor-bearing COS cells. An RSG3/glutathione-S-transferase fusion protein bound more 35S-Gqα than any other member of the G protein family tested. Adenoviral-mediated RGS3 gene transfer in pituitary gonadotropes inhibited gonadotropin releasing hormone-stimulated luteinizing hormone secretion in a dose-related fashion. Adeno-RGS3 also inhibited gonadotropin releasing hormone stimulated 3H-inositol phosphate accumulation, consistent with a molecular site of action at the Gqα protein. Conclusions RGS3 inhibits gonadotropin releasing hormone-stimulated second messenger production (inositol trisphosphate as well as luteinizing hormone secretion from rat pituitary gonadotropes apparently by binding and suppressing the transduction properties of Gqα protein function. A version of RGS3 that is amino

  2. Structure of the Regulator of G Protein Signaling 8 (RGS8)-Gαq Complex: MOLECULAR BASIS FOR Gα SELECTIVITY.

    Science.gov (United States)

    Taylor, Veronica G; Bommarito, Paige A; Tesmer, John J G

    2016-03-04

    Regulator of G protein signaling (RGS) proteins interact with activated Gα subunits via their RGS domains and accelerate the hydrolysis of GTP. Although the R4 subfamily of RGS proteins generally accepts both Gαi/o and Gαq/11 subunits as substrates, the R7 and R12 subfamilies select against Gαq/11. In contrast, only one RGS protein, RGS2, is known to be selective for Gαq/11. The molecular basis for this selectivity is not clear. Previously, the crystal structure of RGS2 in complex with Gαq revealed a non-canonical interaction that could be due to interfacial differences imposed by RGS2, the Gα subunit, or both. To resolve this ambiguity, the 2.6 Å crystal structure of RGS8, an R4 subfamily member, was determined in complex with Gαq. RGS8 adopts the same pose on Gαq as it does when bound to Gαi3, indicating that the non-canonical interaction of RGS2 with Gαq is due to unique features of RGS2. Based on the RGS8-Gαq structure, residues in RGS8 that contact a unique α-helical domain loop of Gαq were converted to those typically found in R12 subfamily members, and the reverse substitutions were introduced into RGS10, an R12 subfamily member. Although these substitutions perturbed their ability to stimulate GTP hydrolysis, they did not reverse selectivity. Instead, selectivity for Gαq seems more likely determined by whether strong contacts can be maintained between α6 of the RGS domain and Switch III of Gαq, regions of high sequence and conformational diversity in both protein families. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  3. [(35)S]-GTPgammaS autoradiography reveals alpha(2) adrenoceptor-mediated G-protein activation in amygdala and lateral septum.

    Science.gov (United States)

    Newman-Tancredi, A; Chaput, C; Touzard, M; Millan, M J

    2000-04-03

    alpha(2)-adrenoceptor-mediated G-protein activation was examined by [(35)S]-GTPgammaS autoradiography. In alpha(2)-adrenoceptor-rich regions (amygdala, lateral septum), noradrenaline stimulated [(35)S]-GTPgammaS binding. These actions were abolished by the selective alpha(2) antagonist, atipamezole. Conversely, in caudate nucleus, which expresses few alpha(2) receptors, noradrenaline-induced stimulation was not inhibited by atipamezole, suggesting that it is not mediated by alpha(2)-adrenoceptors.

  4. Signaling governed by G proteins and cAMP is crucial for growth, secondary metabolism and sexual development in Fusarium fujikuroi.

    Directory of Open Access Journals (Sweden)

    Lena Studt

    Full Text Available The plant-pathogenic fungus Fusarium fujikuroi is a notorious rice pathogen causing hyper-elongation of infected plants due to the production of gibberellic acids (GAs. In addition to GAs, F. fujikuroi produces a wide range of other secondary metabolites, such as fusarins, fusaric acid or the red polyketides bikaverins and fusarubins. The recent availability of the fungal genome sequence for this species has revealed the potential of many more putative secondary metabolite gene clusters whose products remain to be identified. However, the complex regulation of secondary metabolism is far from being understood. Here we studied the impact of the heterotrimeric G protein and the cAMP-mediated signaling network, including the regulatory subunits of the cAMP-dependent protein kinase (PKA, to study their effect on colony morphology, sexual development and regulation of bikaverins, fusarubins and GAs. We demonstrated that fusarubin biosynthesis is negatively regulated by at least two Gα subunits, FfG1 and FfG3, which both function as stimulators of the adenylyl cyclase FfAC. Surprisingly, the primary downstream target of the adenylyl cyclase, the PKA, is not involved in the regulation of fusarubins, suggesting that additional, yet unidentified, cAMP-binding protein(s exist. In contrast, bikaverin biosynthesis is significantly reduced in ffg1 and ffg3 deletion mutants and positively regulated by FfAC and FfPKA1, while GA biosynthesis depends on the active FfAC and FfPKA2 in an FfG1- and FfG3-independent manner. In addition, we provide evidence that G Protein-mediated/cAMP signaling is important for growth in F. fujikuroi because deletion of ffg3, ffac and ffpka1 resulted in impaired growth on minimal and rich media. Finally, sexual crosses of ffg1 mutants showed the importance of a functional FfG1 protein for development of perithecia in the mating strain that carries the MAT1-1 idiomorph.

  5. The p110beta isoform of phosphoinositide 3-kinase signals downstream of G protein-coupled receptors and is functionally redundant with p110gamma.

    Science.gov (United States)

    Guillermet-Guibert, Julie; Bjorklof, Katja; Salpekar, Ashreena; Gonella, Cristiano; Ramadani, Faruk; Bilancio, Antonio; Meek, Stephen; Smith, Andrew J H; Okkenhaug, Klaus; Vanhaesebroeck, Bart

    2008-06-17

    The p110 isoforms of phosphoinositide 3-kinase (PI3K) are acutely regulated by extracellular stimuli. The class IA PI3K catalytic subunits (p110alpha, p110beta, and p110delta) occur in complex with a Src homology 2 (SH2) domain-containing p85 regulatory subunit, which has been shown to link p110alpha and p110delta to Tyr kinase signaling pathways. The p84/p101 regulatory subunits of the p110gamma class IB PI3K lack SH2 domains and instead couple p110gamma to G protein-coupled receptors (GPCRs). Here, we show, using small-molecule inhibitors with selectivity for p110beta and cells derived from a p110beta-deficient mouse line, that p110beta is not a major effector of Tyr kinase signaling but couples to GPCRs. In macrophages, both p110beta and p110gamma contributed to Akt activation induced by the GPCR agonist complement 5a, but not by the Tyr kinase ligand colony-stimulating factor-1. In fibroblasts, which express p110beta but not p110gamma, p110beta mediated Akt activation by the GPCR ligands stromal cell-derived factor, sphingosine-1-phosphate, and lysophosphatidic acid but not by the Tyr kinase ligands PDGF, insulin, and insulin-like growth factor 1. Introduction of p110gamma in these cells reduced the contribution of p110beta to GPCR signaling. Taken together, these data show that p110beta and p110gamma can couple redundantly to the same GPCR agonists. p110beta, which shows a much broader tissue distribution than the leukocyte-restricted p110gamma, could thus provide a conduit for GPCR-linked PI3K signaling in the many cell types where p110gamma expression is low or absent.

  6. The p110β isoform of phosphoinositide 3-kinase signals downstream of G protein-coupled receptors and is functionally redundant with p110γ

    Science.gov (United States)

    Guillermet-Guibert, Julie; Bjorklof, Katja; Salpekar, Ashreena; Gonella, Cristiano; Ramadani, Faruk; Bilancio, Antonio; Meek, Stephen; Smith, Andrew J. H.; Okkenhaug, Klaus; Vanhaesebroeck, Bart

    2008-01-01

    The p110 isoforms of phosphoinositide 3-kinase (PI3K) are acutely regulated by extracellular stimuli. The class IA PI3K catalytic subunits (p110α, p110β, and p110δ) occur in complex with a Src homology 2 (SH2) domain-containing p85 regulatory subunit, which has been shown to link p110α and p110δ to Tyr kinase signaling pathways. The p84/p101 regulatory subunits of the p110γ class IB PI3K lack SH2 domains and instead couple p110γ to G protein-coupled receptors (GPCRs). Here, we show, using small-molecule inhibitors with selectivity for p110β and cells derived from a p110β-deficient mouse line, that p110β is not a major effector of Tyr kinase signaling but couples to GPCRs. In macrophages, both p110β and p110γ contributed to Akt activation induced by the GPCR agonist complement 5a, but not by the Tyr kinase ligand colony-stimulating factor-1. In fibroblasts, which express p110β but not p110γ, p110β mediated Akt activation by the GPCR ligands stromal cell-derived factor, sphingosine-1-phosphate, and lysophosphatidic acid but not by the Tyr kinase ligands PDGF, insulin, and insulin-like growth factor 1. Introduction of p110γ in these cells reduced the contribution of p110β to GPCR signaling. Taken together, these data show that p110β and p110γ can couple redundantly to the same GPCR agonists. p110β, which shows a much broader tissue distribution than the leukocyte-restricted p110γ, could thus provide a conduit for GPCR-linked PI3K signaling in the many cell types where p110γ expression is low or absent. PMID:18544649

  7. Local anesthetic inhibition of G protein-coupled receptor signaling by interference with Galpha(q) protein function

    NARCIS (Netherlands)

    Hollmann, M. W.; Wieczorek, K. S.; Berger, A.; Durieux, M. E.

    2001-01-01

    Although local anesthetics are considered primarily Na(+) channel blockers, previous studies suggest a common intracellular site of action on different G protein-coupled receptors. In the present study, we characterized this site for the LPA, m1 muscarinic, and trypsin receptor. Xenopus laevis

  8. The Multiple Faces of Prostaglandin E2 G-Protein Coupled Receptor Signaling during the Dendritic Cell Life Cycle

    NARCIS (Netherlands)

    de Keijzer, Sandra; Meddens, Marjolein B.M.; Torensma, Ruurd; Cambi, A.

    2013-01-01

    Many processes regulating immune responses are initiated by G-protein coupled receptors (GPCRs) and report biochemical changes in the microenvironment. Dendritic cells (DCs) are the most potent antigen-presenting cells and crucial for the regulation of innate and adaptive immune responses. The lipid

  9. Regulator of G-protein signaling 5 (RGS5) inhibits cell proliferation and enhances radiosensitivity of human lung cancer cells

    International Nuclear Information System (INIS)

    Xu Zumin; Wang Jin; Zuo Yufang; Yu Zhonghua; Peng Fang; Hu Xiao; Zhou Qichao; Ma Honglian; Bao Yong; Chen Ming

    2014-01-01

    Objective: To investigate the effects of regulator and the underlying molecular mechanisms of G-protein signaling 5 (RGS5) on radiation response in human lung cancer cells. Methods: The effects of RGS5 on viability were determined by MTT assay, and apoptosis rate was detected by flow cytometry, in human lung cancer cells. The combined effect of ionizing radiation and RGS5 on tumor cells was detected by colony formation assay. The protein expression was detected by Western blot. Results: RGS5 overexpression remarkably inhibited the survival of human lung cancer cells, and the growth inhibition rate of RGS5 overexpression on A549 and Calu-3 cells were 44.4% (F = 29.18, P < 0.05) and 39.27% (F = 23.04, P < 0.05) at 48 h, and 54.3%(F = 103.45, P < 0.05), 44.7%(F = 108.02, P < 0.05) at 72 h post-irradiation, respectively. RGS5 might exert its inhibitory effects on human lung cancer cells by inducing tumor cell apoptosis, while the apoptotic cells rate in A549 and Calu-3 cells in control group, pTRiEX group and pTRiEX-RGS5 group were (1.3±0.2)%, (3.4±0.6)%, (19.6±2.3)% (F = 86.62, P < 0.05), and (3.2±0.8)%, (3.0±0.9)%, (12.8±1.8)% (F = 28.80, P < 0.05) at 36 h post-irradiation, respectively. Furthermore, RGS5 could sensitize the lung cancer cells to radiation. Conclusions: RGS5 might play an inhibitory role in human lung cancer cell proliferation, which may explain the pathoclinical observation thet high expression of RGSS is a favorable prognostic factor in NSCLC patients. In addition, RGS5 also enhance the anti-tumor effects of radiation in human lung cancer cells. (authors)

  10. Gα and regulator of G-protein signaling (RGS) protein pairs maintain functional compatibility and conserved interaction interfaces throughout evolution despite frequent loss of RGS proteins in plants.

    Science.gov (United States)

    Hackenberg, Dieter; McKain, Michael R; Lee, Soon Goo; Roy Choudhury, Swarup; McCann, Tyler; Schreier, Spencer; Harkess, Alex; Pires, J Chris; Wong, Gane Ka-Shu; Jez, Joseph M; Kellogg, Elizabeth A; Pandey, Sona

    2017-10-01

    Signaling pathways regulated by heterotrimeric G-proteins exist in all eukaryotes. The regulator of G-protein signaling (RGS) proteins are key interactors and critical modulators of the Gα protein of the heterotrimer. However, while G-proteins are widespread in plants, RGS proteins have been reported to be missing from the entire monocot lineage, with two exceptions. A single amino acid substitution-based adaptive coevolution of the Gα:RGS proteins was proposed to enable the loss of RGS in monocots. We used a combination of evolutionary and biochemical analyses and homology modeling of the Gα and RGS proteins to address their expansion and its potential effects on the G-protein cycle in plants. Our results show that RGS proteins are widely distributed in the monocot lineage, despite their frequent loss. There is no support for the adaptive coevolution of the Gα:RGS protein pair based on single amino acid substitutions. RGS proteins interact with, and affect the activity of, Gα proteins from species with or without endogenous RGS. This cross-functional compatibility expands between the metazoan and plant kingdoms, illustrating striking conservation of their interaction interface. We propose that additional proteins or alternative mechanisms may exist which compensate for the loss of RGS in certain plant species. © 2016 The Authors. New Phytologist © 2016 New Phytologist Trust.

  11. Yeast G-proteins mediate directional sensing and polarization behaviors in response to changes in pheromone gradient direction

    Science.gov (United States)

    Moore, Travis I.; Tanaka, Hiromasa; Kim, Hyung Joon; Jeon, Noo Li; Yi, Tau-Mu

    2013-01-01

    Yeast cells polarize by projecting up mating pheromone gradients, a classic cell polarity behavior. However, these chemical gradients may shift direction. We examine how yeast cells sense and respond to a 180o switch in the direction of microfluidically generated pheromone gradients. We identify two behaviors: at low concentrations of α-factor, the initial projection grows by bending, whereas at high concentrations, cells form a second projection toward the new source. Mutations that increase heterotrimeric G-protein activity expand the bending-growth morphology to high concentrations; mutations that increase Cdc42 activity result in second projections at low concentrations. Gradient-sensing projection bending requires interaction between Gβγ and Cdc24, whereas gradient-nonsensing projection extension is stimulated by Bem1 and hyperactivated Cdc42. Of interest, a mutation in Gα affects both bending and extension. Finally, we find a genetic perturbation that exhibits both behaviors. Overexpression of the formin Bni1, a component of the polarisome, makes both bending-growth projections and second projections at low and high α-factor concentrations, suggesting a role for Bni1 downstream of the heterotrimeric G-protein and Cdc42 during gradient sensing and response. Thus we demonstrate that G-proteins modulate in a ligand-dependent manner two fundamental cell-polarity behaviors in response to gradient directional change. PMID:23242998

  12. On the mechanism of aluminum ion-induced neurotoxicity: The effects of aluminum species on G-protein-mediated processes and on drug interactions with the N-methyl-D-aspartate modulated ionophore

    International Nuclear Information System (INIS)

    Hubbard, C.M.

    1989-01-01

    To establish what effects Al 3+ may have on G-protein mediate signal transduction, the effects of Al 3+ on the signal-coupling G-protein from retinal rod outer segments (G t or transducin) have been investigated as a model for the effects of Al 3+ on signal transduction by G-proteins in general. In this investigation, we have studied the effects of Al 3+ on the isolated, light-dependent rhodopsin catalyzed GTP-GDP exchange on G t ; the light-dependent GTPase activity of G t ; the light-independent cGMP hydrolysis by PDE; and the light activated, rhodopsin catalyzed, cGMP hydrolysis by PDE in vitro. To determine the effects of two defined species of aluminum on N-methyl-D-aspartic acid (NMDA) receptor-channel modulation we utilized a specific radioligand binding assay. This allowed us to compare the effects of aluminum to other metal ions on specific [ 3 H]MK-801 binding to the NMDA receptor-channel complex. This complex is involved in long-term potentiation, which is currently being investigated as the mechanism by which learning and memory occur and has been implicated in the pathology of Alzheimer's disease. We have investigated the effects of two different species of aluminum, as well as Ca 2+ , Zn 2+ , Mg 2+ , and Li + on the specific binding of [ 3 H]MK-801 to the NMDA receptor-channel complex under depolarized conditions

  13. Effects of gender on locomotor sensitivity to amphetamine, body weight, and fat mass in regulator of G protein signaling 9 (RGS9) knockout mice.

    Science.gov (United States)

    Walker, Paul D; Jarosz, Patricia A; Bouhamdan, Mohamad; MacKenzie, Robert G

    2015-01-01

    Regulator of G-protein signaling (RGS) protein 9-2 is enriched in the striatum where it modulates dopamine and opioid receptor-mediated signaling. RGS9 knockout (KO) mice show increased psychostimulant-induced behavioral sensitization, as well as exhibit higher body weights and greater fat accumulation compared to wild-type (WT) littermates. In the present study, we found gender influences on each of these phenotypic characteristics. Female RGS9 KO mice exhibited greater locomotor sensitization to amphetamine (1.0mg/kg) treatment as compared to male RGS9 KO mice. Male RGS9 KO mice showed increased body weights as compared to male WT littermates, while no such differences were detected in female mice. Quantitative magnetic resonance showed that male RGS9 KO mice accumulated greater fat mass vs. WT littermates at 5months of age. Such observations could not be explained by increased caloric consumption since male and female RGS9 KO mice demonstrated equivalent daily food intake as compared to their respective WT littermates. Although indirect calorimetry methods found decreased oxygen consumption and carbon dioxide production during the 12-hour dark phase in male RGS9 KO vs. WT mice which are indicative of less energy expenditure, male RGS9 KO mice exhibited lower levels of locomotor activity during this period. Genotype had no effect on metabolic activities when KO and WT groups were compared under fasting vs. feeding treatments. In summary, these results highlight the importance of factoring gender into the experimental design since many studies conducted in RGS9 KO mice utilize locomotor activity as a measured outcome. Copyright © 2014. Published by Elsevier Inc.

  14. A monoclonal antibody for G protein-coupled receptor crystallography

    DEFF Research Database (Denmark)

    Day, Peter W; Rasmussen, Søren Gøgsig Faarup; Parnot, Charles

    2007-01-01

    G protein-coupled receptors (GPCRs) constitute the largest family of signaling proteins in mammals, mediating responses to hormones, neurotransmitters, and senses of sight, smell and taste. Mechanistic insight into GPCR signal transduction is limited by a paucity of high-resolution structural inf...

  15. A monomeric G protein-coupled receptor isolated in a high-density lipoprotein particle efficiently activates its G protein

    DEFF Research Database (Denmark)

    Whorton, Matthew R; Bokoch, Michael P; Rasmussen, Søren Gøgsig Faarup

    2007-01-01

    G protein-coupled receptors (GPCRs) respond to a diverse array of ligands, mediating cellular responses to hormones and neurotransmitters, as well as the senses of smell and taste. The structures of the GPCR rhodopsin and several G proteins have been determined by x-ray crystallography, yet...... the organization of the signaling complex between GPCRs and G proteins is poorly understood. The observations that some GPCRs are obligate heterodimers, and that many GPCRs form both homo- and heterodimers, has led to speculation that GPCR dimers may be required for efficient activation of G proteins. However......, technical limitations have precluded a definitive analysis of G protein coupling to monomeric GPCRs in a biochemically defined and membrane-bound system. Here we demonstrate that a prototypical GPCR, the beta2-adrenergic receptor (beta2AR), can be incorporated into a reconstituted high-density lipoprotein...

  16. Involvement of a putative intercellular signal-recognizing G protein-coupled receptor in the engulfment of Salmonella by the protozoan Tetrahymena

    Directory of Open Access Journals (Sweden)

    P.N. Agbedanu

    2013-07-01

    Full Text Available In an effort to investigate the molecular basis of protozoa engulfment-mediated hypervirulence of Salmonella in cattle, we evaluated protozoan G protein-coupled receptors (GPCRs as transducers of Salmonella engulfment by the model protozoan Tetrahymena. Our laboratory previously demonstrated that non-pathogenic protozoa (including Tetrahymena engulf Salmonella and then exacerbate its virulence in cattle, but the mechanistic details of the phenomenon are not fully understood. GPCRs were investigated since these receptors facilitate phagocytosis of particulates by Tetrahymena, and a GPCR apparently modulates bacterial engulfment for the pathogenic protozoan Entamoeba histolytica. A database search identified three putative Tetrahymena GPCRs, based on sequence homologies and predicted transmembrane domains, that were the focus of this study. Salmonella engulfment by Tetrahymena was assessed in the presence of suramin, a non-specific GPCR inhibitor. Salmonella engulfment was also assessed in Tetrahymena in which expression of putative GPCRs was knocked-down using RNAi. A candidate GPCR was then expressed in a heterologous yeast expression system for further characterization. Our results revealed that Tetrahymena were less efficient at engulfing Salmonella in the presence of suramin. Engulfment was reduced concordantly with a reduction in the density of protozoa. RNAi-based studies revealed that knock-down of one the Tetrahymena GPCRs caused diminished engulfment of Salmonella. Tetrahymena lysates activated this receptor in the heterologous expression system. These data demonstrate that the Tetrahymena receptor is a putative GPCR that facilitates bacterial engulfment by Tetrahymena. Activation of the putative GPCR seemed to be related to protozoan cell density, suggesting that its cognate ligand is an intercellular signaling molecule.

  17. G-protein coupling of cannabinoid receptors

    International Nuclear Information System (INIS)

    Glass, M.

    2001-01-01

    Full text: Since the cloning of the cannabinoid CB1 and CB2 receptors in the early 1990's extensive research has focused on understanding their signal transduction pathways. While it has been known for sometime that both receptors can couple to intracellular signalling via pertussis toxin sensitive G-proteins (Gi/Go), the specificity and kinetics of these interactions have only recently been elucidated. We have developed an in situ reconstitution approach to investigating receptor-G-protein interactions. This approach involves chaotropic extraction of receptor containing membranes in order to inactivate or remove endogenous G-proteins. Recombinant or isolated brain G-proteins can then be added back to the receptors, and their activation monitored through the binding of [ 35 S]-GTPγS. This technique has been utilised for an extensive study of cannabinoid receptor mediated activation of G-proteins. In these studies we have established that CB1 couples with high affinity to both Gi and Go type G-proteins. In contrast, CB2 couples strongly to Gi, but has a very low affinity for Go. This finding correlated well with the previous findings that while CB1 and CB2 both couple to the inhibition of adenylate cyclase, CB1 but not CB2 could also inhibit calcium channels. We then examined the ability of a range of cannabinoid agonists to activate the Gi and Go via CB1. Conventional receptor theory suggests that a receptor is either active or inactive with regard to a G-protein and that the active receptor activates all relevant G-proteins equally. However, in this study we found that agonists could produce different degrees of activation, depending on which G-protein was present. Further studies have compared the ability of the two endocannabinoids to drive the activation of Gi or Go. These studies show that agonists can induce multiple forms of activated receptor that differ in their ability to catalyse the activation of Gi or Go. The ability of an agonist to drive a receptor

  18. Generation of Trichoderma atroviride mutants with constitutively activated G protein signaling by using geneticin resistance as selection marker

    Directory of Open Access Journals (Sweden)

    Gruber Sabine

    2012-11-01

    Full Text Available Abstract Background Species of the fungal genus Trichoderma are important industrial producers of cellulases and hemicellulases, but also widely used as biocontrol agents (BCAs in agriculture. In the latter function Trichoderma species stimulate plant growth, induce plant defense and directly antagonize plant pathogenic fungi through their mycoparasitic capabilities. The recent release of the genome sequences of four mycoparasitic Trichoderma species now forms the basis for large-scale genetic manipulations of these important BCAs. Thus far, only a limited number of dominant selection markers, including Hygromycin B resistance (hph and the acetamidase-encoding amdS gene, have been available for transformation of Trichoderma spp. For more extensive functional genomics studies the utilization of additional dominant markers will be essential. Results We established the Escherichia coli neomycin phosphotransferase II-encoding nptII gene as a novel selectable marker for the transformation of Trichoderma atroviride conferring geneticin resistance. The nptII marker cassette was stably integrated into the fungal genome and transformants exhibited unaltered phenotypes compared to the wild-type. Co-transformation of T. atroviride with nptII and a constitutively activated version of the Gα subunit-encoding tga3 gene (tga3Q207L resulted in a high number of mitotically stable, geneticin-resistant transformants. Further analyses revealed a co-transformation frequency of 68% with 15 transformants having additionally integrated tga3Q207L into their genome. Constitutive activation of the Tga3-mediated signaling pathway resulted in increased vegetative growth and an enhanced ability to antagonize plant pathogenic host fungi. Conclusion The neomycin phosphotransferase II-encoding nptII gene from Escherichia coli proved to be a valuable tool for conferring geneticin resistance to the filamentous fungus T. atroviride thereby contributing to an enhanced genetic

  19. Functional analysis of a regulator of G-protein signaling CgRGS1 in the rubber tree anthracnose fungus Colletotrichum gloeosporioides.

    Science.gov (United States)

    Liu, Zhi-Qiang; Wu, Man-Li; Ke, Zhi-Jian; Liu, Wen-Bo; Li, Xiao-Yu

    2018-04-01

    Colletotrichum gloeosporioides is the causal agent of rubber anthracnose, which is also one of the important biological factors threatening the development of natural rubber industry in the world. Regulators of G-protein signaling (RGS) are key negative regulators of G-proteins, which play important roles in growth, development and pathogenic processes of plant pathogens. In this study, a RGS gene CgRGS1 was functionally characterized in C. gloeosporioides. Compared to the wild type, the CgRGS1 deletion mutant had slow vegetative growth, reduced conidia with multi-end germination, low appressorium formation rate, high resistance to oxidative stress and SDS. Moreover, the mutant was sensitive to osmotic pressure and showed decreased virulence. In conclusion, CgRGS1 is involved in regulation of vegetative growth, conidiation, germination, appressorium formation, oxidative stress, osmotic pressure response and pathogenicity in C. gloeosporioides.

  20. Reflections on: "A general role for adaptations in G-Proteins and the cyclic AMP system in mediating the chronic actions of morphine and cocaine on neuronal function".

    Science.gov (United States)

    Nestler, Eric J

    2016-08-15

    In 1991 we demonstrated that chronic morphine exposure increased levels of adenylyl cyclase and protein kinase A (PKA) in several regions of the rat central nervous system as inferred from measures of enzyme activity in crude extracts (Terwilliger et al., 1991). These findings led us to hypothesize that a concerted upregulation of the cAMP pathway is a general mechanism of opiate tolerance and dependence. Moreover, in the same study we showed similar induction of adenylyl cyclase and PKA activity in nucleus accumbens (NAc) in response to chronic administration of cocaine, but not of several non-abused psychoactive drugs. Morphine and cocaine also induced equivalent changes in inhibitory G protein subunits in this brain region. We thus extended our hypothesis to suggest that, particularly within brain reward regions such as NAc, cAMP pathway upregulation represents a common mechanism of reward tolerance and dependence shared by several classes of drugs of abuse. Research since that time, by many laboratories, has provided substantial support for these hypotheses. Specifically, opiates in several CNS regions including NAc, and cocaine more selectively in NAc, induce expression of certain adenylyl cyclase isoforms and PKA subunits via the transcription factor, CREB, and these transcriptional adaptations serve a homeostatic function to oppose drug action. In certain brain regions, such as locus coeruleus, these adaptations mediate aspects of physical opiate dependence and withdrawal, whereas in NAc they mediate reward tolerance and dependence that drives increased drug self-administration. This work has had important implications for understanding the molecular basis of addiction. "A general role for adaptations in G-proteins and the cyclic AMP system in mediating the chronic actions of morphine and cocaine on neuronal function". Previous studies have shown that chronic morphine increases levels of the G-protein subunits Giα and Goα, adenylate cyclase, cyclic AMP

  1. Gene expression profiling reveals different molecular patterns in G-protein coupled receptor signaling pathways between early- and late-onset preeclampsia.

    Science.gov (United States)

    Liang, Mengmeng; Niu, Jianmin; Zhang, Liang; Deng, Hua; Ma, Jian; Zhou, Weiping; Duan, Dongmei; Zhou, Yuheng; Xu, Huikun; Chen, Longding

    2016-04-01

    Early-onset preeclampsia and late-onset preeclampsia have been regarded as two different phenotypes with heterogeneous manifestations; To gain insights into the pathogenesis of the two traits, we analyzed the gene expression profiles in preeclamptic placentas. A whole genome-wide microarray was used to determine the gene expression profiles in placental tissues from patients with early-onset (n = 7; 36 weeks) preeclampsia and their controls who delivered preterm (n = 5; 36 weeks). Genes were termed differentially expressed if they showed a fold-change ≥ 2 and q-value preeclampsia (177 genes were up-regulated and 450 were down-regulated). Gene ontology analysis identified significant alterations in several biological processes; the top two were immune response and cell surface receptor linked signal transduction. Among the cell surface receptor linked signal transduction-related, differentially expressed genes, those involved in the G-protein coupled receptor protein signaling pathway were significantly enriched. G-protein coupled receptor signaling pathway related genes, such as GPR124 and MRGPRF, were both found to be down-regulated in early-onset preeclampsia. The results were consistent with those of western blotting that the abundance of GPR124 was lower in early-onset compared with late-onset preeclampsia. The different gene expression profiles reflect the different levels of transcription regulation between the two conditions and supported the hypothesis that they are separate disease entities. Moreover, the G-protein coupled receptor signaling pathway related genes may contribute to the mechanism underlying early- and late-onset preeclampsia. Copyright © 2016 Elsevier Ltd. All rights reserved.

  2. A quantitative characterization of the yeast heterotrimeric G protein cycle

    Science.gov (United States)

    Yi, Tau-Mu; Kitano, Hiroaki; Simon, Melvin I.

    2003-01-01

    The yeast mating response is one of the best understood heterotrimeric G protein signaling pathways. Yet, most descriptions of this system have been qualitative. We have quantitatively characterized the heterotrimeric G protein cycle in yeast based on direct in vivo measurements. We used fluorescence resonance energy transfer to monitor the association state of cyan fluorescent protein (CFP)-Gα and Gβγ-yellow fluorescent protein (YFP), and we found that receptor-mediated G protein activation produced a loss of fluorescence resonance energy transfer. Quantitative time course and dose–response data were obtained for both wild-type and mutant cells possessing an altered pheromone response. These results paint a quantitative portrait of how regulators such as Sst2p and the C-terminal tail of α-factor receptor modulate the kinetics and sensitivity of G protein signaling. We have explored critical features of the dynamics including the rapid rise and subsequent decline of active G proteins during the early response, and the relationship between the G protein activation dose–response curve and the downstream dose–response curves for cell-cycle arrest and transcriptional induction. Fitting the data to a mathematical model produced estimates of the in vivo rates of heterotrimeric G protein activation and deactivation in yeast. PMID:12960402

  3. Mas-related G protein coupled receptor-X2: A potential new target for modulating mast cell-mediated allergic and inflammatory diseases.

    Science.gov (United States)

    Ali, Hydar

    2016-12-01

    Mast cells (MCs) are tissue resident immune cells that are best known for their roles in allergic and inflammatory diseases. In addition to the high affinity IgE receptor (FcεRI), MCs express numerous G protein coupled receptors (GPCRs), which are the most common targets of drug therapy. Neurokinin 1 receptor (NK-1R) is expressed on MCs and contributes to IgE and non-IgE-mediated responses in mice. Although NK-1R antagonists are highly effective in modulating experimental allergic and inflammatory responses in mice they lack efficacy in humans. This article reviews recent findings that demonstrate that while neuropeptides (NPs) activate murine MCs via NK-1R and Mas related G protein coupled receptor B2 (MrgprB2), they activate human MCs via Mas-related G protein coupled receptor X2 (MRGPRX2). Interestingly, conventional NK-1R antagonists have off-target activity against mouse MrgprB2 but not human MRGPRX2. These findings suggest that the failure to translate studies with NK-1R antagonists from in vivo mouse studies to the clinic likely reflects their lack of effect on human MRGPRX2. A unique feature of MRGPRX2 that distinguishes it from other GPCRs is that it is activated by a diverse group of ligands that include; neuropeptides, cysteine proteases, antimicrobial peptides and cationic proteins released from activated eosinophils. Thus, the development of small molecule MRGPRX2-specific antagonists or neutralizing antibodies may provide new targets for the treatment of MC-mediated allergic and inflammatory diseases.

  4. G-protein mediated signaling pathways in myogenic responsiveness of mouse mesenteric artery

    DEFF Research Database (Denmark)

    Jensen, Lars Jørn; Joseph, Philomeena Daphne; Haanes, Kristian Agmund

    2015-01-01

    of PLA2 (AACOCF3, 5 µM), DAG lipase (RHC80267, 20 µM), PI3-kinase (wortmannin, 0.03 µM), CYP4A (HET0016, 10 µM), and TRPC channels (SKF96365, 10 µM) had no effects. Gq/11 and G12 mRNA and protein were expressed in MA. The Gα/q inhibitor YM-254890 (0.1 µM) and the AT1-R blocker valsartan (0.3 µ...

  5. Dopamine D2 receptor-mediated G-protein activation in rat striatum: functional autoradiography and influence of unilateral 6-hydroxydopamine lesions of the substantia nigra.

    Science.gov (United States)

    Newman-Tancredi, A; Cussac, D; Brocco, M; Rivet, J M; Chaput, C; Touzard, M; Pasteau, V; Millan, M J

    2001-11-30

    , the present data indicate that, in rat striatum, the actions of quinelorane are mediated primarily by D2 receptors, and suggest that behavioural hypersensitivity to this agonist, induced by unilateral SNPC lesions, is associated with an increase in D2, but not D3 or D4, receptor-mediated G-protein activation.

  6. Shuttling of G protein subunits between the plasma membrane and intracellular membranes.

    Science.gov (United States)

    Chisari, Mariangela; Saini, Deepak Kumar; Kalyanaraman, Vani; Gautam, Narasimhan

    2007-08-17

    Heterotrimeric G proteins (alphabetagamma) mediate the majority of signaling pathways in mammalian cells. It is long held that G protein function is localized to the plasma membrane. Here we examined the spatiotemporal dynamics of G protein localization using fluorescence recovery after photobleaching, fluorescence loss in photobleaching, and a photoswitchable fluorescent protein, Dronpa. Unexpectedly, G protein subunits shuttle rapidly (t1/2 plasma membrane and intracellular membranes. We show that consistent with such shuttling, G proteins constitutively reside in endomembranes. Furthermore, we show that shuttling is inhibited by 2-bromopalmitate. Thus, contrary to present thought, G proteins do not reside permanently on the plasma membrane but are constantly testing the cytoplasmic surfaces of the plasma membrane and endomembranes to maintain G protein pools in intracellular membranes to establish direct communication between receptors and endomembranes.

  7. G-protein-mediated interconversions of cell-surface cAMP receptors and their involvement in excitation and desensitization of guanylate cyclase in Dictyostelium discoideum

    International Nuclear Information System (INIS)

    van Haastert, P.J.; de Wit, R.J.; Janssens, P.M.; Kesbeke, F.; DeGoede, J.

    1986-01-01

    In Dictyostelium discoideum cells, extracellular cAMP induces the rapid (within 2 s) activation of guanylate cyclase, which is followed by complete desensitization after about 10 s. cAMP binding to these cells is heterogeneous, showing a subclass of fast dissociating sites coupled to adenylate cyclase (A-sites) and a subclass of slowly dissociating sites coupled to guanylate cyclase (B-sites). The kinetics of the B-sites were further investigated on a seconds time scale. Statistical analysis of the association of [ 3 H]cAMP to the B-sites and dissociation of the complex revealed that the receptor can exist in three states which interconvert according to the following scheme. cAMP binds to the BF-state (off-rate 2.5 s) which rapidly (t1/2 = 3 s) converts to the BS-state (off-rate 15 s) and subsequently (without a detectable delay) into the BSS-state (off-rate 150 s). In membranes, both the BS- and BSS-states are converted to the BF-state by GTP and GDP, suggesting the involvement of a G-protein. Densensitized cells show a 80% reduction of the formation of the BSS-state, but no reduction of the BF- or BS-state. These data are combined into a model in which the transitions of the B-sites are mediated by a G-protein; activation of the G-protein and guanylate cyclase is associated with the transition of the BS- to the BSS-state of the receptor, whereas desensitization is associated with the inhibition of this transition

  8. Mutations in the VLGR1 Gene Implicate G-Protein Signaling in the Pathogenesis of Usher Syndrome Type II

    Science.gov (United States)

    Weston, Michael D.; Luijendijk, Mirjam W. J.; Humphrey, Kurt D.; Möller, Claes; Kimberling, William J.

    2004-01-01

    Usher syndrome type II (USH2) is a genetically heterogeneous autosomal recessive disorder with at least three genetic subtypes (USH2A, USH2B, and USH2C) and is classified phenotypically as congenital hearing loss and progressive retinitis pigmentosa. The VLGR1 (MASS1) gene in the 5q14.3-q21.1 USH2C locus was considered a likely candidate on the basis of its protein motif structure and expressed-sequence-tag representation from both cochlear and retinal subtracted libraries. Denaturing high-performance liquid chromatography and direct sequencing of polymerase-chain-reaction products amplified from 10 genetically independent patients with USH2C and 156 other patients with USH2 identified four isoform-specific VLGR1 mutations (Q2301X, I2906FS, M2931FS, and T6244X) from three families with USH2C, as well as two sporadic cases. All patients with VLGR1 mutations are female, a significant deviation from random expectations. The ligand(s) for the VLGR1 protein is unknown, but on the basis of its potential extracellular and intracellular protein-protein interaction domains and its wide mRNA expression profile, it is probable that VLGR1 serves diverse cellular and signaling processes. VLGR1 mutations have been previously identified in both humans and mice and are associated with a reflex-seizure phenotype in both species. The identification of additional VLGR1 mutations to test whether a phenotype/genotype correlation exists, akin to that shown for other Usher syndrome disease genes, is warranted. PMID:14740321

  9. International Union of Basic and Clinical Pharmacology Review: WNT/Frizzled signalling: receptor–ligand selectivity with focus on FZD-G protein signalling and its physiological relevance: IUPHAR Review 3

    Science.gov (United States)

    Dijksterhuis, J P; Petersen, J; Schulte, G

    2014-01-01

    The wingless/int1 (WNT)/Frizzled (FZD) signalling pathway controls numerous cellular processes such as proliferation, differentiation, cell-fate decisions, migration and plays a crucial role during embryonic development. Nineteen mammalian WNTs can bind to 10 FZDs thereby activating different downstream pathways such as WNT/β-catenin, WNT/planar cell polarity and WNT/Ca2+. However, the mechanisms of signalling specification and the involvement of heterotrimeric G proteins are still unclear. Disturbances in the pathways can lead to various diseases ranging from cancer, inflammatory diseases to metabolic and neurological disorders. Due to the presence of seven-transmembrane segments, evidence for coupling between FZDs and G proteins and substantial structural differences in class A, B or C GPCRs, FZDs were grouped separately in the IUPHAR GPCR database as the class FZD within the superfamily of GPCRs. Recently, important progress has been made pointing to a direct activation of G proteins after WNT stimulation. WNT/FZD and G protein coupling remain to be fully explored, although the basic observation supporting the nature of FZDs as GPCRs is compelling. Because the involvement of different (i) WNTs; (ii) FZDs; and (iii) intracellular binding partners could selectively affect signalling specification, in this review we present the current understanding of receptor/ligand selectivity of FZDs and WNTs. We pinpoint what is known about signalling specification and the physiological relevance of these interactions with special emphasis on FZD–G protein interactions. LINKED ARTICLESThis article is part of a themed section on Molecular Pharmacology of GPCRs. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2014.171.issue-5 PMID:24032637

  10. Diacylglycerol kinase α mediates 17-β-estradiol-induced proliferation, motility, and anchorage-independent growth of Hec-1A endometrial cancer cell line through the G protein-coupled estrogen receptor GPR30.

    Science.gov (United States)

    Filigheddu, Nicoletta; Sampietro, Sara; Chianale, Federica; Porporato, Paolo E; Gaggianesi, Miriam; Gregnanin, Ilaria; Rainero, Elena; Ferrara, Michele; Perego, Beatrice; Riboni, Francesca; Baldanzi, Gianluca; Graziani, Andrea; Surico, Nicola

    2011-12-01

    Increased levels of endogenous and/or exogenous estrogens are one of the well known risk factors of endometrial cancer. Diacylglycerol kinases (DGKs) are a family of enzymes which phosphorylate diacylglycerol (DAG) to produce phosphatidic acid (PA), thus turning off and on DAG-mediated and PA-mediated signaling pathways, respectively. DGK α activity is stimulated by growth factors and oncogenes and is required for chemotactic, proliferative, and angiogenic signaling in vitro. Herein, using either specific siRNAs or the pharmacological inhibitor R59949, we demonstrate that DGK α activity is required for 17-β-estradiol (E2)-induced proliferation, motility, and anchorage-independent growth of Hec-1A endometrial cancer cell line. Impairment of DGK α activity also influences basal cell proliferation and growth in soft agar of Hec-1A, while it has no effects on basal cell motility. Moreover, we show that DGK α activity induced by E2, as well as its observed effects, are mediated by the G protein-coupled estrogen receptor GPR30 (GPER). These findings suggest that DGK α may be a potential target in endometrial cancer therapy. Copyright © 2011 Elsevier Inc. All rights reserved.

  11. RGS6, but not RGS4, is the dominant regulator of G protein signaling (RGS) modulator of the parasympathetic regulation of mouse heart rate.

    Science.gov (United States)

    Wydeven, Nicole; Posokhova, Ekaterina; Xia, Zhilian; Martemyanov, Kirill A; Wickman, Kevin

    2014-01-24

    Parasympathetic activity decreases heart rate (HR) by inhibiting pacemaker cells in the sinoatrial node (SAN). Dysregulation of parasympathetic influence has been linked to sinus node dysfunction and arrhythmia. RGS (regulator of G protein signaling) proteins are negative modulators of the parasympathetic regulation of HR and the prototypical M2 muscarinic receptor (M2R)-dependent signaling pathway in the SAN that involves the muscarinic-gated atrial K(+) channel IKACh. Both RGS4 and RGS6-Gβ5 have been implicated in these processes. Here, we used Rgs4(-/-), Rgs6(-/-), and Rgs4(-/-):Rgs6(-/-) mice to compare the relative influence of RGS4 and RGS6 on parasympathetic regulation of HR and M2R-IKACh-dependent signaling in the SAN. In retrogradely perfused hearts, ablation of RGS6, but not RGS4, correlated with decreased resting HR, increased heart rate variability, and enhanced sensitivity to the negative chronotropic effects of the muscarinic agonist carbachol. Similarly, loss of RGS6, but not RGS4, correlated with enhanced sensitivity of the M2R-IKACh signaling pathway in SAN cells to carbachol and a significant slowing of M2R-IKACh deactivation rate. Surprisingly, concurrent genetic ablation of RGS4 partially rescued some deficits observed in Rgs6(-/-) mice. These findings, together with those from an acute pharmacologic approach in SAN cells from Rgs6(-/-) and Gβ5(-/-) mice, suggest that the partial rescue of phenotypes in Rgs4(-/-):Rgs6(-/-) mice is attributable to another R7 RGS protein whose influence on M2R-IKACh signaling is masked by RGS4. Thus, RGS6-Gβ5, but not RGS4, is the primary RGS modulator of parasympathetic HR regulation and SAN M2R-IKACh signaling in mice.

  12. Cell adhesion controlled by adhesion G protein-coupled receptor GPR124/ADGRA2 is mediated by a protein complex comprising intersectins and Elmo-Dock.

    Science.gov (United States)

    Hernández-Vásquez, Magda Nohemí; Adame-García, Sendi Rafael; Hamoud, Noumeira; Chidiac, Rony; Reyes-Cruz, Guadalupe; Gratton, Jean Philippe; Côté, Jean-François; Vázquez-Prado, José

    2017-07-21

    Developmental angiogenesis and the maintenance of the blood-brain barrier involve endothelial cell adhesion, which is linked to cytoskeletal dynamics. GPR124 (also known as TEM5/ADGRA2) is an adhesion G protein-coupled receptor family member that plays a pivotal role in brain angiogenesis and in ensuring a tight blood-brain barrier. However, the signaling properties of GPR124 remain poorly defined. Here, we show that ectopic expression of GPR124 promotes cell adhesion, additive to extracellular matrix-dependent effect, coupled with filopodia and lamellipodia formation and an enrichment of a pool of the G protein-coupled receptor at actin-rich cellular protrusions containing VASP, a filopodial marker. Accordingly, GPR124-expressing cells also displayed increased activation of both Rac and Cdc42 GTPases. Mechanistically, we uncover novel direct interactions between endogenous GPR124 and the Rho guanine nucleotide exchange factors Elmo/Dock and intersectin (ITSN). Small fragments of either Elmo or ITSN1 that bind GPR124 blocked GPR124-induced cell adhesion. In addition, Gβγ interacts with the C-terminal tail of GPR124 and promotes the formation of a GPR124-Elmo complex. Furthermore, GPR124 also promotes the activation of the Elmo-Dock complex, as measured by Elmo phosphorylation on a conserved C-terminal tyrosine residue. Interestingly, Elmo and ITSN1 also interact with each other independently of their GPR124-recognition regions. Moreover, endogenous phospho-Elmo and ITSN1 co-localize with GPR124 at lamellipodia of adhering endothelial cells, where GPR124 expression contributes to polarity acquisition during wound healing. Collectively, our results indicate that GPR124 promotes cell adhesion via Elmo-Dock and ITSN. This constitutes a previously unrecognized complex formed of atypical and conventional Rho guanine nucleotide exchange factors for Rac and Cdc42 that is putatively involved in GPR124-dependent angiogenic responses. © 2017 by The American Society for

  13. Plant G-Proteins Come of Age: Breaking the Bond with Animal Models.

    Science.gov (United States)

    Trusov, Yuri; Botella, José R

    2016-01-01

    G-proteins are universal signal transducers mediating many cellular responses. Plant G-protein signaling has been modeled on the well-established animal paradigm but accumulated experimental evidence indicates that G-protein-dependent signaling in plants has taken a very different evolutionary path. Here we review the differences between plant and animal G-proteins reported over past two decades. Most importantly, while in animal systems the G-protein signaling cycle is activated by seven transmembrane-spanning G-protein coupled receptors, the existence of these type of receptors in plants is highly controversial. Instead plant G-proteins have been proven to be functionally associated with atypical receptors such as the Arabidopsis RGS1 and a number of receptor-like kinases. We propose that, instead of the GTP/GDP cycle used in animals, plant G-proteins are activated/de-activated by phosphorylation/de-phosphorylation. We discuss the need of a fresh new look at these signaling molecules and provide a hypothetical model that departs from the accepted animal paradigm.

  14. Arabidopsis N-MYC DOWNREGULATED-LIKE1, a positive regulator of auxin transport in a G protein-mediated pathway.

    Science.gov (United States)

    Mudgil, Yashwanti; Uhrig, Joachm F; Zhou, Jiping; Temple, Brenda; Jiang, Kun; Jones, Alan M

    2009-11-01

    Root architecture results from coordinated cell division and expansion in spatially distinct cells of the root and is established and maintained by gradients of auxin and nutrients such as sugars. Auxin is transported acropetally through the root within the central stele and then, upon reaching the root apex, auxin is transported basipetally through the outer cortical and epidermal cells. The two Gbetagamma dimers of the Arabidopsis thaliana heterotrimeric G protein complex are differentially localized to the central and cortical tissues of the Arabidopsis roots. A null mutation in either the single beta (AGB1) or the two gamma (AGG1 and AGG2) subunits confers phenotypes that disrupt the proper architecture of Arabidopsis roots and are consistent with altered auxin transport. Here, we describe an evolutionarily conserved interaction between AGB1/AGG dimers and a protein designated N-MYC DOWNREGULATED-LIKE1 (NDL1). The Arabidopsis genome encodes two homologs of NDL1 (NDL2 and NDL3), which also interact with AGB1/AGG1 and AGB1/AGG2 dimers. We show that NDL proteins act in a signaling pathway that modulates root auxin transport and auxin gradients in part by affecting the levels of at least two auxin transport facilitators. Reduction of NDL family gene expression and overexpression of NDL1 alter root architecture, auxin transport, and auxin maxima. AGB1, auxin, and sugars are required for NDL1 protein stability in regions of the root where auxin gradients are established; thus, the signaling mechanism contains feedback loops.

  15. Small G proteins in insulin action: Rab and Rho families at the crossroads of signal transduction and GLUT4 vesicle traffic.

    Science.gov (United States)

    Ishikura, S; Koshkina, A; Klip, A

    2008-01-01

    Insulin stimulates glucose uptake into muscle and adipose tissues through glucose transporter 4 (GLUT4). GLUT4 cycles between the intracellular compartments and the plasma membrane. GLUT4 traffic-regulating insulin signals are largely within the insulin receptor-insulin receptor substrate-phosphatidylinositol 3-kinase (IR-IRS-PI3K) axis. In muscle cells, insulin signal bifurcates downstream of the PI3K into one arm leading to the activation of the Ser/Thr kinases Akt and atypical protein kinase C, and another leading to the activation of Rho family protein Rac1 leading to actin remodelling. Activated Akt inactivates AS160, a GTPase-activating protein for Rab family small G proteins. Here we review the roles of Rab and Rho proteins, particularly Rab substrates of AS160 and Rac1, in insulin-stimulated GLUT4 traffic. We discuss: (1) how distinct steps in GLUT4 traffic may be regulated by discrete Rab proteins, and (2) the importance of Rac1 activation in insulin-induced actin remodelling in muscle cells, a key element for the net gain in surface GLUT4.

  16. Stalk-dependent and Stalk-independent Signaling by the Adhesion G Protein-coupled Receptors GPR56 (ADGRG1) and BAI1 (ADGRB1).

    Science.gov (United States)

    Kishore, Ayush; Purcell, Ryan H; Nassiri-Toosi, Zahra; Hall, Randy A

    2016-02-12

    The adhesion G protein-coupled receptors (aGPCRs) are a large yet poorly understood family of seven-transmembrane proteins. A defining characteristic of the aGPCR family is the conserved GAIN domain, which has autoproteolytic activity and can cleave the receptors near the first transmembrane domain. Several aGPCRs, including ADGRB1 (BAI1 or B1) and ADGRG1 (GPR56 or G1), have been found to exhibit significantly increased constitutive activity when truncated to mimic GAIN domain cleavage (ΔNT). Recent reports have suggested that the new N-terminal stalk, which is revealed by GAIN domain cleavage, can directly activate aGPCRs as a tethered agonist. We tested this hypothesis in studies on two distinct aGPCRs, B1 and G1, by engineering mutant receptors lacking the entire NT including the stalk (B1- and G1-SL, with "SL" indicating "stalkless"). These receptors were evaluated in a battery of signaling assays and compared with full-length wild-type and cleavage-mimicking (ΔNT) forms of the two receptors. We found that B1-SL, in multiple assays, exhibited robust signaling activity, suggesting that the membrane-proximal stalk region is not necessary for its activation. For G1, however, the results were mixed, with the SL mutant exhibiting robust activity in several signaling assays (including TGFα shedding, activation of NFAT luciferase, and β-arrestin recruitment) but reduced activity relative to ΔNT in a distinct assay (activation of SRF luciferase). These data support a model in which the activation of certain pathways downstream of aGPCRs is stalk-dependent, whereas signaling to other pathways is stalk-independent. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  17. Emerging Roles for MAS-Related G Protein-Coupled Receptor-X2 in Host Defense Peptide, Opioid, and Neuropeptide-Mediated Inflammatory Reactions.

    Science.gov (United States)

    Ali, Hydar

    2017-01-01

    Mast cells (MCs) are tissue-resident immune cells that contribute to host defense but are best known for their roles in allergic and inflammatory diseases. In humans, MCs are divided into two subtypes based on the protease content of their secretory granules. Thus, human lung MCs contain only tryptase and are known as MC T , whereas skin MCs contain both tryptase and chymase and are known as MC TC . Patients with severe asthma display elevated MCs in the lung, which undergo phenotypic change from MC T to MC TC . Although the human genome contains four Mas related G protein coupled receptor X (MRGPRX) genes, an important feature of MC TC is that they selectively express MRGPRX2. It is activated by antimicrobial host defense peptides such as human β-defensins and the cathelicidin LL-37 and likely contributes to host defense. MRGPRX2 is also a receptor for the neuropeptide substance P, major basic protein, eosinophil peroxidase, opioids, and many FDA-approved cationic drugs. Increased expression of MRGPRX2 or enhanced downstream signaling likely contributes to chronic inflammatory diseases such as rosacea, atopic dermatitis, chronic urticaria, and severe asthma. In this chapter, I will discuss the expression profile and function of MRGPRX1-4 and review the emerging roles of MRGPRX2 on host defense, chronic inflammatory diseases, and drug-induced pseudoallergic reactions. I will also examine the novel aspects of MRGPRX2 signaling in MCs as it related to degranulation and review the mechanisms of its regulation. © 2017 Elsevier Inc. All rights reserved.

  18. Detection of Side Chain Rearrangements Mediating the Motions of Transmembrane Helices in Molecular Dynamics Simulations of G Protein-Coupled Receptors

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    Zied Gaieb

    Full Text Available Structure and dynamics are essential elements of protein function. Protein structure is constantly fluctuating and undergoing conformational changes, which are captured by molecular dynamics (MD simulations. We introduce a computational framework that provides a compact representation of the dynamic conformational space of biomolecular simulations. This method presents a systematic approach designed to reduce the large MD simulation spatiotemporal datasets into a manageable set in order to guide our understanding of how protein mechanics emerge from side chain organization and dynamic reorganization. We focus on the detection of side chain interactions that undergo rearrangements mediating global domain motions and vice versa. Side chain rearrangements are extracted from side chain interactions that undergo well-defined abrupt and persistent changes in distance time series using Gaussian mixture models, whereas global domain motions are detected using dynamic cross-correlation. Both side chain rearrangements and global domain motions represent the dynamic components of the protein MD simulation, and are both mapped into a network where they are connected based on their degree of coupling. This method allows for the study of allosteric communication in proteins by mapping out the protein dynamics into an intramolecular network to reduce the large simulation data into a manageable set of communities composed of coupled side chain rearrangements and global domain motions. This computational framework is suitable for the study of tightly packed proteins, such as G protein-coupled receptors, and we present an application on a seven microseconds MD trajectory of CC chemokine receptor 7 (CCR7 bound to its ligand CCL21. Keywords: Molecular dynamics, Change-point detection, Side chain reorganization, Helical domain motion, Intramolecular network, Membrane proteins, GPCR, GPCR computational modeling, GPCR allostery

  19. Differential Regulation of Mas-Related G Protein-Coupled Receptor X2-Mediated Mast Cell Degranulation by Antimicrobial Host Defense Peptides and Porphyromonas gingivalis Lipopolysaccharide.

    Science.gov (United States)

    Gupta, Kshitij; Idahosa, Chizobam; Roy, Saptarshi; Lee, Donguk; Subramanian, Hariharan; Dhingra, Anuradha; Boesze-Battaglia, Kathleen; Korostoff, Jonathan; Ali, Hydar

    2017-10-01

    Porphyromonas gingivalis is a keystone pathogen that contributes to periodontal pathogenesis by disrupting host-microbe homeostasis and promoting dysbiosis. The virulence of P. gingivalis likely reflects an alteration in the lipid A composition of its lipopolysaccharide (LPS) from the penta-acylated ( Pg LPS 1690 ) to the tetra-acylated ( Pg LPS 1435/1449 ) form. Mast cells play an important role in periodontitis, but the mechanisms of their activation and regulation remain unknown. The expression of epithelium- and neutrophil-derived host defense peptides (HDPs) (LL-37 and human β-defensin-3), which activate mast cells via Mas-related G protein-coupled receptor X2 (MRGPRX2), is increased in periodontitis. We found that MRGPRX2-expressing mast cells are present in normal gingiva and that their numbers are elevated in patients with chronic periodontitis. Furthermore, HDPs stimulated degranulation in a human mast cell line (LAD2) and in RBL-2H3 cells stably expressing MRGPRX2 (RBL-MRGPRX2). Pg LPS 1690 caused substantial inhibition of HDP-induced mast cell degranulation, but Pg LPS 1435/1449 had no effect. A fluorescently labeled HDP (FAM-LL-37) bound to RBL-MRGPRX2 cells, and Pg LPS 1690 inhibited this binding, but Pg LPS 1435/1449 had no effect. These findings suggest that low-level inflammation induced by HDP/MRGPRX2-mediated mast cell degranulation contributes to gingival homeostasis but that sustained inflammation due to elevated levels of both HDPs and MRGPRX2-expressing mast cells promotes periodontal disease. Furthermore, differential regulation of HDP-induced mast cell degranulation by Pg LPS 1690 and Pg LPS 1435/1449 may contribute to the modulation of disease progression. Copyright © 2017 American Society for Microbiology.

  20. G Protein-Coupled Receptors in Cancer

    Directory of Open Access Journals (Sweden)

    Rachel Bar-Shavit

    2016-08-01

    Full Text Available Despite the fact that G protein-coupled receptors (GPCRs are the largest signal-conveying receptor family and mediate many physiological processes, their role in tumor biology is underappreciated. Numerous lines of evidence now associate GPCRs and their downstream signaling targets in cancer growth and development. Indeed, GPCRs control many features of tumorigenesis, including immune cell-mediated functions, proliferation, invasion and survival at the secondary site. Technological advances have further substantiated GPCR modifications in human tumors. Among these are point mutations, gene overexpression, GPCR silencing by promoter methylation and the number of gene copies. At this point, it is imperative to elucidate specific signaling pathways of “cancer driver” GPCRs. Emerging data on GPCR biology point to functional selectivity and “biased agonism”; hence, there is a diminishing enthusiasm for the concept of “one drug per GPCR target” and increasing interest in the identification of several drug options. Therefore, determining the appropriate context-dependent conformation of a functional GPCR as well as the contribution of GPCR alterations to cancer development remain significant challenges for the discovery of dominant cancer genes and the development of targeted therapeutics.

  1. Regulator of G Protein Signaling 7 (RGS7) Can Exist in a Homo-oligomeric Form That Is Regulated by Gαo and R7-binding Protein.

    Science.gov (United States)

    Tayou, Junior; Wang, Qiang; Jang, Geeng-Fu; Pronin, Alexey N; Orlandi, Cesare; Martemyanov, Kirill A; Crabb, John W; Slepak, Vladlen Z

    2016-04-22

    RGS (regulator of G protein signaling) proteins of the R7 subfamily (RGS6, -7, -9, and -11) are highly expressed in neurons where they regulate many physiological processes. R7 RGS proteins contain several distinct domains and form obligatory dimers with the atypical Gβ subunit, Gβ5 They also interact with other proteins such as R7-binding protein, R9-anchoring protein, and the orphan receptors GPR158 and GPR179. These interactions facilitate plasma membrane targeting and stability of R7 proteins and modulate their activity. Here, we investigated RGS7 complexes using in situ chemical cross-linking. We found that in mouse brain and transfected cells cross-linking causes formation of distinct RGS7 complexes. One of the products had the apparent molecular mass of ∼150 kDa on SDS-PAGE and did not contain Gβ5 Mass spectrometry analysis showed no other proteins to be present within the 150-kDa complex in the amount close to stoichiometric with RGS7. This finding suggested that RGS7 could form a homo-oligomer. Indeed, co-immunoprecipitation of differentially tagged RGS7 constructs, with or without chemical cross-linking, demonstrated RGS7 self-association. RGS7-RGS7 interaction required the DEP domain but not the RGS and DHEX domains or the Gβ5 subunit. Using transfected cells and knock-out mice, we demonstrated that R7-binding protein had a strong inhibitory effect on homo-oligomerization of RGS7. In contrast, our data indicated that GPR158 could bind to the RGS7 homo-oligomer without causing its dissociation. Co-expression of constitutively active Gαo prevented the RGS7-RGS7 interaction. These results reveal the existence of RGS protein homo-oligomers and show regulation of their assembly by R7 RGS-binding partners. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  2. Comparative proteomic analyses reveal that the regulators of G-protein signaling proteins regulate amino acid metabolism of the rice blast fungus Magnaporthe oryzae.

    Science.gov (United States)

    Zhang, Haifeng; Ma, Hongyu; Xie, Xin; Ji, Jun; Dong, Yanhan; Du, Yan; Tang, Wei; Zheng, Xiaobo; Wang, Ping; Zhang, Zhengguang

    2014-11-01

    The rice blast fungus Magnaporthe oryzae encodes eight regulators of G-protein (GTP-binding protein) signaling (RGS) proteins MoRgs1-MoRgs8 that orchestrate the growth, asexual/sexual production, appressorium differentiation, and pathogenicity. To address the mechanisms by which MoRgs proteins function, we conducted a 2DE proteome study and identified 82 differentially expressed proteins by comparing five ∆Morgs mutants with wild-type Guy11 strain. We found that the abundances of eight amino acid (AA) biosynthesis or degradation associated proteins were markedly altered in five ∆Morgs mutants, indicating one of the main collective roles for the MoRgs proteins is to influence AA metabolism. We showed that MoRgs proteins have distinct roles in AA metabolism and nutrient responses from growth assays. In addition, we characterized MoLys20 (Lys is lysine), a homocitrate synthase, whose abundance was significantly decreased in the ∆Morgs mutants. The ∆Molys20 mutant is auxotrophic for lys and exogenous lys could partially rescue its auxotrophic defects. Deletion of MoLYS20 resulted in defects in conidiation and infection, as well as pathogenicity on rice. Overall, our results indicate that one of the critical roles for MoRgs proteins is to regulate AA metabolism, and that MoLys20 may be directly or indirectly regulated by MoRgs and participated in lys biosynthesis, thereby affecting fungal development and pathogenicity. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  3. Evolutionary hierarchy of vertebrate-like heterotrimeric G protein families.

    Science.gov (United States)

    Krishnan, Arunkumar; Mustafa, Arshi; Almén, Markus Sällman; Fredriksson, Robert; Williams, Michael J; Schiöth, Helgi B

    2015-10-01

    Heterotrimeric G proteins perform a crucial role as molecular switches controlling various cellular responses mediated by G protein-coupled receptor (GPCR) signaling pathway. Recent data have shown that the vertebrate-like G protein families are found across metazoans and their closest unicellular relatives. However, an overall evolutionary hierarchy of vertebrate-like G proteins, including gene family annotations and in particular mapping individual gene gain/loss events across diverse holozoan lineages is still incomplete. Here, with more expanded invertebrate taxon sampling, we have reconstructed phylogenetic trees for each of the G protein classes/families and provide a robust classification and hierarchy of vertebrate-like heterotrimeric G proteins. Our results further extend the evidence that the common ancestor (CA) of holozoans had at least five ancestral Gα genes corresponding to all major vertebrate Gα classes and contain a total of eight genes including two Gβ and one Gγ. Our results also indicate that the GNAI/O-like gene likely duplicated in the last CA of metazoans to give rise to GNAI- and GNAO-like genes, which are conserved across invertebrates. Moreover, homologs of GNB1-4 paralogon- and GNB5 family-like genes are found in most metazoans and that the unicellular holozoans encode two ancestral Gβ genes. Similarly, most bilaterian invertebrates encode two Gγ genes which include a representative of the GNG gene cluster and a putative homolog of GNG13. Interestingly, our results also revealed key evolutionary events such as the Drosophila melanogaster eye specific Gβ subunit that is found conserved in most arthropods and several previously unidentified species specific expansions within Gαi/o, Gαs, Gαq, Gα12/13 classes and the GNB1-4 paralogon. Also, we provide an overall proposed evolutionary scenario on the expansions of all G protein families in vertebrate tetraploidizations. Our robust classification/hierarchy is essential to further

  4. The viral G protein-coupled receptor ORF74 unmasks phospholipase C signaling of the receptor tyrosine kinase IGF-1R.

    NARCIS (Netherlands)

    de Munnik, S.M.; van der Lee, R.; Velders, D.M.; van Offenbeek, J.; Smits-de Vries, L.; Leurs, R.; Smit, M.J.; Vischer, H.F.

    2016-01-01

    Kaposi's sarcoma-associated herpesvirus (KSHV) encodes the constitutively active G protein-coupled receptor ORF74, which is expressed on the surface of infected host cells and has been linked to the development of the angioproliferative tumor Kaposi's sarcoma. Furthermore, the insulin-like growth

  5. Estrogen and pure antiestrogen fulvestrant (ICI 182 780) augment cell–matrigel adhesion of MCF-7 breast cancer cells through a novel G protein coupled estrogen receptor (GPR30)-to-calpain signaling axis

    Energy Technology Data Exchange (ETDEWEB)

    Chen, Yan; Li, Zheng; He, Yan; Shang, Dandan; Pan, Jigang; Wang, Hongmei; Chen, Huamei; Zhu, Zhuxia [Department of Physiology/Cancer Research Group, Guiyang Medical University School of Basic Medicine, 9 Beijing Road, Guiyang 550004, Guizhou (China); Wan, Lei [Department of Pharmacology, Guiyang Medical University School of Basic Medicine, 9 Beijing Road, Guiyang 550004, Guizhou (China); Wang, Xudong, E-mail: xdwang@gmc.edu.cn [Department of Physiology/Cancer Research Group, Guiyang Medical University School of Basic Medicine, 9 Beijing Road, Guiyang 550004, Guizhou (China)

    2014-03-01

    Fulvestrant (ICI 182 780, ICI) has been used in treating patients with hormone-sensitive breast cancer, yet initial or acquired resistance to endocrine therapies frequently arises and, in particular, cancer recurs as metastasis. We demonstrate here that both 17-beta-estradiol (E2) and ICI enhance cell adhesion to matrigel in MCF-7 breast cancer cells, with increased autolysis of calpain 1 (large subunit) and proteolysis of focal adhesion kinase (FAK), indicating calpain activation. Additionally, either E2 or ICI induced down-regulation of estrogen receptor α without affecting G protein coupled estrogen receptor 30 (GPR30) expression. Interestingly, GPR30 agonist G1 triggered calpain 1 autolysis but not calpain 2, whereas ER agonist diethylstilbestrol caused no apparent calpain autolysis. Furthermore, the actions of E2 and ICI on calpain and cell adhesion were tremendously suppressed by G15, or knockdown of GPR30. E2 and ICI also induced phosphorylation of extracellular regulated protein kinases 1 and 2 (ERK1/2), and suppression of ERK1/2 phosphorylation by U0126 profoundly impeded calpain activation triggered by estrogenic and antiestrogenic stimulations indicating implication of ERK1/2 in the GPR30-mediated action. Lastly, the E2- or ICI-induced cell adhesion was dramatically impaired by calpain-specific inhibitors, ALLN or calpeptin, suggesting requirement of calpain in the GPR30-associated action. These data show that enhanced cell adhesion by E2 and ICI occurs via a novel GPR30-ERK1/2-calpain pathway. Our results indicate that targeting the GPR30 signaling may be a potential strategy to reduce metastasis and improve the efficacy of antiestrogens in treatment of advanced breast cancer. - Highlights: • Estrogen and ICI augment adhesion to matrigel with calpain activation in MCF-7 cells. • GPR30 mediates cell–matrigel adhesion and calpain activation via ERK1/2. • Calpain is required in the cell–matrigel adhesion induced by E2 and ICI.

  6. Estrogen and pure antiestrogen fulvestrant (ICI 182 780) augment cell–matrigel adhesion of MCF-7 breast cancer cells through a novel G protein coupled estrogen receptor (GPR30)-to-calpain signaling axis

    International Nuclear Information System (INIS)

    Chen, Yan; Li, Zheng; He, Yan; Shang, Dandan; Pan, Jigang; Wang, Hongmei; Chen, Huamei; Zhu, Zhuxia; Wan, Lei; Wang, Xudong

    2014-01-01

    Fulvestrant (ICI 182 780, ICI) has been used in treating patients with hormone-sensitive breast cancer, yet initial or acquired resistance to endocrine therapies frequently arises and, in particular, cancer recurs as metastasis. We demonstrate here that both 17-beta-estradiol (E2) and ICI enhance cell adhesion to matrigel in MCF-7 breast cancer cells, with increased autolysis of calpain 1 (large subunit) and proteolysis of focal adhesion kinase (FAK), indicating calpain activation. Additionally, either E2 or ICI induced down-regulation of estrogen receptor α without affecting G protein coupled estrogen receptor 30 (GPR30) expression. Interestingly, GPR30 agonist G1 triggered calpain 1 autolysis but not calpain 2, whereas ER agonist diethylstilbestrol caused no apparent calpain autolysis. Furthermore, the actions of E2 and ICI on calpain and cell adhesion were tremendously suppressed by G15, or knockdown of GPR30. E2 and ICI also induced phosphorylation of extracellular regulated protein kinases 1 and 2 (ERK1/2), and suppression of ERK1/2 phosphorylation by U0126 profoundly impeded calpain activation triggered by estrogenic and antiestrogenic stimulations indicating implication of ERK1/2 in the GPR30-mediated action. Lastly, the E2- or ICI-induced cell adhesion was dramatically impaired by calpain-specific inhibitors, ALLN or calpeptin, suggesting requirement of calpain in the GPR30-associated action. These data show that enhanced cell adhesion by E2 and ICI occurs via a novel GPR30-ERK1/2-calpain pathway. Our results indicate that targeting the GPR30 signaling may be a potential strategy to reduce metastasis and improve the efficacy of antiestrogens in treatment of advanced breast cancer. - Highlights: • Estrogen and ICI augment adhesion to matrigel with calpain activation in MCF-7 cells. • GPR30 mediates cell–matrigel adhesion and calpain activation via ERK1/2. • Calpain is required in the cell–matrigel adhesion induced by E2 and ICI

  7. Site-directed mutagenesis of the Arabidopsis heterotrimeric G protein β subunit suggests divergent mechanisms of effector activation between plant and animal G proteins.

    Science.gov (United States)

    Chakravorty, David; Trusov, Yuri; Botella, José Ramón

    2012-03-01

    Heterotrimeric G proteins are integral components of signal transduction in humans and other mammals and have been therefore extensively studied. However, while they are known to mediate many processes, much less is currently known about the effector pathways and molecular mechanisms used by these proteins to regulate effectors in plants. We designed a complementation strategy to study G protein signaling in Arabidopsis thaliana, particularly the mechanism of action of AGB1, the sole identified β subunit. We used biochemical and effector regulation data from human G protein studies to identify four potentially important residues for site-directed mutagenesis (T65, M111, D250 and W361 of AGB1). Each residue was individually mutated and the resulting mutated protein introduced in the agb1-2 mutant background under the control of the native AGB1 promoter. Interestingly, even though these mutations have been shown to have profound effects on effector signaling in humans, all the mutated subunits were able to restore thirteen of the fifteen Gβ-deficient phenotypes characterized in this study. Only one mutated protein, T65A was unable to complement the hypersensitivity to mannitol during germination observed in agb1 mutants; while only D250A failed to restore lateral root numbers in the agb1 mutant to wild-type levels. Our results suggest that the mechanisms used in mammalian G protein signaling are not well conserved in plant G protein signaling, and that either the effectors used by plant G proteins, or the mechanisms used to activate them, are at least partially divergent from the well-studied mammalian G proteins.

  8. A single extracellular amino acid in Free Fatty Acid Receptor 2 defines antagonist species selectivity and G protein selection bias

    DEFF Research Database (Denmark)

    Sergeev, Eugenia; Hansen, Anders Højgaard; Bolognini, Daniele

    2017-01-01

    selectivity and mutational swap studies confirmed this hypothesis. Extending these studies to agonist function indicated that although the lysine - arginine variation between human and mouse orthologs had limited effect on G protein-mediated signal transduction, removal of positive charge from this residue...... produced a signalling-biased variant of Free Fatty Acid Receptor 2 in which Gi-mediated signalling by both short chain fatty acids and synthetic agonists was maintained whilst there was marked loss of agonist potency for signalling via Gq/11 and G12/13 G proteins. A single residue at the extracellular face...

  9. Distinctive G Protein-Dependent Signaling by Protease-Activated Receptor 2 (PAR2 in Smooth Muscle: Feedback Inhibition of RhoA by cAMP-Independent PKA.

    Directory of Open Access Journals (Sweden)

    Wimolpak Sriwai

    Full Text Available We examined expression of protease-activated receptors 2 (PAR2 and characterized their signaling pathways in rabbit gastric muscle cells. The PAR2 activating peptide SLIGRL (PAR2-AP stimulated Gq, G13, Gi1, PI hydrolysis, and Rho kinase activity, and inhibited cAMP formation. Stimulation of PI hydrolysis was partly inhibited in cells expressing PAR2 siRNA, Gaq or Gai minigene and in cells treated with pertussis toxin, and augmented by expression of dominant negative regulator of G protein signaling (RGS4(N88S. Stimulation of Rho kinase activity was abolished by PAR-2 or Ga13 siRNA, and by Ga13 minigene. PAR2-AP induced a biphasic contraction; initial contraction was selectively blocked by the inhibitor of PI hydrolysis (U73122 or MLC kinase (ML-9, whereas sustained contraction was selectively blocked by the Rho kinase inhibitor (Y27632. PAR2-AP induced phosphorylation of MLC20, MYPT1 but not CPI-17. PAR2-AP also caused a decrease in the association of NF-kB and PKA catalytic subunit: the effect of PAR2-AP was blocked by PAR2 siRNA or phosphorylation-deficient RhoA (RhoA(S188A. PAR2-AP-induced degradation of IkBa and activation of NF-kB were abolished by the blockade of RhoA activity by Clostridium botulinum C3 exoenzyme suggesting RhoA-dependent activation of NF-kB. PAR2-AP-stimulated Rho kinase activity was significantly augmented by the inhibitors of PKA (myristoylated PKI, IKK2 (IKKIV or NF-kB (MG132, and in cells expressing dominant negative mutants of IKK (IKK(K44A, IkBa (IkBa (S32A/S36A or RhoA(S188A, suggesting feedback inhibition of Rho kinase activity via PKA derived from NF-kB pathway. PAR2-AP induced phosphorylation of RhoA and the phosphorylation was attenuated in cells expressing phosphorylation-deficient RhoA(S188A. Our results identified signaling pathways activated by PAR2 to mediate smooth muscle contraction and a novel pathway for feedback inhibition of PAR2-stimulated RhoA. The pathway involves activation of the NF-kB to

  10. The angiotensin type 1 receptor activates extracellular signal-regulated kinases 1 and 2 by G protein-dependent and -independent pathways in cardiac myocytes and langendorff-perfused hearts

    DEFF Research Database (Denmark)

    Aplin, Mark; Christensen, Gitte Lund; Schneider, Mikael

    2007-01-01

    The angiotensin II (AngII) type 1 receptor (AT(1)R) has been shown to activate extracellular signal-regulated kinases 1 and 2 (ERK1/2) through G proteins or G protein-independently through beta-arrestin2 in cellular expression systems. As activation mechanisms may greatly influence the biological...... effects of ERK1/2 activity, differential activation of the AT(1)R in its native cellular context could have important biological and pharmacological implications. To examine if AT(1)R activates ERK1/2 by G protein-independent mechanisms in the heart, we used the [Sar(1), Ile(4), Ile(8)]-AngII ([SII] Ang......II) analogue in native preparations of cardiac myocytes and beating hearts. We found that [SII] AngII does not activate G(q)-coupling, yet stimulates the beta-arrestin2-dependent ERK1/2. The G(q)-activated pool of ERK1/2 rapidly translocates to the nucleus, while the beta-arrestin2-scaffolded pool remains...

  11. Dynamic Coupling and Allosteric Networks in the α Subunit of Heterotrimeric G Proteins.

    Science.gov (United States)

    Yao, Xin-Qiu; Malik, Rabia U; Griggs, Nicholas W; Skjærven, Lars; Traynor, John R; Sivaramakrishnan, Sivaraj; Grant, Barry J

    2016-02-26

    G protein α subunits cycle between active and inactive conformations to regulate a multitude of intracellular signaling cascades. Important structural transitions occurring during this cycle have been characterized from extensive crystallographic studies. However, the link between observed conformations and the allosteric regulation of binding events at distal sites critical for signaling through G proteins remain unclear. Here we describe molecular dynamics simulations, bioinformatics analysis, and experimental mutagenesis that identifies residues involved in mediating the allosteric coupling of receptor, nucleotide, and helical domain interfaces of Gαi. Most notably, we predict and characterize novel allosteric decoupling mutants, which display enhanced helical domain opening, increased rates of nucleotide exchange, and constitutive activity in the absence of receptor activation. Collectively, our results provide a framework for explaining how binding events and mutations can alter internal dynamic couplings critical for G protein function. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  12. [Roles of G protein-coupled estrogen receptor in the male reproductive system].

    Science.gov (United States)

    Chen, Kai-hong; Zhang, Xian; Jiang, Xue-wu

    2016-02-01

    The G protein-coupled estrogen receptor (GPER), also known as G protein-coupled receptor 30 (GPR30), was identified in the recent years as a functional membrane receptor different from the classical nuclear estrogen receptors. This receptor is widely expressed in the cortex, cerebellum, hippocampus, heart, lung, liver, skeletal muscle, and the urogenital system. It is responsible for the mediation of nongenomic effects associated with estrogen and its derivatives, participating in the physiological activities of the body. The present study reviews the molecular structure, subcellular localization, signaling pathways, distribution, and function of GPER in the male reproductive system.

  13. Baicalein suppresses 17-β-estradiol-induced migration, adhesion and invasion of breast cancer cells via the G protein-coupled receptor 30 signaling pathway.

    Science.gov (United States)

    Shang, Dandan; Li, Zheng; Zhu, Zhuxia; Chen, Huamei; Zhao, Lujun; Wang, Xudong; Chen, Yan

    2015-04-01

    Flavonoids are structurally similar to steroid hormones, particularly estrogens, and therefore have been studied for their potential effects on hormone-dependent cancers. Baicalein is the primary flavonoid derived from the root of Scutellaria baicalensis Georgi. In the present study, we investigated the effects of baicalein on 17β-estradiol (E2)-induced migration, adhesion and invasion of MCF-7 and SK-BR-3 breast cancer cells. The results demonstrated that baicalein suppressed E2-stimulated wound-healing migration and cell‑Matrigel adhesion, and ameliorated E2-promoted invasion across a Matrigel-coated Transwell membrane. Furthermore, baicalein interfered with E2-induced novel G protein-coupled estrogen receptor (GPR30)-related signaling, including a decrease in tyrosine phosphorylation of epidermal growth factor receptor (EGFR) as well as phosphorylation of extracellular signal-regulated kinase (ERK) and serine/threonine kinase Akt, without affecting GPR30 expression. The results also showed that baicalein suppressed the expression of GPR30 target genes, cysteine-rich 61 (CYR61) and connective tissue growth factor (CTGF) induced by E2. Furthermore, baicalein prevented GPR30-related signaling activation and upregulation of CYR61 and CTGF mRNA levels induced by G1, a specific GPR 30 agonist. The results suggest that baicalein inhibits E2-induced migration, adhesion and invasion through interfering with GPR30 signaling pathway activation, which indicates that it may act as a therapeutic candidate for the treatment of GPR30-positive breast cancer metastasis.

  14. Tyrosine phosphorylation switching of a G protein.

    Science.gov (United States)

    Li, Bo; Tunc-Ozdemir, Meral; Urano, Daisuke; Jia, Haiyan; Werth, Emily G; Mowrey, David D; Hicks, Leslie M; Dokholyan, Nikolay V; Torres, Matthew P; Jones, Alan M

    2018-03-30

    Heterotrimeric G protein complexes are molecular switches relaying extracellular signals sensed by G protein-coupled receptors (GPCRs) to downstream targets in the cytoplasm, which effect cellular responses. In the plant heterotrimeric GTPase cycle, GTP hydrolysis, rather than nucleotide exchange, is the rate-limiting reaction and is accelerated by a receptor-like regulator of G signaling (RGS) protein. We hypothesized that posttranslational modification of the Gα subunit in the G protein complex regulates the RGS-dependent GTPase cycle. Our structural analyses identified an invariant phosphorylated tyrosine residue (Tyr 166 in the Arabidopsis Gα subunit AtGPA1) located in the intramolecular domain interface where nucleotide binding and hydrolysis occur. We also identified a receptor-like kinase that phosphorylates AtGPA1 in a Tyr 166 -dependent manner. Discrete molecular dynamics simulations predicted that phosphorylated Tyr 166 forms a salt bridge in this interface and potentially affects the RGS protein-accelerated GTPase cycle. Using a Tyr 166 phosphomimetic substitution, we found that the cognate RGS protein binds more tightly to the GDP-bound Gα substrate, consequently reducing its ability to accelerate GTPase activity. In conclusion, we propose that phosphorylation of Tyr 166 in AtGPA1 changes the binding pattern with AtRGS1 and thereby attenuates the steady-state rate of the GTPase cycle. We coin this newly identified mechanism "substrate phosphoswitching." © 2018 by The American Society for Biochemistry and Molecular Biology, Inc.

  15. Different protein kinase C isoenzymes mediate inhibition of cardiac rapidly activating delayed rectifier K+ current by different G-protein coupled receptors.

    Science.gov (United States)

    Liu, Xueli; Wang, Yuhong; Zhang, Hua; Shen, Li; Xu, Yanfang

    2017-12-01

    Elevated angiotensin II (Ang II) and sympathetic activity contributes to a high risk of ventricular arrhythmias in heart disease. The rapidly activating delayed rectifier K + current (I Kr ) carried by the hERG channels plays a critical role in cardiac repolarization, and decreased I Kr is involved in increased cardiac arrhythmogenicity. Stimulation of α 1A -adrenoreceptors or angiotensin II AT 1 receptors is known to inhibit I Kr via PKC. Here, we have identified the PKC isoenzymes mediating the inhibition of I Kr by activation of these two different GPCRs. The whole-cell patch-clamp technique was used to record I Kr in guinea pig cardiomyocytes and HEK293 cells co-transfected with hERG and α 1A -adrenoreceptor or AT 1 receptor genes. A broad spectrum PKC inhibitor Gö6983 (not inhibiting PKCε), a selective cPKC inhibitor Gö6976 and a PKCα-specific inhibitor peptide, blocked the inhibition of I Kr by the α 1A -adrenoreceptor agonist A61603. However, these inhibitors did not affect the reduction of I Kr by activation of AT 1 receptors, whereas the PKCε-selective inhibitor peptide did block the effect. The effects of angiotensin II and the PKCε activator peptide were inhibited in mutant hERG channels in which 17 of the 18 PKC phosphorylation sites were deleted, whereas a deletion of the N-terminus of the hERG channels selectively prevented the inhibition elicited by A61603 and the cPKC activator peptide. Our results indicated that inhibition of I Kr by activation of α 1A -adrenoreceptors or AT 1 receptors were mediated by PKCα and PKCε isoforms respectively, through different molecular mechanisms. © 2017 The British Pharmacological Society.

  16. Cyclosporin A inhibits nucleotide excision repair via downregulation of the xeroderma pigmentosum group A and G proteins, which is mediated by calcineurin inhibition.

    Science.gov (United States)

    Kuschal, Christiane; Thoms, Kai-Martin; Boeckmann, Lars; Laspe, Petra; Apel, Antje; Schön, Michael P; Emmert, Steffen

    2011-10-01

    Cyclosporin A (CsA) inhibits nucleotide excision repair (NER) in human cells, a process that contributes to the skin cancer proneness in organ transplant patients. We investigated the mechanisms of CsA-induced NER reduction by assessing all xeroderma pigmentosum (XP) genes (XPA-XPG). Western blot analyses revealed that XPA and XPG protein expression was reduced in normal human GM00637 fibroblasts exposed to 0.1 and 0.5 μm CsA. Interestingly, the CsA treatment reduced XPG, but not XPA, mRNA expression. Calcineurin knockdown in GM00637 fibroblasts using RNAi led to similar results suggesting that calcineurin-dependent signalling is involved in XPA and XPG protein regulation. CsA-induced reduction in NER could be complemented by the overexpression of either XPA or XPG protein. Likewise, XPA-deficient fibroblasts with stable overexpression of XPA (XP2OS-pCAH19WS) did not show the inhibitory effect of CsA on NER. In contrast, XPC-deficient fibroblasts overexpressing XPC showed CsA-reduced NER. Our data indicate that the CsA-induced inhibition of NER is a result of downregulation of XPA and XPG protein in a calcineurin-dependent manner. © 2011 John Wiley & Sons A/S.

  17. Toll-like receptor 3 signalling up-regulates expression of the HIV co-receptor G-protein coupled receptor 15 on human CD4+ T cells.

    Directory of Open Access Journals (Sweden)

    Miriam Kiene

    Full Text Available BACKGROUND: Many HIV-2 and SIV isolates, as well as some HIV-1 strains, can use the orphan 7-transmembrane receptor GPR15 as co-receptor for efficient entry into host cells. GPR15 is expressed on central memory and effector memory CD4(+ T cells in healthy individuals and a subset of these cells is susceptible to HIV-1 and SIV infection. However, it has not been determined whether GPR15 expression is altered in the context of HIV-1 infection. RESULTS: Here, we show that GPR15 expression in CD4(+ T cells is markedly up-regulated in some HIV-1 infected individuals compared to the rest of the infected patients and to healthy controls. Infection of the PM1 T cell line with primary HIV-1 isolates was found to up-regulate GPR15 expression on the infected cells, indicating that viral components can induce GPR15 expression. Up-regulation of GPR15 expression on CD4(+ T cells was induced by activation of Toll-like receptor 3 signalling via TIR-domain-containing adapter-inducing interferon-β (TRIF and was more prominent on gut-homing compared to lymph node-homing CD4(+ T cells. CONCLUSION: These results suggest that infection-induced up-regulation of GPR15 expression could increase susceptibility of CD4(+ T cells to HIV infection and target cell availability in the gut in some infected individuals.

  18. Inhibitory effects of two G protein-coupled receptor kinases on the cell surface expression and signaling of the human adrenomedullin receptor

    Energy Technology Data Exchange (ETDEWEB)

    Kuwasako, Kenji, E-mail: kuwasako@med.miyazaki-u.ac.jp [Frontier Science Research Center, University of Miyazaki, Miyazaki, 889-1692 (Japan); Sekiguchi, Toshio [Noto Marine Laboratory, Division of Marine Environmental Studies, Institute of Nature and Environmental Technology, Kanazawa University, Ishikawa, 927-0553 (Japan); Nagata, Sayaka [Division of Circulatory and Body Fluid Regulation, Faculty of Medicine, University of Miyazaki, Miyazaki, 889-1692 (Japan); Jiang, Danfeng; Hayashi, Hidetaka [Frontier Science Research Center, University of Miyazaki, Miyazaki, 889-1692 (Japan); Murakami, Manabu [Department of Pharmacology, Hirosaki University, Graduate School of Medicine, Hirosaki, 036-8562 (Japan); Hattori, Yuichi [Department of Molecular and Medical Pharmacology, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, Toyama, 930-0194 (Japan); Kitamura, Kazuo [Division of Circulatory and Body Fluid Regulation, Faculty of Medicine, University of Miyazaki, Miyazaki, 889-1692 (Japan); Kato, Johji [Frontier Science Research Center, University of Miyazaki, Miyazaki, 889-1692 (Japan)

    2016-02-19

    Receptor activity-modifying protein 2 (RAMP2) enables the calcitonin receptor-like receptor (CLR, a family B GPCR) to form the type 1 adrenomedullin receptor (AM{sub 1} receptor). Here, we investigated the effects of the five non-visual GPCR kinases (GRKs 2 through 6) on the cell surface expression of the human (h)AM{sub 1} receptor by cotransfecting each of these GRKs into HEK-293 cells that stably expressed hRAMP2. Flow cytometric analysis revealed that when coexpressed with GRK4 or GRK5, the cell surface expression of the AM{sub 1} receptor was markedly decreased prior to stimulation with AM, thereby attenuating both the specific [{sup 125}I]AM binding and AM-induced cAMP production. These inhibitory effects of both GRKs were abolished by the replacement of the cytoplasmic C-terminal tail (C-tail) of CLR with that of the calcitonin receptor (a family B GPCR) or β{sub 2}-adrenergic receptor (a family A GPCR). Among the sequentially truncated CLR C-tail mutants, those lacking the five residues 449–453 (Ser-Phe-Ser-Asn-Ser) abolished the inhibition of the cell surface expression of CLR via the overexpression of GRK4 or GRK5. Thus, we provided new insight into the function of GRKs in agonist-unstimulated GPCR trafficking using a recombinant AM{sub 1} receptor and further determined the region of the CLR C-tail responsible for this GRK function. - Highlights: • We discovered a novel function of GRKs in GPCR trafficking using human CLR/RAMP2. • GRKs 4 and 5 markedly inhibited the cell surface expression of human CLR/RAMP2. • Both GRKs exhibited highly significant receptor signaling inhibition. • Five residues of the C-terminal tail of CLR govern this function of GRKs.

  19. Inhibitory effects of two G protein-coupled receptor kinases on the cell surface expression and signaling of the human adrenomedullin receptor

    International Nuclear Information System (INIS)

    Kuwasako, Kenji; Sekiguchi, Toshio; Nagata, Sayaka; Jiang, Danfeng; Hayashi, Hidetaka; Murakami, Manabu; Hattori, Yuichi; Kitamura, Kazuo; Kato, Johji

    2016-01-01

    Receptor activity-modifying protein 2 (RAMP2) enables the calcitonin receptor-like receptor (CLR, a family B GPCR) to form the type 1 adrenomedullin receptor (AM_1 receptor). Here, we investigated the effects of the five non-visual GPCR kinases (GRKs 2 through 6) on the cell surface expression of the human (h)AM_1 receptor by cotransfecting each of these GRKs into HEK-293 cells that stably expressed hRAMP2. Flow cytometric analysis revealed that when coexpressed with GRK4 or GRK5, the cell surface expression of the AM_1 receptor was markedly decreased prior to stimulation with AM, thereby attenuating both the specific ["1"2"5I]AM binding and AM-induced cAMP production. These inhibitory effects of both GRKs were abolished by the replacement of the cytoplasmic C-terminal tail (C-tail) of CLR with that of the calcitonin receptor (a family B GPCR) or β_2-adrenergic receptor (a family A GPCR). Among the sequentially truncated CLR C-tail mutants, those lacking the five residues 449–453 (Ser-Phe-Ser-Asn-Ser) abolished the inhibition of the cell surface expression of CLR via the overexpression of GRK4 or GRK5. Thus, we provided new insight into the function of GRKs in agonist-unstimulated GPCR trafficking using a recombinant AM_1 receptor and further determined the region of the CLR C-tail responsible for this GRK function. - Highlights: • We discovered a novel function of GRKs in GPCR trafficking using human CLR/RAMP2. • GRKs 4 and 5 markedly inhibited the cell surface expression of human CLR/RAMP2. • Both GRKs exhibited highly significant receptor signaling inhibition. • Five residues of the C-terminal tail of CLR govern this function of GRKs.

  20. Tumor cell invasion of collagen matrices requires coordinate lipid agonist-induced G-protein and membrane-type matrix metalloproteinase-1-dependent signaling

    Directory of Open Access Journals (Sweden)

    Anthis Nicholas J

    2006-12-01

    Full Text Available Abstract Background Lysophosphatidic acid (LPA and sphingosine 1-phosphate (S1P are bioactive lipid signaling molecules implicated in tumor dissemination. Membrane-type matrix metalloproteinase 1 (MT1-MMP is a membrane-tethered collagenase thought to be involved in tumor invasion via extracellular matrix degradation. In this study, we investigated the molecular requirements for LPA- and S1P-regulated tumor cell migration in two dimensions (2D and invasion of three-dimensional (3D collagen matrices and, in particular, evaluated the role of MT1-MMP in this process. Results LPA stimulated while S1P inhibited migration of most tumor lines in Boyden chamber assays. Conversely, HT1080 fibrosarcoma cells migrated in response to both lipids. HT1080 cells also markedly invaded 3D collagen matrices (~700 μm over 48 hours in response to either lipid. siRNA targeting of LPA1 and Rac1, or S1P1, Rac1, and Cdc42 specifically inhibited LPA- or S1P-induced HT1080 invasion, respectively. Analysis of LPA-induced HT1080 motility on 2D substrates vs. 3D matrices revealed that synthetic MMP inhibitors markedly reduced the distance (~125 μm vs. ~45 μm and velocity of invasion (~0.09 μm/min vs. ~0.03 μm/min only when cells navigated 3D matrices signifying a role for MMPs exclusively in invasion. Additionally, tissue inhibitors of metalloproteinases (TIMPs-2, -3, and -4, but not TIMP-1, blocked lipid agonist-induced invasion indicating a role for membrane-type (MT-MMPs. Furthermore, MT1-MMP expression in several tumor lines directly correlated with LPA-induced invasion. HEK293s, which neither express MT1-MMP nor invade in the presence of LPA, were transfected with MT1-MMP cDNA, and subsequently invaded in response to LPA. When HT1080 cells were seeded on top of or within collagen matrices, siRNA targeting of MT1-MMP, but not other MMPs, inhibited lipid agonist-induced invasion establishing a requisite role for MT1-MMP in this process. Conclusion LPA is a

  1. Interaction of Protease-Activated Receptor 2 with G Proteins and Beta-Arrestin 1 Studied by Bioluminescence Resonance Energy Transfer

    Directory of Open Access Journals (Sweden)

    Mohammed Akli eAyoub

    2013-12-01

    Full Text Available G protein-coupled receptors (GPCRs are well recognized as being able to activate several signaling pathways through the activation of different G proteins as well as other signaling proteins such as beta-arrestins. Therefore, understanding how such multiple GPCR-mediated signaling can be integrated constitute an important aspect. Here, we applied bioluminescence resonance energy transfer (BRET to shed more light on the G protein coupling profile of trypsin receptor, or protease-activated receptor 2 (PAR2, and its interaction with beta-arrestin1. Using YFP and Rluc fusion constructs expressed in COS-7 cells, BRET data revealed a pre-assembly of PAR2 with both Galphai1 and Galphao and a rapid and transient activation of these G proteins upon receptor activation. In contrast, no preassembly of PAR2 with Galpha12 could be detected and their physical association can be measured with a very slow and sustained kinetics similar to that of beta-arrestin1 recruitment. These data demonstrate the coupling of PAR2 with Galphai1, Galphao and Galpha12 in COS-7 cells with differences in the kinetics of GPCR-G protein coupling, a parameter that very likely influences the cellular response. Moreover, this further illustrates that preassembly or agonist-induced G protein interaction depends on receptor-G protein pairs indicating another level of complexity and regulation of the signaling of GPCR-G protein complexes and its multiplicity.

  2. The G-protein-coupled receptor, GPR84, is important for eye development in Xenopus laevis

    OpenAIRE

    Perry, Kimberly J.; Johnson, Verity R.; Malloch, Erica L.; Fukui, Lisa; Wever, Jason; Thomas, Alvin G.; Hamilton, Paul W.; Henry, Jonathan J.

    2010-01-01

    G-protein-coupled receptors (GPCRs) represent diverse, multifamily groups of cell signaling receptors involved in many cellular processes. We identified Xenopus laevis GPR84 as a member of the A18 subfamily of GPCRs. During development, GPR84 is detected in the embryonic lens placode, differentiating lens fiber cells, retina and cornea. Anti-sense morpholino oligonucleotide-mediated knockdown and RNA rescue experiments demonstrate GPR84’s importance in lens, cornea and retinal development. Ex...

  3. Activation of G-proteins by receptor-stimulated nucleoside diphosphate kinase in Dictyostelium.

    Science.gov (United States)

    Bominaar, A A; Molijn, A C; Pestel, M; Veron, M; Van Haastert, P J

    1993-01-01

    Recently, interest in the enzyme nucleoside diphosphate kinase (EC2.7.4.6) has increased as a result of its possible involvement in cell proliferation and development. Since NDP kinase is one of the major sources of GTP in cells, it has been suggested that the effects of an altered NDP kinase activity on cellular processes might be the result of altered transmembrane signal transduction via guanine nucleotide-binding proteins (G-proteins). In the cellular slime mould Dictyostelium discoideum, extracellular cAMP induces an increase of phospholipase C activity via a surface cAMP receptor and G-proteins. In this paper it is demonstrated that part of the cellular NDP kinase is associated with the membrane and stimulated by cell surface cAMP receptors. The GTP produced by the action of NDP kinase is capable of activating G-proteins as monitored by altered G-protein-receptor interaction and the activation of the effector enzyme phospholipase C. Furthermore, specific monoclonal antibodies inhibit the effect of NDP kinase on G-protein activation. These results suggest that receptor-stimulated NDP kinase contributes to the mediation of hormone action by producing GTP for the activation of GTP-binding proteins. Images PMID:8389692

  4. Reduced expression of G protein-coupled receptor kinases in schizophrenia but not in schizoaffective disorder

    Science.gov (United States)

    Bychkov, ER; Ahmed, MR; Gurevich, VV; Benovic, JL; Gurevich, EV

    2011-01-01

    Alterations of multiple G protein-mediated signaling pathways are detected in schizophrenia. G protein-coupled receptor kinases (GRKs) and arrestins terminate signaling by G protein-coupled receptors exerting powerful influence on receptor functions. Modifications of arrestin and/or GRKs expression may contribute to schizophrenia pathology. Cortical expression of arrestins and GRKs was measured postmortem in control and subjects with schizophrenia or schizoaffective disorder. Additionally, arrestin/GRK expression was determined in elderly patients with schizophrenia and age-matched control. Patients with schizophrenia, but not schizoaffective disorder, displayed reduced concentration of arrestin and GRK mRNAs and GRK3 protein. Arrestins and GRK significantly decreased with age. In elderly patients, GRK6 was reduced, with other GRKs and arrestins unchanged. Reduced cortical concentration of GRKs in schizophrenia (resembling that in aging) may result in altered G protein-dependent signaling, thus contributing to prefrontal deficits in schizophrenia. The data suggest distinct molecular mechanisms underlying schizophrenia and schizoaffective disorder. PMID:21784156

  5. Conformation guides molecular efficacy in docking screens of activated β-2 adrenergic G protein coupled receptor.

    Science.gov (United States)

    Weiss, Dahlia R; Ahn, SeungKirl; Sassano, Maria F; Kleist, Andrew; Zhu, Xiao; Strachan, Ryan; Roth, Bryan L; Lefkowitz, Robert J; Shoichet, Brian K

    2013-05-17

    A prospective, large library virtual screen against an activated β2-adrenergic receptor (β2AR) structure returned potent agonists to the exclusion of inverse-agonists, providing the first complement to the previous virtual screening campaigns against inverse-agonist-bound G protein coupled receptor (GPCR) structures, which predicted only inverse-agonists. In addition, two hits recapitulated the signaling profile of the co-crystal ligand with respect to the G protein and arrestin mediated signaling. This functional fidelity has important implications in drug design, as the ability to predict ligands with predefined signaling properties is highly desirable. However, the agonist-bound state provides an uncertain template for modeling the activated conformation of other GPCRs, as a dopamine D2 receptor (DRD2) activated model templated on the activated β2AR structure returned few hits of only marginal potency.

  6. The Role of Cgrp-Receptor Component Protein (Rcp in Cgrp-Mediated Signal Transduction

    Directory of Open Access Journals (Sweden)

    M. A. Prado

    2001-01-01

    Full Text Available The calcitonin gene-related peptide (CGRP-receptor component protein (RCP is a 17-kDa intracellular peripheral membrane protein required for signal transduction at CGRP receptors. To determine the role of RCP in CGRP-mediated signal transduction, RCP was depleted from NIH3T3 cells using antisense strategy. Loss of RCP protein correlated with loss of cAMP production by CGRP in the antisense cells. In contrast, loss of RCP had no effect on CGRP-mediated binding; therefore RCP is not acting as a chaperone for the CGRP receptor. Instead, RCP is a novel signal transduction molecule that couples the CGRP receptor to the cellular signal transduction machinery. RCP thus represents a prototype for a new class of signal transduction proteins that are required for regulation of G protein-coupled receptors.

  7. The ancient link between G-protein-coupled receptors and C-terminal phospholipid kinase domains

    NARCIS (Netherlands)

    Hoogen, van den D.J.; Meijer, Harold J.G.; Seidl, Michael F.; Govers, Francine

    2018-01-01

    Sensing external signals and transducing these into intracellular responses requires a molecular signaling system that is crucial for every living organism. Two important eukaryotic signal transduction pathways that are often interlinked are G-protein signaling and phospholipid signaling.

  8. Cellular and molecular biology of orphan G protein-coupled receptors.

    Science.gov (United States)

    Oh, Da Young; Kim, Kyungjin; Kwon, Hyuk Bang; Seong, Jae Young

    2006-01-01

    The superfamily of G protein-coupled receptors (GPCRs) is the largest and most diverse group of membrane-spanning proteins. It plays a variety of roles in pathophysiological processes by transmitting extracellular signals to cells via heterotrimeric G proteins. Completion of the human genome project revealed the presence of approximately 168 genes encoding established nonsensory GPCRs, as well as 207 genes predicted to encode novel GPCRs for which the natural ligands remained to be identified, the so-called orphan GPCRs. Eighty-six of these orphans have now been paired to novel or previously known molecules, and 121 remain to be deorphaned. A better understanding of the GPCR structures and classification; knowledge of the receptor activation mechanism, either dependent on or independent of an agonist; increased understanding of the control of GPCR-mediated signal transduction; and development of appropriate ligand screening systems may improve the probability of discovering novel ligands for the remaining orphan GPCRs.

  9. BRI1 and BAK1 interact with G proteins and regulate sugar-responsive growth and development in Arabidopsis.

    Science.gov (United States)

    Peng, Yuancheng; Chen, Liangliang; Li, Shengjun; Zhang, Yueying; Xu, Ran; Liu, Zupei; Liu, Wuxia; Kong, Jingjing; Huang, Xiahe; Wang, Yingchun; Cheng, Beijiu; Zheng, Leiying; Li, Yunhai

    2018-04-18

    Sugars function as signal molecules to regulate growth, development, and gene expression in plants, yeasts, and animals. A coordination of sugar availability with phytohormone signals is crucial for plant growth and development. The molecular link between sugar availability and hormone-dependent plant growth are largely unknown. Here we report that BRI1 and BAK1 are involved in sugar-responsive growth and development. Glucose influences the physical interactions and phosphorylations of BRI1 and BAK1 in a concentration-dependent manner. BRI1 and BAK1 physically interact with G proteins that are essential for mediating sugar signaling. Biochemical data show that BRI1 can phosphorylate G protein β subunit and γ subunits, and BAK1 can phosphorylate G protein γ subunits. Genetic analyses suggest that BRI1 and BAK1 function in a common pathway with G-protein subunits to regulate sugar responses. Thus, our findings reveal an important genetic and molecular mechanism by which BR receptors associate with G proteins to regulate sugar-responsive growth and development.

  10. Regulation and Selectivity of Exchange Factors for G-proteins of the Ras-family

    NARCIS (Netherlands)

    Popovic, M.

    2013-01-01

    Small G-proteins are important regulators of the cellular signaling pathways. Among them, members of the Ras family of small G-proteins regulate processes such as cell differentiation, growth, migration, transport and adhesion, and their deregulation may lead to various diseases. Small G-proteins

  11. BILF1 Mediated Transformation Correlates with Constitutive Signaling

    DEFF Research Database (Denmark)

    Lyngaa, Rikke Birgitte

    2009-01-01

    BIFL1 is a G protein-coupled receptor encoded by human EBV. It signals constitutively through G_alpha_i and is an orphan receptor known to down regulate MHCI expression. BILF1 also engage in dimerization with several chemokine receptors and it induced the activity of NF-kappa beta and inhibits...

  12. ROS and ROS-Mediated Cellular Signaling

    Directory of Open Access Journals (Sweden)

    Jixiang Zhang

    2016-01-01

    Full Text Available It has long been recognized that an increase of reactive oxygen species (ROS can modify the cell-signaling proteins and have functional consequences, which successively mediate pathological processes such as atherosclerosis, diabetes, unchecked growth, neurodegeneration, inflammation, and aging. While numerous articles have demonstrated the impacts of ROS on various signaling pathways and clarify the mechanism of action of cell-signaling proteins, their influence on the level of intracellular ROS, and their complex interactions among multiple ROS associated signaling pathways, the systemic summary is necessary. In this review paper, we particularly focus on the pattern of the generation and homeostasis of intracellular ROS, the mechanisms and targets of ROS impacting on cell-signaling proteins (NF-κB, MAPKs, Keap1-Nrf2-ARE, and PI3K-Akt, ion channels and transporters (Ca2+ and mPTP, and modifying protein kinase and Ubiquitination/Proteasome System.

  13. Membrane-localized extra-large G proteins and Gbg of the heterotrimeric G proteins form functional complexes engaged in plant immunity in Arabidopsis.

    Science.gov (United States)

    Maruta, Natsumi; Trusov, Yuri; Brenya, Eric; Parekh, Urvi; Botella, José Ramón

    2015-03-01

    In animals, heterotrimeric G proteins, comprising Ga, Gb, and Gg subunits, are molecular switches whose function tightly depends on Ga and Gbg interaction. Intriguingly, in Arabidopsis (Arabidopsis thaliana), multiple defense responses involve Gbg, but not Ga. We report here that the Gbg dimer directly partners with extra-large G proteins (XLGs) to mediate plant immunity. Arabidopsis mutants deficient in XLGs, Gb, and Gg are similarly compromised in several pathogen defense responses, including disease development and production of reactive oxygen species. Genetic analysis of double, triple, and quadruple mutants confirmed that XLGs and Gbg functionally interact in the same defense signaling pathways. In addition, mutations in XLG2 suppressed the seedling lethal and cell death phenotypes of BRASSINOSTEROID INSENSITIVE1-associated receptor kinase1-interacting receptor-like kinase1 mutants in an identical way as reported for Arabidopsis Gb-deficient mutants. Yeast (Saccharomyces cerevisiae) three-hybrid and bimolecular fluorescent complementation assays revealed that XLG2 physically interacts with all three possible Gbg dimers at the plasma membrane. Phylogenetic analysis indicated a close relationship between XLGs and plant Ga subunits, placing the divergence point at the dawn of land plant evolution. Based on these findings, we conclude that XLGs form functional complexes with Gbg dimers, although the mechanism of action of these complexes, including activation/deactivation, must be radically different form the one used by the canonical Ga subunit and are not likely to share the same receptors. Accordingly, XLGs expand the repertoire of heterotrimeric G proteins in plants and reveal a higher level of diversity in heterotrimeric G protein signaling.

  14. The structure and function of G-protein-coupled receptors

    DEFF Research Database (Denmark)

    Rosenbaum, Daniel M; Rasmussen, Søren Gøgsig Faarup; Kobilka, Brian K

    2009-01-01

    G-protein-coupled receptors (GPCRs) mediate most of our physiological responses to hormones, neurotransmitters and environmental stimulants, and so have great potential as therapeutic targets for a broad spectrum of diseases. They are also fascinating molecules from the perspective of membrane-protein...

  15. G protein-coupled receptors (GPCRs) That Signal via Protein Kinase A (PKA) Cross-talk at Insulin Receptor Substrate 1 (IRS1) to Activate the phosphatidylinositol 3-kinase (PI3K)/AKT Pathway.

    Science.gov (United States)

    Law, Nathan C; White, Morris F; Hunzicker-Dunn, Mary E

    2016-12-30

    G protein-coupled receptors (GPCRs) activate PI3K/v-AKT thymoma viral oncoprotein (AKT) to regulate many cellular functions that promote cell survival, proliferation, and growth. However, the mechanism by which GPCRs activate PI3K/AKT remains poorly understood. We used ovarian preantral granulosa cells (GCs) to elucidate the mechanism by which the GPCR agonist FSH via PKA activates the PI3K/AKT cascade. Insulin-like growth factor 1 (IGF1) is secreted in an autocrine/paracrine manner by GCs and activates the IGF1 receptor (IGF1R) but, in the absence of FSH, fails to stimulate YXXM phosphorylation of IRS1 (insulin receptor substrate 1) required for PI3K/AKT activation. We show that PKA directly phosphorylates the protein phosphatase 1 (PP1) regulatory subunit myosin phosphatase targeting subunit 1 (MYPT1) to activate PP1 associated with the IGF1R-IRS1 complex. Activated PP1 is sufficient to dephosphorylate at least four IRS1 Ser residues, Ser 318 , Ser 346 , Ser 612 , and Ser 789 , and promotes IRS1 YXXM phosphorylation by the IGF1R to activate the PI3K/AKT cascade. Additional experiments indicate that this mechanism also occurs in breast cancer, thyroid, and preovulatory granulosa cells, suggesting that the PKA-dependent dephosphorylation of IRS1 Ser/Thr residues is a conserved mechanism by which GPCRs signal to activate the PI3K/AKT pathway downstream of the IGF1R. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  16. Heterotrimeric G protein subunits are located on rat liver endosomes

    Directory of Open Access Journals (Sweden)

    Van Dyke Rebecca W

    2004-01-01

    Full Text Available Abstract Background Rat liver endosomes contain activated insulin receptors and downstream signal transduction molecules. We undertook these studies to determine whether endosomes also contain heterotrimeric G proteins that may be involved in signal transduction from G protein-coupled receptors. Results By Western blotting Gsα, Giα1,2, Giα3 and Gβ were enriched in both canalicular (CM and basolateral (BLM membranes but also readily detectable on three types of purified rat liver endosomes in the order recycling receptor compartment (RRC > compartment for uncoupling of receptor and ligand (CURL > multivesicular bodies (MVB >> purified secondary lysosomes. Western blotting with antibodies to Na, K-ATPase and to other proteins associated with plasma membranes and intracellular organelles indicated this was not due to contamination of endosome preparations by CM or BLM. Adenylate cyclase (AC was also identified on purified CM, BLM, RRC, CURL and MVB. Percoll gradient fractionation of liver postnuclear supernatants demonstrated co-occurrence of endosomes and heterotrimeric G protein subunits in fractions with little plasma membrane markers. By confocal microscopy, punctate staining for Gsα, Giα3 and Gβ corresponded to punctate areas of endocytosed Texas red-dextran in hepatocytes from control and cholera toxin-treated livers. Conclusion We conclude that heterotrimeric G protein subunits as well as AC likely traffic into hepatocytes on endosome membranes, possibly generating downstream signals spatially separate from signalling generated at the plasma membrane, analogous to the role(s of internalized insulin receptors.

  17. Inositol trisphosphate receptor mediated spatiotemporal calcium signalling.

    Science.gov (United States)

    Miyazaki, S

    1995-04-01

    Spatiotemporal Ca2+ signalling in the cytoplasm is currently understood as an excitation phenomenon by analogy with electrical excitation in the plasma membrane. In many cell types, Ca2+ waves and Ca2+ oscillations are mediated by inositol 1,4,5-trisphosphate (IP3) receptor/Ca2+ channels in the endoplasmic reticulum membrane, with positive feedback between cytosolic Ca2+ and IP3-induced Ca2+ release creating a regenerative process. Remarkable advances have been made in the past year in the analysis of subcellular Ca2+ microdomains using confocal microscopy and of Ca2+ influx pathways that are functionally coupled to IP3-induced Ca2+ release. Ca2+ signals can be conveyed into the nucleus and mitochondria. Ca2+ entry from outside the cell allows repetitive Ca2+ release by providing Ca2+ to refill the endoplasmic reticulum stores, thus giving rise to frequency-encoded Ca2+ signals.

  18. Noncell- and cell-autonomous G-protein-signaling converges with Ca2+/mitogen-activated protein kinase signaling to regulate str-2 receptor gene expression in Caenorhabditis elegans.

    NARCIS (Netherlands)

    H. Lans (Hannes); G. Jansen (Gert)

    2006-01-01

    textabstractIn the sensory system of C. elegans, the candidate odorant receptor gene str-2 is strongly expressed in one of the two AWC neurons and weakly in both ASI neurons. Asymmetric AWC expression results from suppression of str-2 expression by a Ca2+/MAPK signaling pathway in one of the AWC

  19. Understanding the Added Value of G-Protein-Coupled Receptor Heteromers

    Directory of Open Access Journals (Sweden)

    Nuria Franco

    2014-01-01

    Full Text Available G-protein-coupled receptors (GPCRs constitute the most populated family of proteins within the human genome. Since the early sixties work on GPCRs and on GPCR-mediated signaling has led to a number of awards, the most recent being the Nobel Prize in Chemistry for 2012. The future of GPCRs research is surely based on their capacity for heteromerization. Receptor heteromers offer a series of challenges that will help in providing success in academic/basic research and translation into more effective and safer drugs.

  20. Distinct cellular and subcellular distributions of G protein-coupled receptor kinase and arrestin isoforms in the striatum.

    Directory of Open Access Journals (Sweden)

    Evgeny Bychkov

    Full Text Available G protein-coupled receptor kinases (GRKs and arrestins mediate desensitization of G protein-coupled receptors (GPCR. Arrestins also mediate G protein-independent signaling via GPCRs. Since GRK and arrestins demonstrate no strict receptor specificity, their functions in the brain may depend on their cellular complement, expression level, and subcellular targeting. However, cellular expression and subcellular distribution of GRKs and arrestins in the brain is largely unknown. We show that GRK isoforms GRK2 and GRK5 are similarly expressed in direct and indirect pathway neurons in the rat striatum. Arrestin-2 and arrestin-3 are also expressed in neurons of both pathways. Cholinergic interneurons are enriched in GRK2, arrestin-3, and GRK5. Parvalbumin-positive interneurons express more of GRK2 and less of arrestin-2 than medium spiny neurons. The GRK5 subcellular distribution in the human striatal neurons is altered by its phosphorylation: unphosphorylated enzyme preferentially localizes to synaptic membranes, whereas phosphorylated GRK5 is found in plasma membrane and cytosolic fractions. Both GRK isoforms are abundant in the nucleus of human striatal neurons, whereas the proportion of both arrestins in the nucleus was equally low. However, overall higher expression of arrestin-2 yields high enough concentration in the nucleus to mediate nuclear functions. These data suggest cell type- and subcellular compartment-dependent differences in GRK/arrestin-mediated desensitization and signaling.

  1. Navigating the conformational landscape of G protein-coupled receptor kinases during allosteric activation.

    Science.gov (United States)

    Yao, Xin-Qiu; Cato, M Claire; Labudde, Emily; Beyett, Tyler S; Tesmer, John J G; Grant, Barry J

    2017-09-29

    G protein-coupled receptors (GPCRs) are essential for transferring extracellular signals into carefully choreographed intracellular responses controlling diverse aspects of cell physiology. The duration of GPCR-mediated signaling is primarily regulated via GPCR kinase (GRK)-mediated phosphorylation of activated receptors. Although many GRK structures have been reported, the mechanisms underlying GRK activation are not well-understood, in part because it is unknown how these structures map to the conformational landscape available to this enzyme family. Unlike most other AGC kinases, GRKs rely on their interaction with GPCRs for activation and not phosphorylation. Here, we used principal component analysis of available GRK and protein kinase A crystal structures to identify their dominant domain motions and to provide a framework that helps evaluate how close each GRK structure is to being a catalytically competent state. Our results indicated that disruption of an interface formed between the large lobe of the kinase domain and the regulator of G protein signaling homology domain (RHD) is highly correlated with establishment of the active conformation. By introducing point mutations in the GRK5 RHD-kinase domain interface, we show with both in silico and in vitro experiments that perturbation of this interface leads to higher phosphorylation activity. Navigation of the conformational landscape defined by this bioinformatics-based study is likely common to all GPCR-activated GRKs. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  2. An Autocrine Proliferation Repressor Regulates Dictyostelium discoideum Proliferation and Chemorepulsion Using the G Protein-Coupled Receptor GrlH.

    Science.gov (United States)

    Tang, Yu; Wu, Yuantai; Herlihy, Sarah E; Brito-Aleman, Francisco J; Ting, Jose H; Janetopoulos, Chris; Gomer, Richard H

    2018-02-13

    In eukaryotic microbes, little is known about signals that inhibit the proliferation of the cells that secrete the signal, and little is known about signals (chemorepellents) that cause cells to move away from the source of the signal. Autocrine proliferation repressor protein A (AprA) is a protein secreted by the eukaryotic microbe Dictyostelium discoideum AprA is a chemorepellent for and inhibits the proliferation of D. discoideum We previously found that cells sense AprA using G proteins, suggesting the existence of a G protein-coupled AprA receptor. To identify the AprA receptor, we screened mutants lacking putative G protein-coupled receptors. We found that, compared to the wild-type strain, cells lacking putative receptor GrlH ( grlH¯ cells) show rapid proliferation, do not have large numbers of cells moving away from the edges of colonies, are insensitive to AprA-induced proliferation inhibition and chemorepulsion, and have decreased AprA binding. Expression of GrlH in grlH¯ cells ( grlH¯/grlH OE ) rescues the phenotypes described above. These data indicate that AprA signaling may be mediated by GrlH in D. discoideum IMPORTANCE Little is known about how eukaryotic cells can count themselves and thus regulate the size of a tissue or density of cells. In addition, little is known about how eukaryotic cells can sense a repellant signal and move away from the source of the repellant, for instance, to organize the movement of cells in a developing embryo or to move immune cells out of a tissue. In this study, we found that a eukaryotic microbe uses G protein-coupled receptors to mediate both cell density sensing and chemorepulsion. Copyright © 2018 Tang et al.

  3. G protein-coupled receptors: the inside story.

    Science.gov (United States)

    Jalink, Kees; Moolenaar, Wouter H

    2010-01-01

    Recent findings necessitate revision of the traditional view of G protein-coupled receptor (GPCR) signaling and expand the diversity of mechanisms by which receptor signaling influences cell behavior in general. GPCRs elicit signals at the plasma membrane and are then rapidly removed from the cell surface by endocytosis. Internalization of GPCRs has long been thought to serve as a mechanism to terminate the production of second messengers such as cAMP. However, recent studies show that internalized GPCRs can continue to either stimulate or inhibit cAMP production in a sustained manner. They do so by remaining associated with their cognate G protein subunit and adenylyl cyclase at endosomal compartments. Once internalized, the GPCRs produce cellular responses distinct from those elicited at the cell surface.

  4. Phytomelatonin receptor PMTR1-mediated signaling regulates stomatal closure in Arabidopsis thaliana.

    Science.gov (United States)

    Wei, Jian; Li, Dong-Xu; Zhang, Jia-Rong; Shan, Chi; Rengel, Zed; Song, Zhong-Bang; Chen, Qi

    2018-04-27

    Melatonin has been detected in plants in 1995; however, the function and signaling pathway of this putative phytohormone are largely undetermined due to a lack of knowledge about its receptor. Here, we discovered the first phytomelatonin receptor (CAND2/PMTR1) in Arabidopsis thaliana and found that melatonin governs the receptor-dependent stomatal closure. The application of melatonin induced stomatal closure through the heterotrimeric G protein α subunit-regulated H 2 O 2 and Ca 2+ signals. The Arabidopsis mutant lines lacking AtCand2 that encodes a candidate G protein-coupled receptor were insensitive to melatonin-induced stomatal closure. Accordingly, the melatonin-induced H 2 O 2 production and Ca 2+ influx were completely abolished in cand2. CAND2 is a membrane protein that interacts with GPA1 and the expression of AtCand2 was tightly regulated by melatonin in various organs and guard cells. CAND2 showed saturable and specific 125 I-melatonin binding, with apparent K d (dissociation constant) of 0.73 ± 0.10 nmol/L (r 2  = .99), demonstrating this protein is a phytomelatonin receptor (PMTR1). Our results suggest that the phytomelatonin regulation of stomatal closure is dependent on its receptor CAND2/PMTR1-mediated H 2 O 2 and Ca 2+ signaling transduction cascade. © 2018 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  5. Endocytosis of G protein-coupled receptors is regulated by clathrin light chain phosphorylation.

    Science.gov (United States)

    Ferreira, Filipe; Foley, Matthew; Cooke, Alex; Cunningham, Margaret; Smith, Gemma; Woolley, Robert; Henderson, Graeme; Kelly, Eamonn; Mundell, Stuart; Smythe, Elizabeth

    2012-08-07

    Signaling by transmembrane receptors such as G protein-coupled receptors (GPCRs) occurs at the cell surface and throughout the endocytic pathway, and signaling from the cell surface may differ in magnitude and downstream output from intracellular signaling. As a result, the rate at which signaling molecules traverse the endocytic pathway makes a significant contribution to downstream output. Modulation of the core endocytic machinery facilitates differential uptake of individual cargoes. Clathrin-coated pits are a major entry portal where assembled clathrin forms a lattice around invaginating buds that have captured endocytic cargo. Clathrin assembles into triskelia composed of three clathrin heavy chains and associated clathrin light chains (CLCs). Despite the identification of clathrin-coated pits at the cell surface over 30 years ago, the functions of CLCs in endocytosis have been elusive. In this work, we identify a novel role for CLCs in the regulated endocytosis of specific cargoes. Small interfering RNA-mediated knockdown of either CLCa or CLCb inhibits the uptake of GPCRs. Moreover, we demonstrate that phosphorylation of Ser204 in CLCb is required for efficient endocytosis of a subset of GPCRs and identify G protein-coupled receptor kinase 2 (GRK2) as a kinase that can phosphorylate CLCb on Ser204. Overexpression of CLCb(S204A) specifically inhibits the endocytosis of those GPCRs whose endocytosis is GRK2-dependent. Together, these results indicate that CLCb phosphorylation acts as a discriminator for the endocytosis of specific GPCRs. Copyright © 2012 Elsevier Ltd. All rights reserved.

  6. H2O2-Activated Mitochondrial Phospholipase iPLA2 gamma Prevents Lipotoxic Oxidative Stress in Synergy with UCP2, Amplifies Signaling via G-Protein-Coupled Receptor GPR40, and Regulates Insulin Secretion in Pancreatic beta-Cells

    Czech Academy of Sciences Publication Activity Database

    Ježek, Jan; Dlasková, Andrea; Zelenka, Jaroslav; Jabůrek, Martin; Ježek, Petr

    2015-01-01

    Roč. 23, č. 12 (2015), s. 958-972 ISSN 1523-0864 R&D Projects: GA ČR(CZ) GPP303/11/P320; GA ČR(CZ) GA13-02033S; GA ČR(CZ) GA13-06666S; GA ČR GA15-02051S Institutional support: RVO:67985823 Keywords : mitochondrial phospholipase iPLA2 gamma * uncoupling protein UCP2 * G-protein coupled receptor - 40 * glucose-stimulated insulin secretion * pancreatic beta cells Subject RIV: FB - Endocrinology, Diabetology, Metabolism, Nutrition Impact factor: 7.093, year: 2015

  7. Emerging Paradigm of Intracellular Targeting of G Protein-Coupled Receptors.

    Science.gov (United States)

    Chaturvedi, Madhu; Schilling, Justin; Beautrait, Alexandre; Bouvier, Michel; Benovic, Jeffrey L; Shukla, Arun K

    2018-05-04

    G protein-coupled receptors (GPCRs) recognize a diverse array of extracellular stimuli, and they mediate a broad repertoire of signaling events involved in human physiology. Although the major effort on targeting GPCRs has typically been focused on their extracellular surface, a series of recent developments now unfold the possibility of targeting them from the intracellular side as well. Allosteric modulators binding to the cytoplasmic surface of GPCRs have now been described, and their structural mechanisms are elucidated by high-resolution crystal structures. Furthermore, pepducins, aptamers, and intrabodies targeting the intracellular face of GPCRs have also been successfully utilized to modulate receptor signaling. Moreover, small molecule compounds, aptamers, and synthetic intrabodies targeting β-arrestins have also been discovered to modulate GPCR endocytosis and signaling. Here, we discuss the emerging paradigm of intracellular targeting of GPCRs, and outline the current challenges, potential opportunities, and future outlook in this particular area of GPCR biology. Copyright © 2018 Elsevier Ltd. All rights reserved.

  8. An Autocrine Proliferation Repressor Regulates Dictyostelium discoideum Proliferation and Chemorepulsion Using the G Protein-Coupled Receptor GrlH

    Directory of Open Access Journals (Sweden)

    Yu Tang

    2018-02-01

    Full Text Available In eukaryotic microbes, little is known about signals that inhibit the proliferation of the cells that secrete the signal, and little is known about signals (chemorepellents that cause cells to move away from the source of the signal. Autocrine proliferation repressor protein A (AprA is a protein secreted by the eukaryotic microbe Dictyostelium discoideum. AprA is a chemorepellent for and inhibits the proliferation of D. discoideum. We previously found that cells sense AprA using G proteins, suggesting the existence of a G protein-coupled AprA receptor. To identify the AprA receptor, we screened mutants lacking putative G protein-coupled receptors. We found that, compared to the wild-type strain, cells lacking putative receptor GrlH (grlH{macron} cells show rapid proliferation, do not have large numbers of cells moving away from the edges of colonies, are insensitive to AprA-induced proliferation inhibition and chemorepulsion, and have decreased AprA binding. Expression of GrlH in grlH{macron} cells (grlH{macron}/grlHOE rescues the phenotypes described above. These data indicate that AprA signaling may be mediated by GrlH in D. discoideum.

  9. Applications of molecular replacement to G protein-coupled receptors

    International Nuclear Information System (INIS)

    Kruse, Andrew C.; Manglik, Aashish; Kobilka, Brian K.; Weis, William I.

    2013-01-01

    The use of molecular replacement in solving the structures of G protein-coupled receptors is discussed, with specific examples being described in detail. G protein-coupled receptors (GPCRs) are a large class of integral membrane proteins involved in regulating virtually every aspect of human physiology. Despite their profound importance in human health and disease, structural information regarding GPCRs has been extremely limited until recently. With the advent of a variety of new biochemical and crystallographic techniques, the structural biology of GPCRs has advanced rapidly, offering key molecular insights into GPCR activation and signal transduction. To date, almost all GPCR structures have been solved using molecular-replacement techniques. Here, the unique aspects of molecular replacement as applied to individual GPCRs and to signaling complexes of these important proteins are discussed

  10. Insulin signaling mediates sexual attractiveness in Drosophila.

    Directory of Open Access Journals (Sweden)

    Tsung-Han Kuo

    Full Text Available Sexually attractive characteristics are often thought to reflect an individual's condition or reproductive potential, but the underlying molecular mechanisms through which they do so are generally unknown. Insulin/insulin-like growth factor signaling (IIS is known to modulate aging, reproduction, and stress resistance in several species and to contribute to variability of these traits in natural populations. Here we show that IIS determines sexual attractiveness in Drosophila through transcriptional regulation of genes involved in the production of cuticular hydrocarbons (CHC, many of which function as pheromones. Using traditional gas chromatography/mass spectrometry (GC/MS together with newly introduced laser desorption/ionization orthogonal time-of-flight mass spectrometry (LDI-MS we establish that CHC profiles are significantly affected by genetic manipulations that target IIS. Manipulations that reduce IIS also reduce attractiveness, while females with increased IIS are significantly more attractive than wild-type animals. IIS effects on attractiveness are mediated by changes in CHC profiles. Insulin signaling influences CHC through pathways that are likely independent of dFOXO and that may involve the nutrient-sensing Target of Rapamycin (TOR pathway. These results suggest that the activity of conserved molecular regulators of longevity and reproductive output may manifest in different species as external characteristics that are perceived as honest indicators of fitness potential.

  11. Agonists for G-protein-coupled receptor 84 (GPR84) alter cellular morphology and motility but do not induce pro-inflammatory responses in microglia

    OpenAIRE

    Wei, Li; Tokizane, Kyohei; Konishi, Hiroyuki; Yu, Hua-Rong; Kiyama, Hiroshi

    2017-01-01

    Background Several G-protein-coupled receptors (GPCRs) have been shown to be important signaling mediators between neurons and glia. In our previous screening for identification of nerve injury-associated GPCRs, G-protein-coupled receptor 84 (GPR84) mRNA showed the highest up-regulation by microglia after nerve injury. GPR84 is a pro-inflammatory receptor of macrophages in a neuropathic pain mouse model, yet its function in resident microglia in the central nervous system is poorly understood...

  12. Conformational changes in the G protein Gs induced by the β2 adrenergic receptor

    DEFF Research Database (Denmark)

    Chung, Ka Young; Rasmussen, Søren Gøgsig Faarup; Liu, Tong

    2011-01-01

    and initiation of downstream signalling cascades. Despite a wealth of biochemical and biophysical studies on inactive and active conformations of several heterotrimeric G proteins, the molecular underpinnings of G protein activation remain elusive. To characterize this mechanism, we applied peptide amide...

  13. Analysis of Human TAAR8 and Murine Taar8b Mediated Signaling Pathways and Expression Profile

    Directory of Open Access Journals (Sweden)

    Jessica Mühlhaus

    2014-11-01

    Full Text Available The thyroid hormone derivative 3-iodothyronamine (3-T1AM exerts metabolic effects in vivo that contradict known effects of thyroid hormones. 3-T1AM acts as a trace amine-associated receptor 1 (TAAR1 agonist and activates Gs signaling in vitro. Interestingly, 3-T1AM-meditated in vivo effects persist in Taar1 knockout-mice indicating that further targets of 3-T1AM might exist. Here, we investigated another member of the TAAR family, the only scarcely studied mouse and human trace-amine-associated receptor 8 (Taar8b, TAAR8. By RT-qPCR and locked-nucleic-acid (LNA in situ hybridization, Taar8b expression in different mouse tissues was analyzed. Functionally, we characterized TAAR8 and Taar8b with regard to cell surface expression and signaling via different G-protein-mediated pathways. Cell surface expression was verified by ELISA, and cAMP accumulation was quantified by AlphaScreen for detection of Gs and/or Gi/o signaling. Activation of G-proteins Gq/11 and G12/13 was analyzed by reporter gene assays. Expression analyses revealed at most marginal Taar8b expression and no gender differences for almost all analyzed tissues. In heart, LNA-in situ hybridization demonstrated the absence of Taar8b expression. We could not identify 3-T1AM as a ligand for TAAR8 and Taar8b, but both receptors were characterized by a basal Gi/o signaling activity, a so far unknown signaling pathway for TAARs.

  14. G-Protein Coupled Receptors: Surface Display and Biosensor Technology

    Science.gov (United States)

    McMurchie, Edward; Leifert, Wayne

    Signal transduction by G-protein coupled receptors (GPCRs) underpins a multitude of physiological processes. Ligand recognition by the receptor leads to the activation of a generic molecular switch involving heterotrimeric G-proteins and guanine nucleotides. With growing interest and commercial investment in GPCRs in areas such as drug targets, orphan receptors, high-throughput screening of drugs and biosensors, greater attention will focus on assay development to allow for miniaturization, ultrahigh-throughput and, eventually, microarray/biochip assay formats that will require nanotechnology-based approaches. Stable, robust, cell-free signaling assemblies comprising receptor and appropriate molecular switching components will form the basis of future GPCR/G-protein platforms, which should be able to be adapted to such applications as microarrays and biosensors. This chapter focuses on cell-free GPCR assay nanotechnologies and describes some molecular biological approaches for the construction of more sophisticated, surface-immobilized, homogeneous, functional GPCR sensors. The latter points should greatly extend the range of applications to which technologies based on GPCRs could be applied.

  15. Regulation of neuronal communication by G protein-coupled receptors.

    Science.gov (United States)

    Huang, Yunhong; Thathiah, Amantha

    2015-06-22

    Neuronal communication plays an essential role in the propagation of information in the brain and requires a precisely orchestrated connectivity between neurons. Synaptic transmission is the mechanism through which neurons communicate with each other. It is a strictly regulated process which involves membrane depolarization, the cellular exocytosis machinery, neurotransmitter release from synaptic vesicles into the synaptic cleft, and the interaction between ion channels, G protein-coupled receptors (GPCRs), and downstream effector molecules. The focus of this review is to explore the role of GPCRs and G protein-signaling in neurotransmission, to highlight the function of GPCRs, which are localized in both presynaptic and postsynaptic membrane terminals, in regulation of intrasynaptic and intersynaptic communication, and to discuss the involvement of astrocytic GPCRs in the regulation of neuronal communication. Copyright © 2015 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

  16. The dopamine D2 receptor can directly recruit and activate GRK2 without G protein activation.

    Science.gov (United States)

    Pack, Thomas F; Orlen, Margo I; Ray, Caroline; Peterson, Sean M; Caron, Marc G

    2018-04-20

    The dopamine D2 receptor (D2R) is a G protein-coupled receptor (GPCR) that is critical for many central nervous system functions. The D2R carries out these functions by signaling through two transducers: G proteins and β-arrestins (βarrs). Selectively engaging either the G protein or βarr pathway may be a way to improve drugs targeting GPCRs. The current model of GPCR signal transduction posits a chain of events where G protein activation ultimately leads to βarr recruitment. GPCR kinases (GRKs), which are regulated by G proteins and whose kinase action facilitates βarr recruitment, bridge these pathways. Therefore, βarr recruitment appears to be intimately tied to G protein activation via GRKs. Here we sought to understand how GRK2 action at the D2R would be disrupted when G protein activation is eliminated and the effect of this on βarr recruitment. We used two recently developed biased D2R mutants that can preferentially interact either with G proteins or βarrs as well as a βarr-biased D2R ligand, UNC9994. With these functionally selective tools, we investigated the mechanism whereby the βarr-preferring D2R achieves βarr pathway activation in the complete absence of G protein activation. We describe how direct, G protein-independent recruitment of GRK2 drives interactions at the βarr-preferring D2R and also contributes to βarr recruitment at the WT D2R. Additionally, we found an additive interaction between the βarr-preferring D2R mutant and UNC9994. These results reveal that the D2R can directly recruit GRK2 without G protein activation and that this mechanism may have relevance to achieving βarr-biased signaling. © 2018 by The American Society for Biochemistry and Molecular Biology, Inc.

  17. Activation of G protein-coupled estrogen receptor 1 induces coronary artery relaxation via Epac/Rap1-mediated inhibition of RhoA/Rho kinase pathway in parallel with PKA.

    Directory of Open Access Journals (Sweden)

    Xuan Yu

    Full Text Available Previously, we reported that cAMP/PKA signaling is involved in GPER-mediated coronary relaxation by activating MLCP via inhibition of RhoA pathway. In the current study, we tested the hypothesis that activation of GPER induces coronary artery relaxation via inhibition of RhoA/Rho kinase pathway by cAMP downstream targets, exchange proteins directly activated by cAMP (Epac as well as PKA. Our results show that Epac inhibitors, brefeldin A (BFA, 50 μM, or ESI-09 (20 μM, or CE3F4 (100 μM, all partially inhibited porcine coronary artery relaxation response to the selective GPER agonist, G-1 (0.3-3 μM; while concurrent administration of BFA and PKI (5 μM, a PKA inhibitor, almost completely blocked the relaxation effect of G-1. The Epac specific agonist, 8-CPT-2Me-cAMP (007, 1-100 μM, induced a concentration-dependent relaxation response. Furthermore, the activity of Ras-related protein 1 (Rap1 was up regulated by G-1 (1 μM treatment of porcine coronary artery smooth muscle cells (CASMCs. Phosphorylation of vasodilator-stimulated phosphoprotein (p-VASP was elevated by G-1 (1 μM treatment, but not by 007 (50 μM; and the effect of G-1 on p-VASP was blocked by PKI, but not by ESI-09, an Epac antagonist. RhoA activity was similarly down regulated by G-1 and 007, whereas ESI-09 restored most of the reduced RhoA activity by G-1 treatment. Furthermore, G-1 decreased PGF2α-induced p-MYPT1, which was partially reversed with either ESI-09 or PKI; whereas, concurrent administration of ESI-09 and PKI totally prevented the inhibitory effect of G-1. The inhibitory effects of G-1 on p- MLC levels in CASMCs were mostly restored by either ESI-09 or PKI. These results demonstrate that activation of GPER induces coronary artery relaxation via concurrent inhibition of RhoA/Rho kinase by Epac/Rap1 and PKA. GPER could be a potential drug target for preventing and treating cardiovascular diseases.

  18. Mechanism of the G-protein mimetic nanobody binding to a muscarinic G-protein-coupled receptor.

    Science.gov (United States)

    Miao, Yinglong; McCammon, J Andrew

    2018-03-20

    Protein-protein binding is key in cellular signaling processes. Molecular dynamics (MD) simulations of protein-protein binding, however, are challenging due to limited timescales. In particular, binding of the medically important G-protein-coupled receptors (GPCRs) with intracellular signaling proteins has not been simulated with MD to date. Here, we report a successful simulation of the binding of a G-protein mimetic nanobody to the M 2 muscarinic GPCR using the robust Gaussian accelerated MD (GaMD) method. Through long-timescale GaMD simulations over 4,500 ns, the nanobody was observed to bind the receptor intracellular G-protein-coupling site, with a minimum rmsd of 2.48 Å in the nanobody core domain compared with the X-ray structure. Binding of the nanobody allosterically closed the orthosteric ligand-binding pocket, being consistent with the recent experimental finding. In the absence of nanobody binding, the receptor orthosteric pocket sampled open and fully open conformations. The GaMD simulations revealed two low-energy intermediate states during nanobody binding to the M 2 receptor. The flexible receptor intracellular loops contribute remarkable electrostatic, polar, and hydrophobic residue interactions in recognition and binding of the nanobody. These simulations provided important insights into the mechanism of GPCR-nanobody binding and demonstrated the applicability of GaMD in modeling dynamic protein-protein interactions.

  19. Sequence analysis reveals how G protein-coupled receptors transduce the signal to the G protein.

    NARCIS (Netherlands)

    Oliveira, L.; Paiva, P.B.; Paiva, A.C.; Vriend, G.

    2003-01-01

    Sequence entropy-variability plots based on alignments of very large numbers of sequences-can indicate the location in proteins of the main active site and modulator sites. In the previous article in this issue, we applied this observation to a series of well-studied proteins and concluded that it

  20. Modeling structure of G protein-coupled receptors in huan genome

    KAUST Repository

    Zhang, Yang

    2016-01-01

    G protein-coupled receptors (or GPCRs) are integral transmembrane proteins responsible to various cellular signal transductions. Human GPCR proteins are encoded by 5% of human genes but account for the targets of 40% of the FDA approved drugs. Due

  1. Conformational Fluctuations in G-Protein-Coupled Receptors

    Science.gov (United States)

    Brown, Michael F.

    2014-03-01

    G-protein-coupled receptors (GPCRs) comprise almost 50% of pharmaceutical drug targets, where rhodopsin is an important prototype and occurs naturally in a lipid membrane. Rhodopsin photoactivation entails 11-cis to all-trans isomerization of the retinal cofactor, yielding an equilibrium between inactive Meta-I and active Meta-II states. Two important questions are: (1) Is rhodopsin is a simple two-state switch? Or (2) does isomerization of retinal unlock an activated conformational ensemble? For an ensemble-based activation mechanism (EAM) a role for conformational fluctuations is clearly indicated. Solid-state NMR data together with theoretical molecular dynamics (MD) simulations detect increased local mobility of retinal after light activation. Resultant changes in local dynamics of the cofactor initiate large-scale fluctuations of transmembrane helices that expose recognition sites for the signal-transducing G-protein. Time-resolved FTIR studies and electronic spectroscopy further show the conformational ensemble is strongly biased by the membrane lipid composition, as well as pH and osmotic pressure. A new flexible surface model (FSM) describes how the curvature stress field of the membrane governs the energetics of active rhodopsin, due to the spontaneous monolayer curvature of the lipids. Furthermore, influences of osmotic pressure dictate that a large number of bulk water molecules are implicated in rhodopsin activation. Around 60 bulk water molecules activate rhodopsin, which is much larger than the number of structural waters seen in X-ray crystallography, or inferred from studies of bulk hydrostatic pressure. Conformational selection and promoting vibrational motions of rhodopsin lead to activation of the G-protein (transducin). Our biophysical data give a paradigm shift in understanding GPCR activation. The new view is: dynamics and conformational fluctuations involve an ensemble of substates that activate the cognate G-protein in the amplified visual

  2. microRNA-146a inhibits G protein-coupled receptor-mediated activation of NF-κB by targeting CARD10 and COPS8 in gastric cancer

    DEFF Research Database (Denmark)

    Crone, Stephanie Geisler; Jacobsen, Anders; Federspiel, Birgitte

    2012-01-01

    Gastric cancer is the second most common cause of cancer-related death in the world. Inflammatory signals originating from gastric cancer cells are important for recruiting inflammatory cells and regulation of metastasis of gastric cancer. Several microRNAs (miRNA) have been shown to be involved...... in development and progression of gastric cancer. miRNA-146a (miR-146a) is a modulator of inflammatory signals, but little is known about its importance in gastric cancer. We therefore wanted to identify targets of miR-146a in gastric cancer and examine its biological roles....

  3. G protein abnormalities in pituitary adenomas.

    Science.gov (United States)

    Spada, A; Lania, A; Ballarè, E

    1998-07-25

    It has been demonstrated that the majority of secreting and nonsecreting adenomas is monoclonal in origin suggesting that these neoplasia arise from the replication of a single mutated cell, in which growth advantage results from either activation of protooncogenes or inactivation of antioncogenes. Although a large number of genes has been screened for mutations, only few genetic abnormalities have been found in pituitary tumors such as allelic deletion of chromosome 11q13 where the MEN-1 gene has been localised, and mutations in the gene encoding the alpha subunit of the stimulatory Gs and Gi2 protein. These mutations constitutively activate the alpha subunit of the Gs and Gi2 protein by inhibiting their intrinsic GTPase activity. Both Gs alpha and Gi2alpha can be considered products of protooncogenes (gsp and gip2, respectively) since gain of function mutations that activate mitogenic signals have been recognized in human tumors. Gsp oncogene is found in 30-40% of GH-secreting adenomas, in a low percentage of nonfunctioning and ACTH-secreting pituitary adenomas, in toxic thyroid adenomas and differentiated thyroid carcinomas. The same mutations, occurred early in embriogenesis, have been also identified in tissues from patients affected with the McCune Albright syndrome. These mutations result in an increased cAMP production and in the subsequent overactivation of specific pathways involved in both cell growth and specific programmes of cell differentiation. By consequence, the endocrine tumors expressing gsp oncogene retain differentiated functions. The gip2 oncogene has been identified in about 10% of nonfunctioning pituitary adenomas, in tumors of the ovary and the adrenal cortex. However, it remains to be established whether Gi proteins activate mitogenic signals in pituitary cells. Since Gi proteins are involved in mediating the effect of inhibitory neurohormones on intracellular effectors, it has been proposed that in pituitary tumors the low expression of

  4. The G protein Gi1 exhibits basal coupling but not preassembly with G protein-coupled receptors.

    Science.gov (United States)

    Bondar, Alexey; Lazar, Josef

    2017-06-09

    The G i/o protein family transduces signals from a diverse group of G protein-coupled receptors (GPCRs). The observed specificity of G i/o -GPCR coupling and the high rate of G i/o signal transduction have been hypothesized to be enabled by the existence of stable associates between G i/o proteins and their cognate GPCRs in the inactive state (G i/o -GPCR preassembly). To test this hypothesis, we applied the recently developed technique of two-photon polarization microscopy (2PPM) to Gα i1 subunits labeled with fluorescent proteins and four GPCRs: the α 2A -adrenergic receptor, GABA B , cannabinoid receptor type 1 (CB 1 R), and dopamine receptor type 2. Our experiments with non-dissociating mutants of fluorescently labeled Gα i1 subunits (exhibiting impaired dissociation from activated GPCRs) showed that 2PPM is capable of detecting GPCR-G protein interactions. 2PPM experiments with non-mutated fluorescently labeled Gα i1 subunits and α 2A -adrenergic receptor, GABA B , or dopamine receptor type 2 receptors did not reveal any interaction between the G i1 protein and the non-stimulated GPCRs. In contrast, non-stimulated CB 1 R exhibited an interaction with the G i1 protein. Further experiments revealed that this interaction is caused solely by CB 1 R basal activity; no preassembly between CB 1 R and the G i1 protein could be observed. Our results demonstrate that four diverse GPCRs do not preassemble with non-active G i1 However, we also show that basal GPCR activity allows interactions between non-stimulated GPCRs and G i1 (basal coupling). These findings suggest that G i1 interacts only with active GPCRs and that the well known high speed of GPCR signal transduction does not require preassembly between G proteins and GPCRs. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  5. Phosphorylation of G Protein-Coupled Receptors: From the Barcode Hypothesis to the Flute Model.

    Science.gov (United States)

    Yang, Zhao; Yang, Fan; Zhang, Daolai; Liu, Zhixin; Lin, Amy; Liu, Chuan; Xiao, Peng; Yu, Xiao; Sun, Jin-Peng

    2017-09-01

    Seven transmembrane G protein-coupled receptors (GPCRs) are often phosphorylated at the C terminus and on intracellular loops in response to various extracellular stimuli. Phosphorylation of GPCRs by GPCR kinases and certain other kinases can promote the recruitment of arrestin molecules. The arrestins critically regulate GPCR functions not only by mediating receptor desensitization and internalization, but also by redirecting signaling to G protein-independent pathways via interactions with numerous downstream effector molecules. Accumulating evidence over the past decade has given rise to the phospho-barcode hypothesis, which states that ligand-specific phosphorylation patterns of a receptor direct its distinct functional outcomes. Our recent work using unnatural amino acid incorporation and fluorine-19 nuclear magnetic resonance ( 19 F-NMR) spectroscopy led to the flute model, which provides preliminary insight into the receptor phospho-coding mechanism, by which receptor phosphorylation patterns are recognized by an array of phosphate-binding pockets on arrestin and are translated into distinct conformations. These selective conformations are recognized by various effector molecules downstream of arrestin. The phospho-barcoding mechanism enables arrestin to recognize a wide range of phosphorylation patterns of GPCRs, contributing to their diverse functions. Copyright © 2017 by The Author(s).

  6. Arabidopsis G-protein interactome reveals connections to cell wall carbohydrates and morphogenesis.

    Science.gov (United States)

    Klopffleisch, Karsten; Phan, Nguyen; Augustin, Kelsey; Bayne, Robert S; Booker, Katherine S; Botella, Jose R; Carpita, Nicholas C; Carr, Tyrell; Chen, Jin-Gui; Cooke, Thomas Ryan; Frick-Cheng, Arwen; Friedman, Erin J; Fulk, Brandon; Hahn, Michael G; Jiang, Kun; Jorda, Lucia; Kruppe, Lydia; Liu, Chenggang; Lorek, Justine; McCann, Maureen C; Molina, Antonio; Moriyama, Etsuko N; Mukhtar, M Shahid; Mudgil, Yashwanti; Pattathil, Sivakumar; Schwarz, John; Seta, Steven; Tan, Matthew; Temp, Ulrike; Trusov, Yuri; Urano, Daisuke; Welter, Bastian; Yang, Jing; Panstruga, Ralph; Uhrig, Joachim F; Jones, Alan M

    2011-09-27

    The heterotrimeric G-protein complex is minimally composed of Gα, Gβ, and Gγ subunits. In the classic scenario, the G-protein complex is the nexus in signaling from the plasma membrane, where the heterotrimeric G-protein associates with heptahelical G-protein-coupled receptors (GPCRs), to cytoplasmic target proteins called effectors. Although a number of effectors are known in metazoans and fungi, none of these are predicted to exist in their canonical forms in plants. To identify ab initio plant G-protein effectors and scaffold proteins, we screened a set of proteins from the G-protein complex using two-hybrid complementation in yeast. After deep and exhaustive interrogation, we detected 544 interactions between 434 proteins, of which 68 highly interconnected proteins form the core G-protein interactome. Within this core, over half of the interactions comprising two-thirds of the nodes were retested and validated as genuine in planta. Co-expression analysis in combination with phenotyping of loss-of-function mutations in a set of core interactome genes revealed a novel role for G-proteins in regulating cell wall modification.

  7. DMPD: IRAK1: a critical signaling mediator of innate immunity. [Dynamic Macrophage Pathway CSML Database

    Lifescience Database Archive (English)

    Full Text Available 17890055 IRAK1: a critical signaling mediator of innate immunity. Gottipati S, Rao ...IRAK1: a critical signaling mediator of innate immunity. PubmedID 17890055 Title IRAK1: a critical signaling mediator

  8. Construction and Deciphering of Human Phosphorylation-Mediated Signaling Transduction Networks.

    Science.gov (United States)

    Zhang, Menghuan; Li, Hong; He, Ying; Sun, Han; Xia, Li; Wang, Lishun; Sun, Bo; Ma, Liangxiao; Zhang, Guoqing; Li, Jing; Li, Yixue; Xie, Lu

    2015-07-02

    Protein phosphorylation is the most abundant reversible covalent modification. Human protein kinases participate in almost all biological pathways, and approximately half of the kinases are associated with disease. PhoSigNet was designed to store and display human phosphorylation-mediated signal transduction networks, with additional information related to cancer. It contains 11 976 experimentally validated directed edges and 216 871 phosphorylation sites. Moreover, 3491 differentially expressed proteins in human cancer from dbDEPC, 18 907 human cancer variation sites from CanProVar, and 388 hyperphosphorylation sites from PhosphoSitePlus were collected as annotation information. Compared with other phosphorylation-related databases, PhoSigNet not only takes the kinase-substrate regulatory relationship pairs into account, but also extends regulatory relationships up- and downstream (e.g., from ligand to receptor, from G protein to kinase, and from transcription factor to targets). Furthermore, PhoSigNet allows the user to investigate the impact of phosphorylation modifications on cancer. By using one set of in-house time series phosphoproteomics data, the reconstruction of a conditional and dynamic phosphorylation-mediated signaling network was exemplified. We expect PhoSigNet to be a useful database and analysis platform benefiting both proteomics and cancer studies.

  9. GPCR-Mediated Signaling of Metabolites

    DEFF Research Database (Denmark)

    Husted, Anna Sofie; Trauelsen, Mette; Rudenko, Olga

    2017-01-01

    microbiota target primarily enteroendocrine, neuronal, and immune cells in the lamina propria of the gut mucosa and the liver and, through these tissues, the rest of the body. In contrast, metabolites from the intermediary metabolism act mainly as metabolic stress-induced autocrine and paracrine signals...... and obesity. The concept of key metabolites as ligands for specific GPCRs has broadened our understanding of metabolic signaling significantly and provides a number of novel potential drug targets....

  10. Internalization of G-protein-coupled receptors: Implication in receptor function, physiology and diseases.

    Science.gov (United States)

    Calebiro, Davide; Godbole, Amod

    2018-04-01

    G protein-coupled receptors (GPCRs) are the largest family of membrane receptors and mediate the effects of numerous hormones and neurotransmitters. The nearly 1000 GPCRs encoded by the human genome regulate virtually all physiological functions and are implicated in the pathogenesis of prevalent human diseases such as thyroid disorders, hypertension or Parkinson's disease. As a result, 30-50% of all currently prescribed drugs are targeting these receptors. Once activated, GPCRs induce signals at the cell surface. This is often followed by internalization, a process that results in the transfer of receptors from the plasma membrane to membranes of the endosomal compartment. Internalization was initially thought to be mainly implicated in signal desensitization, a mechanism of adaptation to prolonged receptor stimulation. However, several unexpected functions have subsequently emerged. Most notably, accumulating evidence indicates that internalization can induce prolonged receptor signaling on intracellular membranes, which is apparently required for at least some biological effects of hormones like TSH, LH and adrenaline. These findings reveal an even stronger connection between receptor internalization and signaling than previously thought. Whereas new studies are just beginning to reveal an important physiological role for GPCR signaling after internalization and ways to exploit it for therapeutic purposes, future investigations will be required to explore its involvement in human disease. Copyright © 2018 Elsevier Ltd. All rights reserved.

  11. Using constitutive activity to define appropriate high-throughput screening assays for orphan g protein-coupled receptors.

    Science.gov (United States)

    Ngo, Tony; Coleman, James L J; Smith, Nicola J

    2015-01-01

    Orphan G protein-coupled receptors represent an underexploited resource for drug discovery but pose a considerable challenge for assay development because their cognate G protein signaling pathways are often unknown. In this methodological chapter, we describe the use of constitutive activity, that is, the inherent ability of receptors to couple to their cognate G proteins in the absence of ligand, to inform the development of high-throughput screening assays for a particular orphan receptor. We specifically focus on a two-step process, whereby constitutive G protein coupling is first determined using yeast Gpa1/human G protein chimeras linked to growth and β-galactosidase generation. Coupling selectivity is then confirmed in mammalian cells expressing endogenous G proteins and driving accumulation of transcription factor-fused luciferase reporters specific to each of the classes of G protein. Based on these findings, high-throughput screening campaigns can be performed on the already miniaturized mammalian reporter system.

  12. Small G proteins Rac1 and Ras regulate serine/threonine protein phosphatase 5 (PP5)·extracellular signal-regulated kinase (ERK) complexes involved in the feedback regulation of Raf1.

    Science.gov (United States)

    Mazalouskas, Matthew D; Godoy-Ruiz, Raquel; Weber, David J; Zimmer, Danna B; Honkanen, Richard E; Wadzinski, Brian E

    2014-02-14

    Serine/threonine protein phosphatase 5 (PP5, PPP5C) is known to interact with the chaperonin heat shock protein 90 (HSP90) and is involved in the regulation of multiple cellular signaling cascades that control diverse cellular processes, such as cell growth, differentiation, proliferation, motility, and apoptosis. Here, we identify PP5 in stable complexes with extracellular signal-regulated kinases (ERKs). Studies using mutant proteins reveal that the formation of PP5·ERK1 and PP5·ERK2 complexes partially depends on HSP90 binding to PP5 but does not require PP5 or ERK1/2 activity. However, PP5 and ERK activity regulates the phosphorylation state of Raf1 kinase, an upstream activator of ERK signaling. Whereas expression of constitutively active Rac1 promotes the assembly of PP5·ERK1/2 complexes, acute activation of ERK1/2 fails to influence the phosphatase-kinase interaction. Introduction of oncogenic HRas (HRas(V12)) has no effect on PP5-ERK1 binding but selectively decreases the interaction of PP5 with ERK2, in a manner that is independent of PP5 and MAPK/ERK kinase (MEK) activity, yet paradoxically requires ERK2 activity. Additional studies conducted with oncogenic variants of KRas4B reveal that KRas(L61), but not KRas(V12), also decreases the PP5-ERK2 interaction. The expression of wild type HRas or KRas proteins fails to reduce PP5-ERK2 binding, indicating that the effect is specific to HRas(V12) and KRas(L61) gain-of-function mutations. These findings reveal a novel, differential responsiveness of PP5-ERK1 and PP5-ERK2 interactions to select oncogenic Ras variants and also support a role for PP5·ERK complexes in regulating the feedback phosphorylation of PP5-associated Raf1.

  13. G protein-coupled receptor kinase-3-deficient mice exhibit WHIM syndrome features and attenuated inflammatory responses

    Science.gov (United States)

    Tarrant, Teresa K.; Billard, Matthew J.; Timoshchenko, Roman G.; McGinnis, Marcus W.; Serafin, D. Stephen; Foreman, Oded; Esserman, Denise A.; Chao, Nelson J.; Lento, William E.; Lee, David M.; Patel, Dhavalkumar; Siderovski, David P.

    2013-01-01

    Chemokine receptor interactions coordinate leukocyte migration in inflammation. Chemokine receptors are GPCRs that when activated, are phosphorylated by GRKs to turn off G protein-mediated signaling yet recruit additional signaling machinery. Recently, GRK3 was identified as a negative regulator of CXCL12/CXCR4 signaling that is defective in human WHIM syndrome. Here, we report that GRK3−/− mice exhibit numerous features of human WHIM, such as impaired CXCL12-mediated desensitization, enhanced CXCR4 signaling to ERK activation, altered granulocyte migration, and a mild myelokathexis. Moreover, GRK3−/− protects mice from two acute models of inflammatory arthritis (K/BxN serum transfer and CAIA). In these granulocyte-dependent disease models, protection of GRK3−/− mice is mediated by retention of cells in the marrow, fewer circulating granulocytes in the peripheral blood, and reduced granulocytes in the joints during active inflammation. In contrast to WHIM, GRK3−/− mice have minimal hypogammaglobulinemia and a peripheral leukocytosis with increased lymphocytes and absent neutropenia. Thus, we conclude that the loss of GRK3-mediated regulation of CXCL12/CXCR4 signaling contributes to some, but not all, of the complete WHIM phenotype and that GRK3 inhibition may be beneficial in the treatment of inflammatory arthritis. PMID:23935208

  14. Mathematical modelling of SERK mediated BR signalling

    NARCIS (Netherlands)

    Esse, van G.W.

    2013-01-01

    Being sessile by nature plants are continuously challenged by biotic and abiotic stress factors. At the cellular level, different stimuli are perceived and translated to the desired response. In order to achieve this, signal transduction cascades have to be interlinked. Complex networks

  15. Presynaptic G Protein-Coupled Receptors: Gatekeepers of Addiction?

    Directory of Open Access Journals (Sweden)

    Kari A Johnson

    2016-11-01

    Full Text Available Drug abuse and addiction cause widespread social and public health problems, and the neurobiology underlying drug actions and drug use and abuse is an area of intensive research. Drugs of abuse alter synaptic transmission, and these actions contribute to acute intoxication as well as the chronic effects of abused substances. Transmission at most mammalian synapses involves neurotransmitter activation of two receptor subtypes, ligand-gated ion channels that mediate fast synaptic responses, and G protein-coupled receptors (GPCRs that have slower neuromodulatory actions. The GPCRs represent a large proportion of neurotransmitter receptors involved in almost all facets of nervous system function. In addition, these receptors are targets for many pharmacotherapeutic agents. Drugs of abuse directly or indirectly affect neuromodulation mediated by GPCRs, with important consequences for intoxication, drug taking and responses to prolonged drug exposure, withdrawal and addiction. Among the GPCRs are several subtypes involved in presynaptic inhibition, most of which are coupled to the Gi/o class of G protein. There is increasing evidence that these presynaptic Gi/o-coupled GPCRs have important roles in the actions of drugs of abuse, as well as behaviors related to these drugs. This topic will be reviewed, with particular emphasis on receptors for three neurotransmitters, dopamine (D1- and D2-like receptors, endocannabinoids (CB1 receptors and glutamate (group II metabotropic glutamate (mGlu receptors. The focus is on recent evidence from laboratory animal models (and some evidence in humans implicating these receptors in the acute and chronic effects of numerous abused drugs, as well as in the control of drug seeking and taking. The ability of drugs targeting these receptors to modify drug seeking behavior has raised the possibility of using compounds targeting these receptors for addiction pharmacotherapy. This topic is also discussed, with emphasis on

  16. Common variants in the G protein beta3 subunit gene and thyroid disorders in a formerly iodine-deficient population.

    Science.gov (United States)

    Völzke, Henry; Bornhorst, Alexa; Rimmbach, Christian; Petersenn, Holger; Geissler, Ingrid; Nauck, Matthias; Wallaschofski, Henri; Kroemer, Heyo K; Rosskopf, Dieter

    2009-10-01

    Heterotrimeric G proteins are key mediators of signals from membrane receptors-including the thyroid-stimulating hormone (TSH) receptor-to cellular effectors. Gain-of-function mutations in the TSH receptor and the Galpha(S) subunit occur frequently in hyperfunctioning thyroid nodules and differentiated thyroid carcinomas, whereby the T allele of a common polymorphism (825C>T, rs5443) in the G protein beta3 subunit gene (GNB3) is associated with increased G protein-mediated signal transduction and a complex phenotype. The aim of this study was to investigate whether this common polymorphism affects key parameters of thyroid function and morphology and influences the pathogenesis of thyroid diseases in the general population. The population-based cross-sectional Study of Health in Pomerania is a general health survey with focus on thyroid diseases in northeast Germany, a formerly iodine-deficient area. Data from 3428 subjects (1800 men and 1628 women) were analyzed for an association of the GNB3 genotype with TSH, free triiodothyronine and thyroxine levels, urine iodine and thiocyanate excretion, and thyroid ultrasound morphology including thyroid volume, presence of goiter, and thyroid nodules. There was no association between GNB3 genotype status and the functional or morphological thyroid parameters investigated, neither in crude analyses nor upon multivariable analyses including known confounders of thyroid disorders. Based on the data from this large population-based survey, we conclude that the GNB3 825C>T polymorphism does not affect key parameters of thyroid function and morphology in the general population of a formerly iodine-deficient area.

  17. Conformational Profiling of the AT1 Angiotensin II Receptor Reflects Biased Agonism, G Protein Coupling, and Cellular Context.

    Science.gov (United States)

    Devost, Dominic; Sleno, Rory; Pétrin, Darlaine; Zhang, Alice; Shinjo, Yuji; Okde, Rakan; Aoki, Junken; Inoue, Asuka; Hébert, Terence E

    2017-03-31

    Here, we report the design and use of G protein-coupled receptor-based biosensors to monitor ligand-mediated conformational changes in receptors in intact cells. These biosensors use bioluminescence resonance energy transfer with Renilla luciferase (RlucII) as an energy donor, placed at the distal end of the receptor C-tail, and the small fluorescent molecule FlAsH as an energy acceptor, its binding site inserted at different positions throughout the intracellular loops and C-terminal tail of the angiotensin II type I receptor. We verified that the modifications did not compromise receptor localization or function before proceeding further. Our biosensors were able to capture effects of both canonical and biased ligands, even to the extent of discriminating between different biased ligands. Using a combination of G protein inhibitors and HEK 293 cell lines that were CRISPR/Cas9-engineered to delete Gα q , Gα 11 , Gα 12 , and Gα 13 or β-arrestins, we showed that Gα q and Gα 11 are required for functional responses in conformational sensors in ICL3 but not ICL2. Loss of β-arrestin did not alter biased ligand effects on ICL2P2. We also demonstrate that such biosensors are portable between different cell types and yield context-dependent readouts of G protein-coupled receptor conformation. Our study provides mechanistic insights into signaling events that depend on either G proteins or β-arrestin. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  18. The Biased G-Protein-Coupled Receptor Agonism Bridges the Gap between the Insulin Receptor and the Metabolic Syndrome

    Science.gov (United States)

    Liauchonak, Iryna; Dawoud, Fady; Riat, Yatin; Sambi, Manpreet; Jain, Justin; Kalaydina, Regina-Veronicka; Mendonza, Nicole; Bajwa, Komal

    2018-01-01

    Insulin signaling, as mediated through the insulin receptor (IR), plays a critical role in metabolism. Aberrations in this signaling cascade lead to several pathologies, the majority of which are classified under the umbrella term “metabolic syndrome”. Although many of these pathologies are associated with insulin resistance, the exact mechanisms are not well understood. One area of current interest is the possibility of G-protein-coupled receptors (GPCRs) influencing or regulating IR signaling. This concept is particularly significant, because GPCRs have been shown to participate in cross-talk with the IR. More importantly, GPCR signaling has also been shown to preferentially regulate specific downstream signaling targets through GPCR agonist bias. A novel study recently demonstrated that this GPCR-biased agonism influences the activity of the IR without the presence of insulin. Although GPCR-IR cross-talk has previously been established, the notion that GPCRs can regulate the activation of the IR is particularly significant in relation to metabolic syndrome and other pathologies that develop as a result of alterations in IR signaling. As such, we aim to provide an overview of the physiological and pathophysiological roles of the IR within metabolic syndrome and its related pathologies, including cardiovascular health, gut microflora composition, gastrointestinal tract functioning, polycystic ovarian syndrome, pancreatic cancer, and neurodegenerative disorders. Furthermore, we propose that the GPCR-biased agonism may perhaps mediate some of the downstream signaling effects that further exacerbate these diseases for which the mechanisms are currently not well understood. PMID:29462993

  19. G Protein-coupled Receptor Kinases of the GRK4 Protein Subfamily Phosphorylate Inactive G Protein-coupled Receptors (GPCRs).

    Science.gov (United States)

    Li, Lingyong; Homan, Kristoff T; Vishnivetskiy, Sergey A; Manglik, Aashish; Tesmer, John J G; Gurevich, Vsevolod V; Gurevich, Eugenia V

    2015-04-24

    G protein-coupled receptor (GPCR) kinases (GRKs) play a key role in homologous desensitization of GPCRs. It is widely assumed that most GRKs selectively phosphorylate only active GPCRs. Here, we show that although this seems to be the case for the GRK2/3 subfamily, GRK5/6 effectively phosphorylate inactive forms of several GPCRs, including β2-adrenergic and M2 muscarinic receptors, which are commonly used as representative models for GPCRs. Agonist-independent GPCR phosphorylation cannot be explained by constitutive activity of the receptor or membrane association of the GRK, suggesting that it is an inherent ability of GRK5/6. Importantly, phosphorylation of the inactive β2-adrenergic receptor enhanced its interactions with arrestins. Arrestin-3 was able to discriminate between phosphorylation of the same receptor by GRK2 and GRK5, demonstrating preference for the latter. Arrestin recruitment to inactive phosphorylated GPCRs suggests that not only agonist activation but also the complement of GRKs in the cell regulate formation of the arrestin-receptor complex and thereby G protein-independent signaling. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  20. X-ray structure of the mammalian GIRK2-βγ G-protein complex

    Energy Technology Data Exchange (ETDEWEB)

    Whorton, Matthew R.; MacKinnon, Roderick [Rockefeller

    2013-07-30

    G-protein-gated inward rectifier K+ (GIRK) channels allow neurotransmitters, through G-protein-coupled receptor stimulation, to control cellular electrical excitability. In cardiac and neuronal cells this control regulates heart rate and neural circuit activity, respectively. Here we present the 3.5Å resolution crystal structure of the mammalian GIRK2 channel in complex with βγ G-protein subunits, the central signalling complex that links G-protein-coupled receptor stimulation to K+ channel activity. Short-range atomic and long-range electrostatic interactions stabilize four βγ G-protein subunits at the interfaces between four K+ channel subunits, inducing a pre-open state of the channel. The pre-open state exhibits a conformation that is intermediate between the closed conformation and the open conformation of the constitutively active mutant. The resultant structural picture is compatible with ‘membrane delimited’ activation of GIRK channels by G proteins and the characteristic burst kinetics of channel gating. The structures also permit a conceptual understanding of how the signalling lipid phosphatidylinositol-4,5-bisphosphate (PIP2) and intracellular Na+ ions participate in multi-ligand regulation of GIRK channels.

  1. Tissue- and subunit-specific regulation of G-protein expression by hypo- and hyperthyroidism

    NARCIS (Netherlands)

    Michel-Reher, M. B.; Gross, G.; Jasper, J. R.; Bernstein, D.; Olbricht, T.; Brodde, O. E.; Michel, M. C.

    1993-01-01

    Thyroid hormone status has profound effects on signal transduction in various tissues throughout the body. Therefore, we quantified the signal transducing G-proteins in the rat heart, cerebral cortex, vas deferens and liver by immunoblotting and pertussis toxin labeling in response to chemically

  2. Growth Factor Mediated Signaling in Pancreatic Pathogenesis

    Energy Technology Data Exchange (ETDEWEB)

    Nandy, Debashis; Mukhopadhyay, Debabrata, E-mail: mukhopadhyay.debabrata@mayo.edu [Department of Biochemistry and Molecular Biology, College of Medicine, Mayo Clinic, 200 First Street SW, Guggenheim 1321C, Rochester, MN 55905 (United States)

    2011-02-24

    Functionally, the pancreas consists of two types of tissues: exocrine and endocrine. Exocrine pancreatic disorders mainly involve acute and chronic pancreatitis. Acute pancreatitis typically is benign, while chronic pancreatitis is considered a risk factor for developing pancreatic cancer. Pancreatic carcinoma is the fourth leading cause of cancer related deaths worldwide. Most pancreatic cancers develop in the exocrine tissues. Endocrine pancreatic tumors are more uncommon, and typically are less aggressive than exocrine tumors. However, the endocrine pancreatic disorder, diabetes, is a dominant cause of morbidity and mortality. Importantly, different growth factors and their receptors play critical roles in pancreatic pathogenesis. Hence, an improved understanding of how various growth factors affect pancreatitis and pancreatic carcinoma is necessary to determine appropriate treatment. This chapter describes the role of different growth factors such as vascular endothelial growth factor (VEGF), insulin-like growth factor (IGF), platelet derived growth factor (PDGF), fibroblast growth factor (FGF), epidermal growth factor (EGF), and transforming growth factor (TGF) in various pancreatic pathophysiologies. Finally, the crosstalk between different growth factor axes and their respective signaling mechanisms, which are involved in pancreatitis and pancreatic carcinoma, are also discussed.

  3. Growth Factor Mediated Signaling in Pancreatic Pathogenesis

    International Nuclear Information System (INIS)

    Nandy, Debashis; Mukhopadhyay, Debabrata

    2011-01-01

    Functionally, the pancreas consists of two types of tissues: exocrine and endocrine. Exocrine pancreatic disorders mainly involve acute and chronic pancreatitis. Acute pancreatitis typically is benign, while chronic pancreatitis is considered a risk factor for developing pancreatic cancer. Pancreatic carcinoma is the fourth leading cause of cancer related deaths worldwide. Most pancreatic cancers develop in the exocrine tissues. Endocrine pancreatic tumors are more uncommon, and typically are less aggressive than exocrine tumors. However, the endocrine pancreatic disorder, diabetes, is a dominant cause of morbidity and mortality. Importantly, different growth factors and their receptors play critical roles in pancreatic pathogenesis. Hence, an improved understanding of how various growth factors affect pancreatitis and pancreatic carcinoma is necessary to determine appropriate treatment. This chapter describes the role of different growth factors such as vascular endothelial growth factor (VEGF), insulin-like growth factor (IGF), platelet derived growth factor (PDGF), fibroblast growth factor (FGF), epidermal growth factor (EGF), and transforming growth factor (TGF) in various pancreatic pathophysiologies. Finally, the crosstalk between different growth factor axes and their respective signaling mechanisms, which are involved in pancreatitis and pancreatic carcinoma, are also discussed

  4. A Dictyostelium chalone uses G proteins to regulate proliferation

    Directory of Open Access Journals (Sweden)

    Hanson Nana E

    2009-07-01

    Full Text Available Abstract Background Several studies have shown that organ size, and the proliferation of tumor metastases, may be regulated by negative feedback loops in which autocrine secreted factors called chalones inhibit proliferation. However, very little is known about chalones, and how cells sense them. We previously identified two secreted proteins, AprA and CfaD, which act as chalones in Dictyostelium. Cells lacking AprA or CfaD proliferate faster than wild-type cells, and adding recombinant AprA or CfaD to cells slows their proliferation. Results We show here that cells lacking the G protein components Galpha8, Galpha9, and Gbeta proliferate faster than wild-type cells despite secreting normal or high levels of AprA and CfaD. Compared with wild-type cells, the proliferation of galpha8-, galpha9- and gbeta- cells are only weakly inhibited by recombinant AprA (rAprA. Like AprA and CfaD, Galpha8 and Gbeta inhibit cell proliferation but not cell growth (the rate of increase in mass and protein per nucleus, whereas Galpha9 inhibits both proliferation and growth. galpha8- cells show normal cell-surface binding of rAprA, whereas galpha9- and gbeta- cells have fewer cell-surface rAprA binding sites, suggesting that Galpha9 and Gbeta regulate the synthesis or processing of the AprA receptor. Like other ligands that activate G proteins, rAprA induces the binding of [3H]GTP to membranes, and GTPgammaS inhibits the binding of rAprA to membranes. Both AprA-induced [3H]GTP binding and the GTPgammaS inhibition of rAprA binding require Galpha8 and Gbeta but not Galpha9. Like aprA- cells, galpha8- cells have reduced spore viability. Conclusion This study shows that Galpha8 and Gbeta are part of the signal transduction pathway used by AprA to inhibit proliferation but not growth in Dictyostelium, whereas Galpha9 is part of a differealnt pathway that regulates both proliferation and growth, and that a chalone signal transduction pathway uses G proteins.

  5. A Dictyostelium chalone uses G proteins to regulate proliferation.

    Science.gov (United States)

    Bakthavatsalam, Deenadayalan; Choe, Jonathan M; Hanson, Nana E; Gomer, Richard H

    2009-07-27

    Several studies have shown that organ size, and the proliferation of tumor metastases, may be regulated by negative feedback loops in which autocrine secreted factors called chalones inhibit proliferation. However, very little is known about chalones, and how cells sense them. We previously identified two secreted proteins, AprA and CfaD, which act as chalones in Dictyostelium. Cells lacking AprA or CfaD proliferate faster than wild-type cells, and adding recombinant AprA or CfaD to cells slows their proliferation. We show here that cells lacking the G protein components Galpha8, Galpha9, and Gbeta proliferate faster than wild-type cells despite secreting normal or high levels of AprA and CfaD. Compared with wild-type cells, the proliferation of galpha8-, galpha9- and gbeta- cells are only weakly inhibited by recombinant AprA (rAprA). Like AprA and CfaD, Galpha8 and Gbeta inhibit cell proliferation but not cell growth (the rate of increase in mass and protein per nucleus), whereas Galpha9 inhibits both proliferation and growth. galpha8- cells show normal cell-surface binding of rAprA, whereas galpha9- and gbeta- cells have fewer cell-surface rAprA binding sites, suggesting that Galpha9 and Gbeta regulate the synthesis or processing of the AprA receptor. Like other ligands that activate G proteins, rAprA induces the binding of [3H]GTP to membranes, and GTPgammaS inhibits the binding of rAprA to membranes. Both AprA-induced [3H]GTP binding and the GTPgammaS inhibition of rAprA binding require Galpha8 and Gbeta but not Galpha9. Like aprA- cells, galpha8- cells have reduced spore viability. This study shows that Galpha8 and Gbeta are part of the signal transduction pathway used by AprA to inhibit proliferation but not growth in Dictyostelium, whereas Galpha9 is part of a differealnt pathway that regulates both proliferation and growth, and that a chalone signal transduction pathway uses G proteins.

  6. Exploring allosteric coupling in the α-subunit of Heterotrimeric G proteins using evolutionary and ensemble-based approaches

    Directory of Open Access Journals (Sweden)

    Hilser Vincent J

    2008-05-01

    Full Text Available Abstract Background Allosteric coupling, which can be defined as propagation of a perturbation at one region of the protein molecule (such as ligand binding to distant sites in the same molecule, constitutes the most general mechanism of regulation of protein function. However, unlike molecular details of ligand binding, structural elements involved in allosteric effects are difficult to diagnose. Here, we identified allosteric linkages in the α-subunits of heterotrimeric G proteins, which were evolved to transmit membrane receptor signals by allosteric mechanisms, by using two different approaches that utilize fundamentally different and independent information. Results We analyzed: 1 correlated mutations in the family of G protein α-subunits, and 2 cooperativity of the native state ensemble of the Gαi1 or transducin. The combination of these approaches not only recovered already-known details such as the switch regions that change conformation upon nucleotide exchange, and those regions that are involved in receptor, effector or Gβγ interactions (indicating that the predictions of the analyses can be viewed with a measure of confidence, but also predicted new sites that are potentially involved in allosteric communication in the Gα protein. A summary of the new sites found in the present analysis, which were not apparent in crystallographic data, is given along with known functional and structural information. Implications of the results are discussed. Conclusion A set of residues and/or structural elements that are potentially involved in allosteric communication in Gα is presented. This information can be used as a guide to structural, spectroscopic, mutational, and theoretical studies on the allosteric network in Gα proteins, which will provide a better understanding of G protein-mediated signal transduction.

  7. G Protein-Coupled Receptors: What a Difference a ‘Partner’ Makes

    Directory of Open Access Journals (Sweden)

    Benoît T. Roux

    2014-01-01

    Full Text Available G protein-coupled receptors (GPCRs are important cell signaling mediators, involved in essential physiological processes. GPCRs respond to a wide variety of ligands from light to large macromolecules, including hormones and small peptides. Unfortunately, mutations and dysregulation of GPCRs that induce a loss of function or alter expression can lead to disorders that are sometimes lethal. Therefore, the expression, trafficking, signaling and desensitization of GPCRs must be tightly regulated by different cellular systems to prevent disease. Although there is substantial knowledge regarding the mechanisms that regulate the desensitization and down-regulation of GPCRs, less is known about the mechanisms that regulate the trafficking and cell-surface expression of newly synthesized GPCRs. More recently, there is accumulating evidence that suggests certain GPCRs are able to interact with specific proteins that can completely change their fate and function. These interactions add on another level of regulation and flexibility between different tissue/cell-types. Here, we review some of the main interacting proteins of GPCRs. A greater understanding of the mechanisms regulating their interactions may lead to the discovery of new drug targets for therapy.

  8. The cAMP-induced G protein subunits dissociation monitored in live Dictyostelium cells by BRET reveals two activation rates, a positive effect of caffeine and potential role of microtubules.

    Science.gov (United States)

    Tariqul Islam, A F M; Yue, Haicen; Scavello, Margarethakay; Haldeman, Pearce; Rappel, Wouter-Jan; Charest, Pascale G

    2018-08-01

    To study the dynamics and mechanisms controlling activation of the heterotrimeric G protein Gα2βγ in Dictyostelium in response to stimulation by the chemoattractant cyclic AMP (cAMP), we monitored the G protein subunit interaction in live cells using bioluminescence resonance energy transfer (BRET). We found that cAMP induces the cAR1-mediated dissociation of the G protein subunits to a similar extent in both undifferentiated and differentiated cells, suggesting that only a small number of cAR1 (as expressed in undifferentiated cells) is necessary to induce the full activation of Gα2βγ. In addition, we found that treating cells with caffeine increases the potency of cAMP-induced Gα2βγ activation; and that disrupting the microtubule network but not F-actin inhibits the cAMP-induced dissociation of Gα2βγ. Thus, microtubules are necessary for efficient cAR1-mediated activation of the heterotrimeric G protein. Finally, kinetics analyses of Gα2βγ subunit dissociation induced by different cAMP concentrations indicate that there are two distinct rates at which the heterotrimeric G protein subunits dissociate when cells are stimulated with cAMP concentrations above 500 nM versus only one rate at lower cAMP concentrations. Quantitative modeling suggests that the kinetics profile of Gα2βγ subunit dissociation results from the presence of both uncoupled and G protein pre-coupled cAR1 that have differential affinities for cAMP and, consequently, induce G protein subunit dissociation through different rates. We suggest that these different signaling kinetic profiles may play an important role in initial chemoattractant gradient sensing. Copyright © 2018 Elsevier Inc. All rights reserved.

  9. Constitutive Activity among Orphan Class-A G Protein Coupled Receptors.

    Directory of Open Access Journals (Sweden)

    Adam L Martin

    Full Text Available The purpose of this study was to evaluate the extent of constitutive activity among orphan class-A G protein coupled receptors within the cAMP signaling pathway. Constitutive signaling was revealed by changes in gene expression under control of the cAMP response element. Gene expression was measured in Chinese hamster ovary cells transiently co-transfected with plasmids containing a luciferase reporter and orphan receptor. Criteria adopted for defining constitutive activation were: 1 200% elevation over baseline reporter gene expression; 2 40% inhibition of baseline expression; and 3 40% inhibition of expression stimulated by 3 μM forskolin. Five patterns of activity were noted: 1 inhibition under both baseline and forskolin stimulated expression (GPR15, GPR17, GPR18, GPR20, GPR25, GPR27, GPR31, GPR32, GPR45, GPR57, GPR68, GPR83, GPR84, GPR132, GPR150, GPR176; 2 no effect on baseline expression, but inhibition of forskolin stimulated expression (GPR4, GPR26, GPR61, GPR62, GPR78, GPR101, GPR119; 3 elevation of baseline signaling coupled with inhibition of forskolin stimulated expression (GPR6, GPR12; 4 elevation of baseline signaling without inhibition of forskolin stimulated expression (GPR3, GPR21, GPR52, GPR65; and 5 no effect on expression (GPR1, GPR19, GPR22, GPR34, GPR35, GPR39, GPR63, GPR82, GPR85, GPR87. Constitutive activity was observed in 75% of the orphan class-A receptors examined (30 of 40. This constitutive signaling cannot be explained by simple overexpression of the receptor. Inhibition of cAMP mediated expression was far more common (65% than stimulation of expression (15%. Orphan receptors that were closely related based on amino acid homology tended to have similar effects on gene expression. These results suggest that identification of inverse agonists may be a fruitful approach for categorizing these orphan receptors and targeting them for pharmacological intervention.

  10. Constitutive Activity among Orphan Class-A G Protein Coupled Receptors.

    Science.gov (United States)

    Martin, Adam L; Steurer, Michael A; Aronstam, Robert S

    2015-01-01

    The purpose of this study was to evaluate the extent of constitutive activity among orphan class-A G protein coupled receptors within the cAMP signaling pathway. Constitutive signaling was revealed by changes in gene expression under control of the cAMP response element. Gene expression was measured in Chinese hamster ovary cells transiently co-transfected with plasmids containing a luciferase reporter and orphan receptor. Criteria adopted for defining constitutive activation were: 1) 200% elevation over baseline reporter gene expression; 2) 40% inhibition of baseline expression; and 3) 40% inhibition of expression stimulated by 3 μM forskolin. Five patterns of activity were noted: 1) inhibition under both baseline and forskolin stimulated expression (GPR15, GPR17, GPR18, GPR20, GPR25, GPR27, GPR31, GPR32, GPR45, GPR57, GPR68, GPR83, GPR84, GPR132, GPR150, GPR176); 2) no effect on baseline expression, but inhibition of forskolin stimulated expression (GPR4, GPR26, GPR61, GPR62, GPR78, GPR101, GPR119); 3) elevation of baseline signaling coupled with inhibition of forskolin stimulated expression (GPR6, GPR12); 4) elevation of baseline signaling without inhibition of forskolin stimulated expression (GPR3, GPR21, GPR52, GPR65); and 5) no effect on expression (GPR1, GPR19, GPR22, GPR34, GPR35, GPR39, GPR63, GPR82, GPR85, GPR87). Constitutive activity was observed in 75% of the orphan class-A receptors examined (30 of 40). This constitutive signaling cannot be explained by simple overexpression of the receptor. Inhibition of cAMP mediated expression was far more common (65%) than stimulation of expression (15%). Orphan receptors that were closely related based on amino acid homology tended to have similar effects on gene expression. These results suggest that identification of inverse agonists may be a fruitful approach for categorizing these orphan receptors and targeting them for pharmacological intervention.

  11. Nanobody-Enabled Reverse Pharmacology on G-Protein-Coupled Receptors.

    Science.gov (United States)

    Pardon, Els; Betti, Cecilia; Laeremans, Toon; Chevillard, Florent; Guillemyn, Karel; Kolb, Peter; Ballet, Steven; Steyaert, Jan

    2018-05-04

    The conformational complexity of transmembrane signaling of G-protein-coupled receptors (GPCRs) is a central hurdle for the design of screens for receptor agonists. In their basal states, GPCRs have lower affinities for agonists compared to their G-protein-bound active state conformations. Moreover, different agonists can stabilize distinct active receptor conformations and do not uniformly activate all cellular signaling pathways linked to a given receptor (agonist bias). Comparative fragment screens were performed on a β 2 -adrenoreceptor-nanobody fusion locked in its active-state conformation by a G-protein-mimicking nanobody, and the same receptor in its basal-state conformation. This simple biophysical assay allowed the identification and ranking of multiple novel agonists and permitted classification of the efficacy of each hit in agonist, antagonist, or inverse agonist categories, thereby opening doors to nanobody-enabled reverse pharmacology. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  12. Structure, function, pharmacology and therapeutic potential of the G protein, Gα/q,11

    Directory of Open Access Journals (Sweden)

    Danielle eKamato

    2015-03-01

    Full Text Available G protein coupled receptors are one of the major classes of cell surface receptors and are associated with a group of G proteins consisting of 3 subunits termed alpha, beta and gamma. G proteins are classified into four families according to their α subunit; Gαi, Gαs, Gα12/13 and Gαq. There are several downstream pathways of Gαq of which the best known is upon activation via GTP, Gαq activates phospholipase Cβ, hydrolysing phosphatidylinositol 4,5-biphosphate into diacylglycerol and inositol triphosphate and activating protein kinase C and increasing calcium efflux from the endoplasmic recticulum. Although G proteins, in particular the Gαq/11 are central elements in GPCR signalling, their actual roles have not yet been thoroughly investigated. The lack of research of the role on Gαq/11 in cell biology is partially due to the obscure nature of the available pharmacological agents. YM-254890 is the most useful Gαq-selective inhibitor with antiplatelet, antithrombotic and thrombolytic effects. YM-254890 inhibits Gαq signalling pathways by preventing the exchange of GDP for GTP. UBO-QIC is a structurally similar compound to YM-254890 which can inhibit platelet aggregation and cause vasorelaxation in rats. Many agents are available for the study of signalling downstream of Gαq/11. The role of G proteins could potentially represents a novel therapeutic target to block all G protein dependent mechanisms. This review will explore the range of pharmacological and molecular tools available for the study of the role of Gαq/11 in GPCR signalling.

  13. Quaternary structure of a G-protein-coupled receptor heterotetramer in complex with Gi and Gs.

    Science.gov (United States)

    Navarro, Gemma; Cordomí, Arnau; Zelman-Femiak, Monika; Brugarolas, Marc; Moreno, Estefania; Aguinaga, David; Perez-Benito, Laura; Cortés, Antoni; Casadó, Vicent; Mallol, Josefa; Canela, Enric I; Lluís, Carme; Pardo, Leonardo; García-Sáez, Ana J; McCormick, Peter J; Franco, Rafael

    2016-04-05

    G-protein-coupled receptors (GPCRs), in the form of monomers or homodimers that bind heterotrimeric G proteins, are fundamental in the transfer of extracellular stimuli to intracellular signaling pathways. Different GPCRs may also interact to form heteromers that are novel signaling units. Despite the exponential growth in the number of solved GPCR crystal structures, the structural properties of heteromers remain unknown. We used single-particle tracking experiments in cells expressing functional adenosine A1-A2A receptors fused to fluorescent proteins to show the loss of Brownian movement of the A1 receptor in the presence of the A2A receptor, and a preponderance of cell surface 2:2 receptor heteromers (dimer of dimers). Using computer modeling, aided by bioluminescence resonance energy transfer assays to monitor receptor homomerization and heteromerization and G-protein coupling, we predict the interacting interfaces and propose a quaternary structure of the GPCR tetramer in complex with two G proteins. The combination of results points to a molecular architecture formed by a rhombus-shaped heterotetramer, which is bound to two different interacting heterotrimeric G proteins (Gi and Gs). These novel results constitute an important advance in understanding the molecular intricacies involved in GPCR function.

  14. Selectivity determinants of GPCR-G-protein binding

    DEFF Research Database (Denmark)

    Flock, Tilman; Hauser, Alexander S; Lund, Nadia

    2017-01-01

    of the G-protein barcode through distinct residues, like multiple keys (receptors) opening the same lock (G protein) using non-identical cuts. Considering the evolutionary history of GPCRs allows the identification of these selectivity-determining residues. These findings lay the foundation...

  15. Molecular basis of cannabinoid CB1 receptor coupling to the G protein heterotrimer Gαiβγ: identification of key CB1 contacts with the C-terminal helix α5 of Gαi.

    Science.gov (United States)

    Shim, Joong-Youn; Ahn, Kwang H; Kendall, Debra A

    2013-11-08

    The cannabinoid (CB1) receptor is a member of the rhodopsin-like G protein-coupled receptor superfamily. The human CB1 receptor, which is among the most expressed receptors in the brain, has been implicated in several disease states, including drug addiction, anxiety, depression, obesity, and chronic pain. Different classes of CB1 agonists evoke signaling pathways through the activation of specific subtypes of G proteins. The molecular basis of CB1 receptor coupling to its cognate G protein is unknown. As a first step toward understanding CB1 receptor-mediated G protein signaling, we have constructed a ternary complex structural model of the CB1 receptor and Gi heterotrimer (CB1-Gi), guided by the x-ray structure of β2-adrenergic receptor (β2AR) in complex with Gs (β2AR-Gs), through 824-ns duration molecular dynamics simulations in a fully hydrated 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine bilayer environment. We identified a group of residues at the juxtamembrane regions of the intracellular loops 2 and 3 (IC2 and IC3) of the CB1 receptor, including Ile-218(3.54), Tyr-224(IC2), Asp-338(6.30), Arg-340(6.32), Leu-341(6.33), and Thr-344(6.36), as potential key contacts with the extreme C-terminal helix α5 of Gαi. Ala mutations of these residues at the receptor-Gi interface resulted in little G protein coupling activity, consistent with the present model of the CB1-Gi complex, which suggests tight interactions between CB1 and the extreme C-terminal helix α5 of Gαi. The model also suggests that unique conformational changes in the extreme C-terminal helix α5 of Gα play a crucial role in the receptor-mediated G protein activation.

  16. Drug discovery opportunities and challenges at G protein coupled receptors for long chain free fatty acids

    Directory of Open Access Journals (Sweden)

    Nicholas D Holliday

    2012-01-01

    Full Text Available Discovery of G protein coupled receptors for long chain free fatty acids (FFAs, FFA1 (GPR40 and GPR120, has expanded our understanding of these nutrients as signalling molecules. These receptors have emerged as important sensors for FFA levels in the circulation or the gut lumen, based on evidence from in vitro and rodent models, and an increasing number of human studies. Here we consider their promise as therapeutic targets for metabolic disease, including type 2 diabetes and obesity. FFA1 directly mediates acute FFA-induced glucose-stimulated insulin secretion in pancreatic beta-cells, while GPR120 and FFA1 trigger release of incretins from intestinal endocrine cells, and so indirectly enhance insulin secretion and promote satiety. GPR120 signalling in adipocytes and macrophages also results in insulin sensitizing and beneficial anti-inflammatory effects. Drug discovery has focussed on agonists to replicate acute benefits of FFA receptor signalling, with promising early results for FFA1 agonists in man. Controversy surrounding chronic effects of FFA1 on beta-cells illustrates that long term benefits of antagonists also need exploring. It has proved challenging to generate highly selective potent ligands for FFA1 or GPR120 subtypes, given that both receptors have hydrophobic orthosteric binding sites, which are not completely defined and have modest ligand affinity. Structure activity relationships are also reliant on functional read outs, in the absence of robust binding assays to provide direct affinity estimates. Nevertheless synthetic ligands have already helped dissect specific contributions of FFA1 and GPR120 signalling from the many possible cellular effects of FFAs. Approaches including use of fluorescent ligand binding assays, and targeting allosteric receptor sites, may improve further preclinical ligand development at these receptors, to exploit their unique potential to target multiple facets of diabetes.

  17. Root signals that mediate mutualistic interactions in the rhizosphere.

    Science.gov (United States)

    Rasmann, Sergio; Turlings, Ted Cj

    2016-08-01

    A recent boom in research on belowground ecology is rapidly revealing a multitude of fascinating interactions, in particular in the rhizosphere. Many of these interactions are mediated by photo-assimilates that are excreted by plant roots. Root exudates are not mere waste products, but serve numerous functions to control abiotic and biotic processes. These functions range from changing the chemical and physical properties of the soil, inhibiting the growth of competing plants, combatting herbivores, and regulating the microbial community. Particularly intriguing are root-released compounds that have evolved to serve mutualistic interactions with soil-dwelling organisms. These mutually beneficial plant-mediated signals are not only of fundamental ecological interest, but also exceedingly important from an agronomical perspective. Here, we attempt to provide an overview of the plant-produced compounds that have so far been implicated in mutualistic interactions. We propose that these mutualistic signals may have evolved from chemical defenses and we point out that they can be (mis)used by specialized pathogens and herbivores. We speculate that many more signals and interactions remain to be uncovered and that a good understanding of the mechanisms and ecological implications can be the basis for exploitation and manipulation of the signals for crop improvement and protection. Copyright © 2016 Elsevier Ltd. All rights reserved.

  18. Conformational biosensors reveal GPCR signalling from endosomes

    DEFF Research Database (Denmark)

    Irannejad, R; Tomshine, Jin C; Tomshine, Jon R

    2013-01-01

    A long-held tenet of molecular pharmacology is that canonical signal transduction mediated by G-protein-coupled receptor (GPCR) coupling to heterotrimeric G proteins is confined to the plasma membrane. Evidence supporting this traditional view is based on analytical methods that provide limited...... or no subcellular resolution. It has been subsequently proposed that signalling by internalized GPCRs is restricted to G-protein-independent mechanisms such as scaffolding by arrestins, or GPCR activation elicits a discrete form of persistent G protein signalling, or that internalized GPCRs can indeed contribute...... in the early endosome membrane, and that internalized receptors contribute to the overall cellular cyclic AMP response within several minutes after agonist application. These findings provide direct support for the hypothesis that canonical GPCR signalling occurs from endosomes as well as the plasma membrane...

  19. G protein-membrane interactions II: Effect of G protein-linked lipids on membrane structure and G protein-membrane interactions.

    Science.gov (United States)

    Casas, Jesús; Ibarguren, Maitane; Álvarez, Rafael; Terés, Silvia; Lladó, Victoria; Piotto, Stefano P; Concilio, Simona; Busquets, Xavier; López, David J; Escribá, Pablo V

    2017-09-01

    G proteins often bear myristoyl, palmitoyl and isoprenyl moieties, which favor their association with the membrane and their accumulation in G Protein Coupled Receptor-rich microdomains. These lipids influence the biophysical properties of membranes and thereby modulate G protein binding to bilayers. In this context, we showed here that geranylgeraniol, but neither myristate nor palmitate, increased the inverted hexagonal (H II ) phase propensity of phosphatidylethanolamine-containing membranes. While myristate and palmitate preferentially associated with phosphatidylcholine membranes, geranylgeraniol favored nonlamellar-prone membranes. In addition, Gαi 1 monomers had a higher affinity for lamellar phases, while Gβγ and Gαβγ showed a marked preference for nonlamellar prone membranes. Moreover, geranylgeraniol enhanced the binding of G protein dimers and trimers to phosphatidylethanolamine-containing membranes, yet it decreased that of monomers. By contrast, both myristate and palmitate increased the Gαi 1 preference for lamellar membranes. Palmitoylation reinforced the binding of the monomer to PC membranes and myristoylation decreased its binding to PE-enriched bilayer. Finally, binding of dimers and trimers to lamellar-prone membranes was decreased by palmitate and myristate, but it was increased in nonlamellar-prone bilayers. These results demonstrate that co/post-translational G protein lipid modifications regulate the membrane lipid structure and that they influence the physico-chemical properties of membranes, which in part explains why G protein subunits sort to different plasma membrane domains. This article is part of a Special Issue entitled: Membrane Lipid Therapy: Drugs Targeting Biomembranes edited by Pablo V. Escribá. Copyright © 2017 Elsevier B.V. All rights reserved.

  20. Regulation of G Protein-Coupled Receptors by Ubiquitination

    Directory of Open Access Journals (Sweden)

    Kamila Skieterska

    2017-04-01

    Full Text Available G protein-coupled receptors (GPCRs comprise the largest family of membrane receptors that control many cellular processes and consequently often serve as drug targets. These receptors undergo a strict regulation by mechanisms such as internalization and desensitization, which are strongly influenced by posttranslational modifications. Ubiquitination is a posttranslational modification with a broad range of functions that is currently gaining increased appreciation as a regulator of GPCR activity. The role of ubiquitination in directing GPCRs for lysosomal degradation has already been well-established. Furthermore, this modification can also play a role in targeting membrane and endoplasmic reticulum-associated receptors to the proteasome. Most recently, ubiquitination was also shown to be involved in GPCR signaling. In this review, we present current knowledge on the molecular basis of GPCR regulation by ubiquitination, and highlight the importance of E3 ubiquitin ligases, deubiquitinating enzymes and β-arrestins. Finally, we discuss classical and newly-discovered functions of ubiquitination in controlling GPCR activity.

  1. Cross-communication between Gi and Gs in a G-protein-coupled receptor heterotetramer guided by a receptor C-terminal domain.

    Science.gov (United States)

    Navarro, Gemma; Cordomí, Arnau; Brugarolas, Marc; Moreno, Estefanía; Aguinaga, David; Pérez-Benito, Laura; Ferre, Sergi; Cortés, Antoni; Casadó, Vicent; Mallol, Josefa; Canela, Enric I; Lluís, Carme; Pardo, Leonardo; McCormick, Peter J; Franco, Rafael

    2018-02-28

    G-protein-coupled receptor (GPCR) heteromeric complexes have distinct properties from homomeric GPCRs, giving rise to new receptor functionalities. Adenosine receptors (A 1 R or A 2A R) can form A 1 R-A 2A R heteromers (A 1 -A 2A Het), and their activation leads to canonical G-protein-dependent (adenylate cyclase mediated) and -independent (β-arrestin mediated) signaling. Adenosine has different affinities for A 1 R and A 2A R, allowing the heteromeric receptor to detect its concentration by integrating the downstream G i - and G s -dependent signals. cAMP accumulation and β-arrestin recruitment assays have shown that, within the complex, activation of A 2A R impedes signaling via A 1 R. We examined the mechanism by which A 1 -A 2A Het integrates G i - and G s -dependent signals. A 1 R blockade by A 2A R in the A 1 -A 2A Het is not observed in the absence of A 2A R activation by agonists, in the absence of the C-terminal domain of A 2A R, or in the presence of synthetic peptides that disrupt the heteromer interface of A 1 -A 2A Het, indicating that signaling mediated by A 1 R and A 2A R is controlled by both G i and G s proteins. We identified a new mechanism of signal transduction that implies a cross-communication between G i and G s proteins guided by the C-terminal tail of the A 2A R. This mechanism provides the molecular basis for the operation of the A 1 -A 2A Het as an adenosine concentration-sensing device that modulates the signals originating at both A 1 R and A 2A R.

  2. Acute engagement of Gq-mediated signaling in the bed nucleus of the stria terminalis induces anxiety-like behavior.

    Science.gov (United States)

    Mazzone, C M; Pati, D; Michaelides, M; DiBerto, J; Fox, J H; Tipton, G; Anderson, C; Duffy, K; McKlveen, J M; Hardaway, J A; Magness, S T; Falls, W A; Hammack, S E; McElligott, Z A; Hurd, Y L; Kash, T L

    2018-01-01

    The bed nucleus of the stria terminalis (BNST) is a brain region important for regulating anxiety-related behavior in both humans and rodents. Here we used a chemogenetic strategy to investigate how engagement of G protein-coupled receptor (GPCR) signaling cascades in genetically defined GABAergic BNST neurons modulates anxiety-related behavior and downstream circuit function. We saw that stimulation of vesicular γ-aminobutyric acid (GABA) transporter (VGAT)-expressing BNST neurons using hM3Dq, but neither hM4Di nor rM3Ds designer receptors exclusively activated by a designer drug (DREADD), promotes anxiety-like behavior. Further, we identified that activation of hM3Dq receptors in BNST VGAT neurons can induce a long-term depression-like state of glutamatergic synaptic transmission, indicating DREADD-induced changes in synaptic plasticity. Further, we used DREADD-assisted metabolic mapping to profile brain-wide network activity following activation of G q -mediated signaling in BNST VGAT neurons and saw increased activity within ventral midbrain structures, including the ventral tegmental area and hindbrain structures such as the locus coeruleus and parabrachial nucleus. These results highlight that G q -mediated signaling in BNST VGAT neurons can drive downstream network activity that correlates with anxiety-like behavior and points to the importance of identifying endogenous GPCRs within genetically defined cell populations. We next used a microfluidics approach to profile the receptorome of single BNST VGAT neurons. This approach yielded multiple G q -coupled receptors that are associated with anxiety-like behavior and several potential novel candidates for regulation of anxiety-like behavior. From this, we identified that stimulation of the G q -coupled receptor 5-HT 2C R in the BNST is sufficient to elevate anxiety-like behavior in an acoustic startle task. Together, these results provide a novel profile of receptors within genetically defined BNST VGAT

  3. DMPD: Negative regulation of cytoplasmic RNA-mediated antiviral signaling. [Dynamic Macrophage Pathway CSML Database

    Lifescience Database Archive (English)

    Full Text Available 18703349 Negative regulation of cytoplasmic RNA-mediated antiviral signaling. Komur...Show Negative regulation of cytoplasmic RNA-mediated antiviral signaling. PubmedID 18703349 Title Negative r...egulation of cytoplasmic RNA-mediated antiviral signaling. Authors Komuro A, Bamm

  4. The G-protein-coupled receptor, GPR84, is important for eye development in Xenopus laevis.

    Science.gov (United States)

    Perry, Kimberly J; Johnson, Verity R; Malloch, Erica L; Fukui, Lisa; Wever, Jason; Thomas, Alvin G; Hamilton, Paul W; Henry, Jonathan J

    2010-11-01

    G-protein-coupled receptors (GPCRs) represent diverse, multifamily groups of cell signaling receptors involved in many cellular processes. We identified Xenopus laevis GPR84 as a member of the A18 subfamily of GPCRs. During development, GPR84 is detected in the embryonic lens placode, differentiating lens fiber cells, retina, and cornea. Anti-sense morpholino oligonucleotide-mediated knockdown and RNA rescue experiments demonstrate GPR84's importance in lens, cornea, and retinal development. Examination of cell proliferation using an antibody against histone H3 S10P reveals significant increases in the lens and retina following GPR84 knockdown. Additionally, there was also an increase in apoptosis in the retina and lens, as revealed by TUNEL assay. Reciprocal transplantation of the presumptive lens ectoderm between uninjected controls and morpholino-injected embryos demonstrates that GPR84 is necessary in the retina for proper development of the retina, as well as other eye tissues including the lens and cornea. © 2010 Wiley-Liss, Inc.

  5. G-protein-coupled inward rectifier potassium channels involved in corticostriatal presynaptic modulation.

    Science.gov (United States)

    Meneses, David; Mateos, Verónica; Islas, Gustavo; Barral, Jaime

    2015-09-01

    Presynaptic modulation has been associated mainly with calcium channels but recent data suggests that inward rectifier potassium channels (K(IR)) also play a role. In this work we set to characterize the role of presynaptic K(IR) channels in corticostriatal synaptic transmission. We elicited synaptic potentials in striatum by stimulating cortical areas and then determined the synaptic responses of corticostriatal synapsis by using paired pulse ratio (PPR) in the presence and absence of several potassium channel blockers. Unspecific potassium channels blockers Ba(2+) and Cs(+) reduced the PPR, suggesting that these channels are presynaptically located. Further pharmacological characterization showed that application of tertiapin-Q, a specific K(IR)3 channel family blocker, also induced a reduction of PPR, suggesting that K(IR)3 channels are present at corticostriatal terminals. In contrast, exposure to Lq2, a specific K(IR)1.1 inward rectifier potassium channel, did not induce any change in PPR suggesting the absence of these channels in the presynaptic corticostriatal terminals. Our results indicate that K(IR)3 channels are functionally expressed at the corticostriatal synapses, since blockage of these channels result in PPR decrease. Our results also help to explain how synaptic activity may become sensitive to extracellular signals mediated by G-protein coupled receptors. A vast repertoire of receptors may influence neurotransmitter release in an indirect manner through regulation of K(IR)3 channels. © 2015 Wiley Periodicals, Inc.

  6. G-protein-coupled receptor 137 accelerates proliferation of urinary bladder cancer cells in vitro.

    Science.gov (United States)

    Du, Yiheng; Bi, Wenhuan; Zhang, Fei; Wu, Wenbo; Xia, Shujie; Liu, Haitao

    2015-01-01

    Urinary bladder cancer is a worldwide concern because of its level of incidence and recurrence. To search an effective therapeutic strategy for urinary bladder cancer, it is important to identify proteins involved in tumorigenesis that could serve as potential targets for diagnosis and treatment. G-protein-coupled receptors (GPRs) constitute a large protein family of receptors that sense molecules outside the cell and activate signal transduction pathways and cellular responses inside the cell. GPR137 is a newly discovered human gene encoding orphan GPRs. In this study, we aimed to investigate the physiological role of GPR137 in urinary bladder cancer. The effect of GPR137 on cell growth was examined via an RNA interference (RNAi) lentivirus system in two human urinary bladder cancer cell lines BT5637 and T24. Lentivirus-mediated RNAi could specifically suppressed GPR137 expression in vitro, resulting in alleviated cell viability and impaired colony formation, as well as blocks G0/G1 and S phases of the cell cycle. These results suggested GPR137 as an essential player in urinary bladder cancer cell growth, and it may serve as a potential target for gene therapy in the treatment of urinary bladder cancer. © 2014 International Union of Biochemistry and Molecular Biology, Inc.

  7. Global analysis of gene expression mediated by OX1 orexin receptor signaling in a hypothalamic cell line.

    Directory of Open Access Journals (Sweden)

    Eric Koesema

    Full Text Available The orexins and their cognate G-protein coupled receptors have been widely studied due to their associations with various behaviors and cellular processes. However, the detailed downstream signaling cascades that mediate these effects are not completely understood. We report the generation of a neuronal model cell line that stably expresses the OX1 orexin receptor (OX1 and an RNA-Seq analysis of changes in gene expression seen upon receptor activation. Upon treatment with orexin, several families of related transcription factors are transcriptionally regulated, including the early growth response genes (Egr, the Kruppel-like factors (Klf, and the Nr4a subgroup of nuclear hormone receptors. Furthermore, some of the transcriptional effects observed have also been seen in data from in vivo sleep deprivation microarray studies, supporting the physiological relevance of the data set. Additionally, inhibition of one of the most highly regulated genes, serum and glucocorticoid-regulated kinase 1 (Sgk1, resulted in the diminished orexin-dependent induction of a subset of genes. These results provide new insight into the molecular signaling events that occur during OX1 signaling and support a role for orexin signaling in the stimulation of wakefulness during sleep deprivation studies.

  8. Molecular Signaling Pathways Mediating Osteoclastogenesis Induced by Prostate Cancer Cells

    International Nuclear Information System (INIS)

    Rafiei, Shahrzad; Komarova, Svetlana V

    2013-01-01

    Advanced prostate cancer commonly metastasizes to bone leading to osteoblastic and osteolytic lesions. Although an osteolytic component governed by activation of bone resorbing osteoclasts is prominent in prostate cancer metastasis, the molecular mechanisms of prostate cancer-induced osteoclastogenesis are not well-understood. We studied the effect of soluble mediators released from human prostate carcinoma cells on osteoclast formation from mouse bone marrow and RAW 264.7 monocytes. Soluble factors released from human prostate carcinoma cells significantly increased viability of naïve bone marrow monocytes, as well as osteoclastogenesis from precursors primed with receptor activator of nuclear factor κ-B ligand (RANKL). The prostate cancer-induced osteoclastogenesis was not mediated by RANKL as it was not inhibited by osteoprotegerin (OPG). However inhibition of TGFβ receptor I (TβRI), or macrophage-colony stimulating factor (MCSF) resulted in attenuation of prostate cancer-induced osteoclastogenesis. We characterized the signaling pathways induced in osteoclast precursors by soluble mediators released from human prostate carcinoma cells. Prostate cancer factors increased basal calcium levels and calcium fluctuations, induced nuclear localization of nuclear factor of activated t-cells (NFAT)c1, and activated prolonged phosphorylation of ERK1/2 in RANKL-primed osteoclast precursors. Inhibition of calcium signaling, NFATc1 activation, and ERK1/2 phosphorylation significantly reduced the ability of prostate cancer mediators to stimulate osteoclastogenesis. This study reveals the molecular mechanisms underlying the direct osteoclastogenic effect of prostate cancer derived factors, which may be beneficial in developing novel osteoclast-targeting therapeutic approaches

  9. Motile cilia of human airway epithelia contain hedgehog signaling components that mediate noncanonical hedgehog signaling.

    Science.gov (United States)

    Mao, Suifang; Shah, Alok S; Moninger, Thomas O; Ostedgaard, Lynda S; Lu, Lin; Tang, Xiao Xiao; Thornell, Ian M; Reznikov, Leah R; Ernst, Sarah E; Karp, Philip H; Tan, Ping; Keshavjee, Shaf; Abou Alaiwa, Mahmoud H; Welsh, Michael J

    2018-02-06

    Differentiated airway epithelia produce sonic hedgehog (SHH), which is found in the thin layer of liquid covering the airway surface. Although previous studies showed that vertebrate HH signaling requires primary cilia, as airway epithelia mature, the cells lose primary cilia and produce hundreds of motile cilia. Thus, whether airway epithelia have apical receptors for SHH has remained unknown. We discovered that motile cilia on airway epithelial cells have HH signaling proteins, including patched and smoothened. These cilia also have proteins affecting cAMP-dependent signaling, including Gα i and adenylyl cyclase 5/6. Apical SHH decreases intracellular levels of cAMP, which reduces ciliary beat frequency and pH in airway surface liquid. These results suggest that apical SHH may mediate noncanonical HH signaling through motile cilia to dampen respiratory defenses at the contact point between the environment and the lung, perhaps counterbalancing processes that stimulate airway defenses. Copyright © 2018 the Author(s). Published by PNAS.

  10. Crosstalk between G protein-coupled receptors (GPCRs and tyrosine kinase receptor (TXR in the heart after morphine withdrawal

    Directory of Open Access Journals (Sweden)

    Pilar eAlmela

    2013-12-01

    Full Text Available G protein-coupled receptors (GPCRs comprise a large family of membrane receptors involved in signal transduction. These receptors are linked to a variety of physiological and biological processes such as regulation of neurotransmission, growth and cell differentiation among others. Some of the effects of GPCRs are known to be mediated by the activation of mitogen-activated extracellular kinase (MAPK pathways. Cross-talk among various signal pathways plays an important role in activation of intracellular and intranuclear signal transduction cascades. Naloxone-induced morphine withdrawal leads to an up-regulation of adenyl cyclase-mediated signalling, resulting in high expression of protein kinase (PK A. In addition, there is also an increased expression of extracellular signal regulated kinase (ERK, one member of MAPK. For this reason, the crosstalk between these GPCRs and receptors with tyrosine kinase activity (TKR can be considered a possible mechanism for adaptive changes that occurs after morphine withdrawal. Morphine withdrawal activates ERK1/2 and phosphorylated tyrosine hydroxylase (TH at Ser31 in the right and left ventricle. When N-(2-guanidinoethyl-5-isoquinolinesulfonamide (HA-1004, a PKA inhibitor was infused, the ability of morphine withdrawal to activate ERK, which phosphorylates TH at Ser31, was reduced. The present finding demonstrated that the enhancement of ERK1/2 expression and the phosphorylation state of TH at Ser31 during morphine withdrawal are dependent on PKA and suggest cross-talk between PKA and ERK1/2 transduction pathway mediating morphine withdrawal-induced activation of TH. Increasing understanding of the mechanisms that interconnect the two pathway regulated by GPCRs and TKRs may facilitate the design of new therapeutic strategies.

  11. Leukotriene D4 activates distinct G-proteins in intestinal epithelial cells to regulate stress fibre formation and to generate intracellular Ca2+ mobilisation and ERK1/2 activation

    International Nuclear Information System (INIS)

    Nielsen, Christian Kamp; Massoumi, Ramin; Sonnerlind, Maria; Sjoelander, Anita

    2005-01-01

    We have shown that the pro-inflammatory mediator LTD 4 , via its G-protein-coupled receptor CysLT 1 , signals through both pertussis-toxin-sensitive and -insensitive G-proteins to induce various cellular responses. To further characterise the initial step of the different signalling pathways emanating from the CysLT 1 receptor, we transfected intestinal epithelial cells, Int 407, with different mini vectors that each express a specific inhibitory peptide directed against a unique α subunit of a specific heterotrimeric G-protein. Our results revealed that LTD 4 -induced stress fibre formation is inhibited approximately 80% by a vector expressing an inhibitory peptide against the pertussis-toxin-insensitive Gα 12 -protein in intestinal epithelial Int 407 cells. Control experiments revealed that the LPA-induced stress fibre formation, mediated via the Gα 12 -protein in other cell types, was blocked by the same peptide in intestinal Int 407 cells. Furthermore, the CysLT 1 -receptor-mediated calcium signal and activation of the proliferative ERK1/2 kinase are blocked in cells transfected with a vector expressing an inhibitory peptide against the Gα i3 -protein, whereas in cells transfected with an empty ECFP-vector or vectors expressing inhibitory peptides against the Gα i1-2 -, Gα 12 -, Gα R -proteins these signals are not significantly affected. Consequently, the CysLT 1 receptor has the capacity to activate at least two distinctly different heterotrimeric G-proteins that transduce activation of unique downstream cellular events

  12. Agonist-mediated activation of Bombyx mori diapause hormone receptor signals to extracellular signal-regulated kinases 1 and 2 through Gq-PLC-PKC-dependent cascade.

    Science.gov (United States)

    Jiang, Xue; Yang, Jingwen; Shen, Zhangfei; Chen, Yajie; Shi, Liangen; Zhou, Naiming

    2016-08-01

    Diapause is a developmental strategy adopted by insects to survive in challenging environments such as the low temperatures of a winter. This unique process is regulated by diapause hormone (DH), which is a neuropeptide hormone that induces egg diapause in Bombyx mori and is involved in terminating pupal diapause in heliothis moths. An G protein-coupled receptor from the silkworm, B. mori, has been identified as a specific cell surface receptor for DH. However, the detailed information on the DH-DHR system and its mechanism(s) involved in the induction of embryonic diapause remains unknown. Here, we combined functional assays with various specific inhibitors to elucidate the DHR-mediated signaling pathways. Upon activation by DH, B. mori DHR is coupled to the Gq protein, leading to a significant increase of intracellular Ca(2+) and cAMP response element-driven luciferase activity in an UBO-QIC, a specific Gq inhibitor, sensitive manner. B. mori DHR elicited ERK1/2 phosphorylation in a dose- and time-dependent manner in response to DH. This effect was almost completely inhibited by co-incubation with UBO-QIC and was also significantly suppressed by PLC inhibitor U73122, PKC inhibitors Gö6983 and the Ca(2+) chelator EGTA. Moreover, DHR-induced activation of ERK1/2 was significantly attenuated by treatment with the Gβγ specific inhibitors gallein and M119K and the PI3K specific inhibitor Wortmannin, but not by the Src specific inhibitor PP2. Our data also demonstrates that the EGFR-transactivation pathway is not involved in the DHR-mediated ERK1/2 phosphorylation. Future efforts are needed to clarify the role of the ERK1/2 signaling pathway in the DH-mediated induction of B. mori embryonic diapause. Copyright © 2016 Elsevier Ltd. All rights reserved.

  13. A modeling strategy for G-protein coupled receptors

    Directory of Open Access Journals (Sweden)

    Anna Kahler

    2016-03-01

    Full Text Available Cell responses can be triggered via G-protein coupled receptors (GPCRs that interact with small molecules, peptides or proteins and transmit the signal over the membrane via structural changes to activate intracellular pathways. GPCRs are characterized by a rather low sequence similarity and exhibit structural differences even for functionally closely related GPCRs. An accurate structure prediction for GPCRs is therefore not straightforward. We propose a computational approach that relies on the generation of several independent models based on different template structures, which are subsequently refined by molecular dynamics simulations. A comparison of their conformational stability and the agreement with GPCR-typical structural features is then used to select a favorable model. This strategy was applied to predict the structure of the herpesviral chemokine receptor US28 by generating three independent models based on the known structures of the chemokine receptors CXCR1, CXCR4, and CCR5. Model refinement and evaluation suggested that the model based on CCR5 exhibits the most favorable structural properties. In particular, the GPCR-typical structural features, such as a conserved water cluster or conserved non-covalent contacts, are present to a larger extent in the model based on CCR5 compared to the other models. A final model validation based on the recently published US28 crystal structure confirms that the CCR5-based model is the most accurate and exhibits 80.8% correctly modeled residues within the transmembrane helices. The structural agreement between the selected model and the crystal structure suggests that our modeling strategy may also be more generally applicable to other GPCRs of unknown structure.

  14. Oligomerization of G protein-coupled receptors: computational methods.

    Science.gov (United States)

    Selent, J; Kaczor, A A

    2011-01-01

    Recent research has unveiled the complexity of mechanisms involved in G protein-coupled receptor (GPCR) functioning in which receptor dimerization/oligomerization may play an important role. Although the first high-resolution X-ray structure for a likely functional chemokine receptor dimer has been deposited in the Protein Data Bank, the interactions and mechanisms of dimer formation are not yet fully understood. In this respect, computational methods play a key role for predicting accurate GPCR complexes. This review outlines computational approaches focusing on sequence- and structure-based methodologies as well as discusses their advantages and limitations. Sequence-based approaches that search for possible protein-protein interfaces in GPCR complexes have been applied with success in several studies, but did not yield always consistent results. Structure-based methodologies are a potent complement to sequence-based approaches. For instance, protein-protein docking is a valuable method especially when guided by experimental constraints. Some disadvantages like limited receptor flexibility and non-consideration of the membrane environment have to be taken into account. Molecular dynamics simulation can overcome these drawbacks giving a detailed description of conformational changes in a native-like membrane. Successful prediction of GPCR complexes using computational approaches combined with experimental efforts may help to understand the role of dimeric/oligomeric GPCR complexes for fine-tuning receptor signaling. Moreover, since such GPCR complexes have attracted interest as potential drug target for diverse diseases, unveiling molecular determinants of dimerization/oligomerization can provide important implications for drug discovery.

  15. Electrochemical DNA biosensor based on MNAzyme-mediated signal amplification

    International Nuclear Information System (INIS)

    Diao, Wei; Tang, Min; Ding, Xiaojuan; Zhang, Ye; Yang, Jianru; Cheng, Wenbin; Mo, Fei; Wen, Bo; Xu, Lulu; Yan, Yurong

    2016-01-01

    The authors describe an electrochemical sensing strategy for highly sensitive and specific detection of target (analyte) DNA based on an amplification scheme mediated by a multicomponent nucleic acid enzyme (MNAzyme). MNAzymes were formed by multicomponent complexes which produce amplified “output” signals in response to specific “input” signal. In the presence of target nucleic acid, multiple partial enzymes (partzymes) oligonucleotides are assembled to form active MNAzymes. These can cleave H0 substrate into two pieces, thereby releasing the activated MNAzyme to undergo an additional cycle of amplification. Here, the two pieces contain a biotin-tagged sequence and a byproduct. The biotin-tagged sequences are specifically captured by the detection probes immobilized on the gold electrode. By employing streptavidinylated alkaline phosphatase as an enzyme label, an electrochemical signal is obtained. The electrode, if operated at a working potential of 0.25 V (vs. Ag/AgCl) in solution of pH 7.5, covers the 100 pM to 0.25 μM DNA concentration range, with a 79 pM detection limit. In our perception, the strategy introduced here has a wider potential in that it may be applied to molecular diagnostics and pathogen detection. (author)

  16. G protein-coupled receptor kinase-2 (GRK-2) regulates serotonin metabolism through the monoamine oxidase AMX-2 in Caenorhabditis elegans.

    Science.gov (United States)

    Wang, Jianjun; Luo, Jiansong; Aryal, Dipendra K; Wetsel, William C; Nass, Richard; Benovic, Jeffrey L

    2017-04-07

    G protein-coupled receptors (GPCRs) regulate many animal behaviors. GPCR signaling is mediated by agonist-promoted interactions of GPCRs with heterotrimeric G proteins, GPCR kinases (GRKs), and arrestins. To further elucidate the role of GRKs in regulating GPCR-mediated behaviors, we utilized the genetic model system Caenorhabditis elegans Our studies demonstrate that grk-2 loss-of-function strains are egg laying-defective and contain low levels of serotonin (5-HT) and high levels of the 5-HT metabolite 5-hydroxyindole acetic acid (5-HIAA). The egg laying defect could be rescued by the expression of wild type but not by catalytically inactive grk-2 or by the selective expression of grk-2 in hermaphrodite-specific neurons. The addition of 5-HT or inhibition of 5-HT metabolism also rescued the egg laying defect. Furthermore, we demonstrate that AMX-2 is the primary monoamine oxidase that metabolizes 5-HT in C. elegans , and we also found that grk-2 loss-of-function strains have abnormally high levels of AMX-2 compared with wild-type nematodes. Interestingly, GRK-2 was also found to interact with and promote the phosphorylation of AMX-2. Additional studies reveal that 5-HIAA functions to inhibit egg laying in a manner dependent on the 5-HT receptor SER-1 and the G protein GOA-1. These results demonstrate that GRK-2 modulates 5-HT metabolism by regulating AMX-2 function and that 5-HIAA may function in the SER-1 signaling pathway. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  17. Multilepton signals of gauge mediated supersymmetry breaking at the LHC

    Energy Technology Data Exchange (ETDEWEB)

    D' Hondt, Jorgen [Theoretische Natuurkunde and IIHE/ELEM, Vrije Universiteit Brussel, Pleinlaan 2, B-1050 Brussels (Belgium); International Solvay Institutes, Brussels (Belgium); De Causmaecker, Karen [Theoretische Natuurkunde and IIHE/ELEM, Vrije Universiteit Brussel, Pleinlaan 2, B-1050 Brussels (Belgium); International Solvay Institutes, Brussels (Belgium); Theory Division, Physics Department, CERN, CH-1211 Geneva 23 (Switzerland); Fuks, Benjamin [Theory Division, Physics Department, CERN, CH-1211 Geneva 23 (Switzerland); Institut Pluridisciplinaire Hubert Curien/Département Recherches Subatomiques, Université de Strasbourg/CNRS-IN2P3, 23 Rue du Loess, F-67037 Strasbourg (France); Mariotti, Alberto [Institute for Particle Physics Phenomenology, Durham University, Durham DH1 3LE (United Kingdom); Mawatari, Kentarou [Theoretische Natuurkunde and IIHE/ELEM, Vrije Universiteit Brussel, Pleinlaan 2, B-1050 Brussels (Belgium); International Solvay Institutes, Brussels (Belgium); Petersson, Christoffer [International Solvay Institutes, Brussels (Belgium); Physique Théorique et Mathématique, Université Libre de Bruxelles, C.P. 231, B-1050 Brussels (Belgium); Department of Fundamental Physics, Chalmers University of Technology, 412 96 Göteborg (Sweden); Redigolo, Diego [International Solvay Institutes, Brussels (Belgium); Physique Théorique et Mathématique, Université Libre de Bruxelles, C.P. 231, B-1050 Brussels (Belgium)

    2014-04-04

    We investigate multilepton LHC signals arising from electroweak processes involving sleptons. We consider the framework of general gauge mediated supersymmetry breaking, focusing on models where the low mass region of the superpartner spectrum consists of the three generations of charged sleptons and the nearly massless gravitino. We demonstrate how such models can provide an explanation for the anomalous four lepton events recently observed by the CMS Collaboration, while satisfying other existing experimental constraints. The best fit to the CMS data is obtained for a selectron/smuon mass of around 145 GeV and a stau mass of around 90 GeV. These models also give rise to final states with more than four leptons, offering alternative channels in which they can be probed and we estimate the corresponding production rates at the LHC.

  18. Intracellular Signaling Mediators in the Circulatory and Ventilatory Systems

    CERN Document Server

    Thiriet, Marc

    2013-01-01

    The volumes in this authoritative series present a multidisciplinary approach to modeling and simulation of flows in the cardiovascular and ventilatory systems, especially multiscale modeling and coupled simulations. The cardiovascular and respiratory systems are tightly coupled, as their primary function is to supply oxygen to and remove carbon dioxide from the body's cells. Because physiological conduits have deformable and reactive walls, macroscopic flow behavior and prediction must be coupled to phenomenological models of nano- and microscopic events in a corrector scheme of regulated mechanisms when the vessel lumen caliber varies markedly. Therefore, investigation of flows of blood and air in physiological conduits requires an understanding of the biology, chemistry, and physics of these systems together with the mathematical tools to describe their functioning. Volume 4 is devoted to major sets of intracellular mediators that transmit signals upon stimulation of cell-surface receptors.  Activation of...

  19. DMPD: Signalling pathways mediating type I interferon gene expression. [Dynamic Macrophage Pathway CSML Database

    Lifescience Database Archive (English)

    Full Text Available 17904888 Signalling pathways mediating type I interferon gene expression. Edwards M...hways mediating type I interferon gene expression. PubmedID 17904888 Title Signalling pathways...R, Slater L, Johnston SL. Microbes Infect. 2007 Sep;9(11):1245-51. Epub 2007 Jul 1. (.png) (.svg) (.html) (.csml) Show Signalling pat

  20. Linear collider signal of anomaly mediated supersymmetry breaking model

    International Nuclear Information System (INIS)

    Ghosh Dilip Kumar; Kundu, Anirban; Roy, Probir; Roy, Sourov

    2001-01-01

    Though the minimal model of anomaly mediated supersymmetry breaking has been significantly constrained by recent experimental and theoretical work, there are still allowed regions of the parameter space for moderate to large values of tan β. We show that these regions will be comprehensively probed in a √s = 1 TeV e + e - linear collider. Diagnostic signals to this end are studied by zeroing in on a unique and distinct feature of a large class of models in this genre: a neutral winolike Lightest Supersymmetric Particle closely degenerate in mass with a winolike chargino. The pair production processes e + e - → e tilde L ± e tilde L ± , e tilde R ± e tilde R ± , e tilde L ± e tilde R ± , ν tilde anti ν tilde, χ tilde 1 0 χ tilde 2 0 , χ tilde 2 0 χ tilde 2 0 are all considered at √s = 1 TeV corresponding to the proposed TESLA linear collider in two natural categories of mass ordering in the sparticle spectra. The signals analysed comprise multiple combinations of fast charged leptons (any of which can act as the trigger) plus displaced vertices X D (any of which can be identified by a heavy ionizing track terminating in the detector) and/or associated soft pions with characteristic momentum distributions. (author)

  1. Possible involvement of G-proteins and cAMP in the induction of progesterone hydroxylating enzyme system in the vascular wilt fungus Fusarium oxysporum.

    Science.gov (United States)

    Poli, Anna; Di Pietro, Antonio; Zigon, Dusan; Lenasi, Helena

    2009-02-01

    Fungi present the ability to hydroxylate steroids. In some filamentous fungi, progesterone induces an enzyme system which converts the compound into a less toxic hydroxylated product. We investigated the progesterone response in the vascular wilt pathogen Fusarium oxysporum, using mass spectrometry and high performance liquid chromatography (HPLC). Progesterone was mainly transformed into 15alpha-hydroxyprogesterone, which was found predominantly in the extracellular medium. The role of two conserved fungal signaling cascades in the induction of the progesterone-transforming enzyme system was studied, using knockout mutants lacking the mitogen-activated protein kinase Fmk1 or the heterotrimeric G-protein beta subunit Fgb1 functioning upstream of the cyclic adenosine monophosphate (cAMP) pathway. No steroid hydroxylation was induced in the Deltafgb1 strain, suggesting a role for the G-protein beta subunit in progesterone signaling. Exogenous cAMP restored the induction of progesterone-transforming activity in the Deltafgb1 strain, suggesting that steroid signaling in F. oxysporum is mediated by the cAMP-PKA pathway.

  2. Bioorthogonal fluorescent labeling of functional G-protein-coupled receptors

    DEFF Research Database (Denmark)

    Tian, He; Naganathan, Saranga; Kazmi, Manija A

    2014-01-01

    Novel methods are required for site-specific, quantitative fluorescent labeling of G-protein-coupled receptors (GPCRs) and other difficult-to-express membrane proteins. Ideally, fluorescent probes should perturb the native structure and function as little as possible. We evaluated bioorthogonal...

  3. Muscarinic supersensitivity and impaired receptor desensitization in G protein-coupled receptor kinase 5-deficient mice.

    Science.gov (United States)

    Gainetdinov, R R; Bohn, L M; Walker, J K; Laporte, S A; Macrae, A D; Caron, M G; Lefkowitz, R J; Premont, R T

    1999-12-01

    G protein-coupled receptor kinase 5 (GRK5) is a member of a family of enzymes that phosphorylate activated G protein-coupled receptors (GPCR). To address the physiological importance of GRK5-mediated regulation of GPCRs, mice bearing targeted deletion of the GRK5 gene (GRK5-KO) were generated. GRK5-KO mice exhibited mild spontaneous hypothermia as well as pronounced behavioral supersensitivity upon challenge with the nonselective muscarinic agonist oxotremorine. Classical cholinergic responses such as hypothermia, hypoactivity, tremor, and salivation were enhanced in GRK5-KO animals. The antinociceptive effect of oxotremorine was also potentiated and prolonged. Muscarinic receptors in brains from GRK5-KO mice resisted oxotremorine-induced desensitization, as assessed by oxotremorine-stimulated [5S]GTPgammaS binding. These data demonstrate that elimination of GRK5 results in cholinergic supersensitivity and impaired muscarinic receptor desensitization and suggest that a deficit of GPCR desensitization may be an underlying cause of behavioral supersensitivity.

  4. Δ9-THC-Caused Synaptic and Memory Impairments Are Mediated through COX-2 Signaling

    OpenAIRE

    Chen, Rongqing; Zhang, Jian; Fan, Ni; Teng, Zhao-qian; Wu, Yan; Yang, Hongwei; Tang, Ya-ping; Sun, Hao; Song, Yunping; Chen, Chu

    2013-01-01

    Marijuana has been used for thousands of years as a treatment for medical conditions. However, untoward side effects limit its medical value. Here we show that synaptic and cognitive impairments following repeated exposure to Δ9-tetrahydrocannabinol (Δ9-THC) are associated with the induction of cyclooxygenase-2 (COX-2), an inducible enzyme that converts arachidonic acid to prostanoids, in the brain. COX-2 induction by Δ9-THC is mediated via CB1 receptor-coupled G-protein βγ subunits. Pharmaco...

  5. Three alpha-subunits of heterotrimeric G proteins and an adenylyl cyclase have distinct roles in fruiting body development in the homothallic fungus Sordaria macrospora.

    Science.gov (United States)

    Kamerewerd, Jens; Jansson, Malin; Nowrousian, Minou; Pöggeler, Stefanie; Kück, Ulrich

    2008-09-01

    Sordaria macrospora, a self-fertile filamentous ascomycete, carries genes encoding three different alpha-subunits of heterotrimeric G proteins (gsa, G protein Sordaria alpha subunit). We generated knockout strains for all three gsa genes (Deltagsa1, Deltagsa2, and Deltagsa3) as well as all combinations of double mutants. Phenotypic analysis of single and double mutants showed that the genes for Galpha-subunits have distinct roles in the sexual life cycle. While single mutants show some reduction of fertility, double mutants Deltagsa1Deltagsa2 and Deltagsa1Deltagsa3 are completely sterile. To test whether the pheromone receptors PRE1 and PRE2 mediate signaling via distinct Galpha-subunits, two recently generated Deltapre strains were crossed with all Deltagsa strains. Analyses of the corresponding double mutants revealed that compared to GSA2, GSA1 is a more predominant regulator of a signal transduction cascade downstream of the pheromone receptors and that GSA3 is involved in another signaling pathway that also contributes to fruiting body development and fertility. We further isolated the gene encoding adenylyl cyclase (AC) (sac1) for construction of a knockout strain. Analyses of the three DeltagsaDeltasac1 double mutants and one Deltagsa2Deltagsa3Deltasac1 triple mutant indicate that SAC1 acts downstream of GSA3, parallel to a GSA1-GSA2-mediated signaling pathway. In addition, the function of STE12 and PRO41, two presumptive signaling components, was investigated in diverse double mutants lacking those developmental genes in combination with the gsa genes. This analysis was further completed by expression studies of the ste12 and pro41 transcripts in wild-type and mutant strains. From the sum of all our data, we propose a model for how different Galpha-subunits interact with pheromone receptors, adenylyl cyclase, and STE12 and thus cooperatively regulate sexual development in S. macrospora.

  6. Enhanced expression of G-protein coupled estrogen receptor (GPER/GPR30) in lung cancer

    International Nuclear Information System (INIS)

    Jala, Venkatakrishna Rao; Radde, Brandie N; Haribabu, Bodduluri; Klinge, Carolyn M

    2012-01-01

    G-protein-coupled estrogen receptor (GPER/GPR30) was reported to bind 17β-estradiol (E 2 ), tamoxifen, and ICI 182,780 (fulvestrant) and promotes activation of epidermal growth factor receptor (EGFR)-mediated signaling in breast, endometrial and thyroid cancer cells. Although lung adenocarcinomas express estrogen receptors α and β (ERα and ERβ), the expression of GPER in lung cancer has not been investigated. The purpose of this study was to examine the expression of GPER in lung cancer. The expression patterns of GPER in various lung cancer lines and lung tumors were investigated using standard quantitative real time PCR (at mRNA levels), Western blot and immunohistochemistry (IHC) methods (at protein levels). The expression of GPER was scored and the pairwise comparisons (cancer vs adjacent tissues as well as cancer vs normal lung tissues) were performed. Analysis by real-time PCR and Western blotting revealed a significantly higher expression of GPER at both mRNA and protein levels in human non small cell lung cancer cell (NSCLC) lines relative to immortalized normal lung bronchial epithelial cells (HBECs). The virally immortalized human small airway epithelial cell line HPL1D showed higher expression than HBECs and similar expression to NSCLC cells. Immunohistochemical analysis of tissue sections of murine lung adenomas as well as human lung adenocarcinomas, squamous cell carcinomas and non-small cell lung carcinomas showed consistently higher expression of GPER in the tumor relative to the surrounding non-tumor tissue. The results from this study demonstrate increased GPER expression in lung cancer cells and tumors compared to normal lung. Further evaluation of the function and regulation of GPER will be necessary to determine if GPER is a marker of lung cancer progression

  7. The role of G-protein receptor 84 in experimental neuropathic pain.

    Science.gov (United States)

    Nicol, Louise S C; Dawes, John M; La Russa, Federica; Didangelos, Athanasios; Clark, Anna K; Gentry, Clive; Grist, John; Davies, John B; Malcangio, Marzia; McMahon, Stephen B

    2015-06-10

    G-protein receptor 84 (GPR84) is an orphan receptor that is induced markedly in monocytes/macrophages and microglia during inflammation, but its pathophysiological function is unknown. Here, we investigate the role of GPR84 in a murine model of traumatic nerve injury. Naive GPR84 knock-out (KO) mice exhibited normal behavioral responses to acute noxious stimuli, but subsequent to partial sciatic nerve ligation (PNL), KOs did not develop mechanical or thermal hypersensitivity, in contrast to wild-type (WT) littermates. Nerve injury increased ionized calcium binding adapter molecule 1 (Iba1) and phosphorylated p38 MAPK immunoreactivity in the dorsal horn and Iba1 and cluster of differentiation 45 expression in the sciatic nerve, with no difference between genotypes. PCR array analysis revealed that Gpr84 expression was upregulated in the spinal cord and sciatic nerve of WT mice. In addition, the expression of arginase-1, a marker for anti-inflammatory macrophages, was upregulated in KO sciatic nerve. Based on this evidence, we investigated whether peripheral macrophages behave differently in the absence of GPR84. We found that lipopolysaccharide-stimulated KO macrophages exhibited attenuated expression of several proinflammatory mediators, including IL-1β, IL-6, and TNF-α. Forskolin-stimulated KO macrophages also showed greater cAMP induction, a second messenger associated with immunosuppression. In summary, our results demonstrate that GPR84 is a proinflammatory receptor that contributes to nociceptive signaling via the modulation of macrophages, whereas in its absence the response of these cells to an inflammatory insult is impaired. Copyright © 2015 Nicol et al.

  8. High content screening for G protein-coupled receptors using cell-based protein translocation assays

    DEFF Research Database (Denmark)

    Grånäs, Charlotta; Lundholt, Betina Kerstin; Heydorn, Arne

    2005-01-01

    G protein-coupled receptors (GPCRs) have been one of the most productive classes of drug targets for several decades, and new technologies for GPCR-based discovery promise to keep this field active for years to come. While molecular screens for GPCR receptor agonist- and antagonist-based drugs...... will continue to be valuable discovery tools, the most exciting developments in the field involve cell-based assays for GPCR function. Some cell-based discovery strategies, such as the use of beta-arrestin as a surrogate marker for GPCR function, have already been reduced to practice, and have been used...... as valuable discovery tools for several years. The application of high content cell-based screening to GPCR discovery has opened up additional possibilities, such as direct tracking of GPCRs, G proteins and other signaling pathway components using intracellular translocation assays. These assays provide...

  9. Cloning and characterization of a G protein-activated human phosphoinositide-3 kinase.

    Science.gov (United States)

    Stoyanov, B; Volinia, S; Hanck, T; Rubio, I; Loubtchenkov, M; Malek, D; Stoyanova, S; Vanhaesebroeck, B; Dhand, R; Nürnberg, B

    1995-08-04

    Phosphoinositide-3 kinase activity is implicated in diverse cellular responses triggered by mammalian cell surface receptors and in the regulation of protein sorting in yeast. Receptors with intrinsic and associated tyrosine kinase activity recruit heterodimeric phosphoinositide-3 kinases that consist of p110 catalytic subunits and p85 adaptor molecules containing Src homology 2 (SH2) domains. A phosphoinositide-3 kinase isotype, p110 gamma, was cloned and characterized. The p110 gamma enzyme was activated in vitro by both the alpha and beta gamma subunits of heterotrimeric guanosine triphosphate (GTP)-binding proteins (G proteins) and did not interact with p85. A potential pleckstrin homology domain is located near its amino terminus. The p110 gamma isotype may link signaling through G protein-coupled receptors to the generation of phosphoinositide second messengers phosphorylated in the D-3 position.

  10. Characterization of NAADP-mediated calcium signaling in human spermatozoa

    Energy Technology Data Exchange (ETDEWEB)

    Sánchez-Tusie, A.A. [Departamento de Genética del Desarrollo y Fisiología Molecular, Instituto de Biotecnología, Universidad Nacional Autónoma de México, Cuernavaca, Morelos (Mexico); Vasudevan, S.R.; Churchill, G.C. [Department of Pharmacology, University of Oxford, Oxford OX1 3QT, England (United Kingdom); Nishigaki, T. [Departamento de Genética del Desarrollo y Fisiología Molecular, Instituto de Biotecnología, Universidad Nacional Autónoma de México, Cuernavaca, Morelos (Mexico); Treviño, C.L., E-mail: ctrevino@ibt.unam.mx [Departamento de Genética del Desarrollo y Fisiología Molecular, Instituto de Biotecnología, Universidad Nacional Autónoma de México, Cuernavaca, Morelos (Mexico)

    2014-01-10

    Highlights: •Human sperm cells synthesize NAADP. •NAADP-AM mediates [Ca{sup 2+}]{sub i} increases in human sperm in the absence of [Ca{sup 2+}]{sub o}. •Human sperm have two acidic compartments located in the head and midpiece. -- Abstract: Ca{sup 2+} signaling in spermatozoa plays a crucial role during processes such as capacitation and release of the acrosome, but the underlying molecular mechanisms still remain unclear. Nicotinic acid adenine dinucleotide phosphate (NAADP) is a potent Ca{sup 2+}-releasing second messenger in a variety of cellular processes. The presence of a NAADP synthesizing enzyme in sea urchin sperm has been previously reported, suggesting a possible role of NAADP in sperm Ca{sup 2+} signaling. In this work we used in vitro enzyme assays to show the presence of a novel NAADP synthesizing enzyme in human sperm, and to characterize its sensitivity to Ca{sup 2+} and pH. Ca{sup 2+} fluorescence imaging studies demonstrated that the permeable form of NAADP (NAADP-AM) induces intracellular [Ca{sup 2+}] increases in human sperm even in the absence of extracellular Ca{sup 2+}. Using LysoTracker®, a fluorescent probe that selectively accumulates in acidic compartments, we identified two such stores in human sperm cells. Their acidic nature was further confirmed by the reduction in staining intensity observed upon inhibition of the endo-lysosomal proton pump with Bafilomycin, or after lysosomal bursting with glycyl-L-phenylalanine-2-naphthylamide. The selective fluorescent NAADP analog, Ned-19, stained the same subcellular regions as LysoTracker®, suggesting that these stores are the targets of NAADP action.

  11. Novel water-soluble curcumin derivative mediating erectile signaling.

    Science.gov (United States)

    Abdel Aziz, Mohamed Talaat; El Asmer, Mohammed F; Rezq, Ameen; Kumosani, Taha Abdullah; Mostafa, Samya; Mostafa, Taymour; Atta, Hazem; Abdel Aziz Wassef, Mohamed; Fouad, Hanan H; Rashed, Laila; Sabry, Dina; Hassouna, Amira A; Senbel, Amira; Abdel Aziz, Ahmed

    2010-08-01

    Curcumin is an inducer of heme oxygenase enzyme-1 (HO-1) that is involved in erectile signaling via elevating cyclic guanosine monophosphate (cGMP)levels. To assess the effect of oral administration of a water-soluble long-acting curcumin derivative on erectile signaling. Two hundred and thirty six male white albino rats were divided into four groups; group 1 (N = 20) includes control. Group 2 (N = 72) was equally divided into four subgroups; subgroup 1 received pure curcumin (10 mg/kg), subgroup 2 received the long-acting curcumin derivative (2 mg/kg), subgroup 3 received the long-acting curcumin derivative (10 mg/kg), and subgroup 4 received sildenafil (4 mg/kg). Subgroups were sacrificed after the first, second, and third hour. Group 3 (N = 72) was equally divided into the same four subgroups already mentioned and were sacrificed after 24 hours, 48 hours, and 1 week. Group 4 (N = 72) was subjected to intracavernosal pressure (ICP) measurements 1 hour following oral administration of the same previous doses in the same rat subgroups. Cavernous tissue HO enzyme activity, cGMP, and ICP. In group 2, there was a significant progressive maintained elevation of HO activity and cGMP tissue levels starting from the first hour in subgroups 3 and 4, whereas, the rise in HO activity and cGMP started from second hour regarding the other rat subgroups. Sildenafil effect decreased after 3 hours. In group 3, there was a significant maintained elevation of HO activity and cGMP tissue levels extended to 1 week as compared to controls for all rat subgroups that received both forms of curcumin. In group 4, long-acting curcumin derivative exhibited more significant potentiation of intracavernosal pressure as compared to control and to the pure curcumin. Water-soluble long-acting curcumin derivative could mediate erectile function via upregulating cavernous tissue cGMP. © 2009 International Society for Sexual Medicine.

  12. Negative regulation of RIG-I-mediated antiviral signaling by TRK-fused gene (TFG) protein

    International Nuclear Information System (INIS)

    Lee, Na-Rae; Shin, Han-Bo; Kim, Hye-In; Choi, Myung-Soo; Inn, Kyung-Soo

    2013-01-01

    Highlights: •TRK-fused gene product (TFG) interacts with TRIM25 upon viral infection. •TFG negatively regulates RIG-I mediated antiviral signaling. •TFG depletion leads to enhanced viral replication. •TFG act downstream of MAVS. -- Abstract: RIG-I (retinoic acid inducible gene I)-mediated antiviral signaling serves as the first line of defense against viral infection. Upon detection of viral RNA, RIG-I undergoes TRIM25 (tripartite motif protein 25)-mediated K63-linked ubiquitination, leading to type I interferon (IFN) production. In this study, we demonstrate that TRK-fused gene (TFG) protein, previously identified as a TRIM25-interacting protein, binds TRIM25 upon virus infection and negatively regulates RIG-I-mediated type-I IFN signaling. RIG-I-mediated IFN production and nuclear factor (NF)-κB signaling pathways were upregulated by the suppression of TFG expression. Furthermore, vesicular stomatitis virus (VSV) replication was significantly inhibited by small inhibitory hairpin RNA (shRNA)-mediated knockdown of TFG, supporting the suppressive role of TFG in RIG-I-mediated antiviral signaling. Interestingly, suppression of TFG expression increased not only RIG-I-mediated signaling but also MAVS (mitochondrial antiviral signaling protein)-induced signaling, suggesting that TFG plays a pivotal role in negative regulation of RNA-sensing, RIG-I-like receptor (RLR) family signaling pathways

  13. The role of GαO-mediated signaling in the rostral ventrolateral medulla oblongata in cardiovascular reflexes and control of cardiac ventricular excitability.

    Science.gov (United States)

    Ang, Richard; Abramowitz, Joel; Birnbaumer, Lutz; Gourine, Alexander V; Tinker, Andrew

    2016-08-01

    The heart is controlled by the sympathetic and parasympathetic limbs of the autonomic nervous system with inhibitory signaling mechanisms recruited in both limbs. The aim of this study was to determine the role of inhibitory heterotrimeric G proteins in the central nervous mechanisms underlying autonomic control of the heart and its potential role in arrhythmogenesis. Mice with conditional deletion of the inhibitory heterotrimeric G protein GαO in the presympathetic area of the rostral ventral lateral medulla (RVLM) were generated to determine the role of GαO-mediated signalling in autonomic control and electrophysiological properties of the heart. GαO deletion within the RVLM was not associated with changes in heart rate (HR) or the arterial blood pressure at rest (home cage, normal behavior). However, exposure to stressful conditions (novel environment, hypoxia, or hypercapnia) in these mice was associated with abnormal HR responses and an increased baroreflex gain when assessed under urethane anesthesia. This was associated with shortening of the ventricular effective refractory period. This phenotype was reversed by systemic beta-adrenoceptor blockade, suggesting that GαO depletion in the RVLM increases central sympathetic drive. The data obtained support the hypothesis that GαO-mediated signaling within the presympathetic circuits of the RVLM contributes to the autonomic control of the heart. GαO deficiency in the RVLM has a significant impact on cardiovascular responses to stress, cardiovascular reflexes and electrical properties of the heart. © 2016 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of the American Physiological Society and The Physiological Society.

  14. Glucagon-like peptide-1 and its class B G Protein-coupled receptors: A long march to therapeutic successes.

    NARCIS (Netherlands)

    de Graaf, C.; Donnely, D.; Wootten, D.; Lau, J.; Sexton, P.M.; Miller, L.J.; Ahn, J.M.; Liao, J.; Fletcher, M.M.; Yang, D.; Brown, A.J.; Zhou, C.; Deng, J.; Wang, M.W.

    2016-01-01

    Theglucagon-likepeptide (GLP)-1receptor (GLP-1R) is a class B G protein-coupled receptor (GPCR) that mediates the action of GLP-1, a peptide hormone secretedfromthreemajor tissues inhumans,enteroendocrine L cells in the distal intestine, a cells in the pancreas, and the central nervous system, which

  15. Mechanisms of estradiol-induced insulin secretion by the G protein-coupled estrogen receptor GPR30/GPER in pancreatic beta-cells.

    Science.gov (United States)

    Sharma, Geetanjali; Prossnitz, Eric R

    2011-08-01

    Sexual dimorphism and supplementation studies suggest an important role for estrogens in the amelioration of glucose intolerance and diabetes. Because little is known regarding the signaling mechanisms involved in estradiol-mediated insulin secretion, we investigated the role of the G protein-coupled receptor 30, now designated G protein-coupled estrogen receptor (GPER), in activating signal transduction cascades in β-cells, leading to secretion of insulin. GPER function in estradiol-induced signaling in the pancreatic β-cell line MIN6 was assessed using small interfering RNA and GPER-selective ligands (G-1 and G15) and in islets isolated from wild-type and GPER knockout mice. GPER is expressed in MIN6 cells, where estradiol and the GPER-selective agonist G-1 mediate calcium mobilization and activation of ERK and phosphatidylinositol 3-kinase. Both estradiol and G-1 induced insulin secretion under low- and high-glucose conditions, which was inhibited by pretreatment with GPER antagonist G15 as well as depletion of GPER by small interfering RNA. Insulin secretion in response to estradiol and G-1 was dependent on epidermal growth factor receptor and ERK activation and further modulated by phosphatidylinositol 3-kinase activity. In islets isolated from wild-type mice, the GPER antagonist G15 inhibited insulin secretion induced by estradiol and G-1, both of which failed to induce insulin secretion in islets obtained from GPER knockout mice. Our results indicate that GPER activation of the epidermal growth factor receptor and ERK in response to estradiol treatment plays a critical role in the secretion of insulin from β-cells. The results of this study suggest that the activation of downstream signaling pathways by the GPER-selective ligand G-1 could represent a novel therapeutic strategy in the treatment of diabetes.

  16. Mechanisms of Estradiol-Induced Insulin Secretion by the G Protein-Coupled Estrogen Receptor GPR30/GPER in Pancreatic β-Cells

    Science.gov (United States)

    Sharma, Geetanjali

    2011-01-01

    Sexual dimorphism and supplementation studies suggest an important role for estrogens in the amelioration of glucose intolerance and diabetes. Because little is known regarding the signaling mechanisms involved in estradiol-mediated insulin secretion, we investigated the role of the G protein-coupled receptor 30, now designated G protein-coupled estrogen receptor (GPER), in activating signal transduction cascades in β-cells, leading to secretion of insulin. GPER function in estradiol-induced signaling in the pancreatic β-cell line MIN6 was assessed using small interfering RNA and GPER-selective ligands (G-1 and G15) and in islets isolated from wild-type and GPER knockout mice. GPER is expressed in MIN6 cells, where estradiol and the GPER-selective agonist G-1 mediate calcium mobilization and activation of ERK and phosphatidylinositol 3-kinase. Both estradiol and G-1 induced insulin secretion under low- and high-glucose conditions, which was inhibited by pretreatment with GPER antagonist G15 as well as depletion of GPER by small interfering RNA. Insulin secretion in response to estradiol and G-1 was dependent on epidermal growth factor receptor and ERK activation and further modulated by phosphatidylinositol 3-kinase activity. In islets isolated from wild-type mice, the GPER antagonist G15 inhibited insulin secretion induced by estradiol and G-1, both of which failed to induce insulin secretion in islets obtained from GPER knockout mice. Our results indicate that GPER activation of the epidermal growth factor receptor and ERK in response to estradiol treatment plays a critical role in the secretion of insulin from β-cells. The results of this study suggest that the activation of downstream signaling pathways by the GPER-selective ligand G-1 could represent a novel therapeutic strategy in the treatment of diabetes. PMID:21673097

  17. Ligand-specific regulation of the extracellular surface of a G-protein-coupled receptor

    Energy Technology Data Exchange (ETDEWEB)

    Bokoch, Michael P.; Zou, Yaozhong; Rasmussen, Søren G.F.; Liu, Corey W.; Nygaard, Rie; Rosenbaum, Daniel M.; Fung, Juan José; Choi, Hee-Jung; Thian, Foon Sun; Kobilka, Tong Sun; Puglisi, Joseph D.; Weis, William I.; Pardo, Leonardo; Prosser, R. Scott; Mueller, Luciano; Kobilka, Brian K. (Stanford-MED); (Toronto); (BMS); (UAB, Spain)

    2010-01-14

    G-protein-coupled receptors (GPCRs) are seven-transmembrane proteins that mediate most cellular responses to hormones and neurotransmitters. They are the largest group of therapeutic targets for a broad spectrum of diseases. Recent crystal structures of GPCRs have revealed structural conservation extending from the orthosteric ligand-binding site in the transmembrane core to the cytoplasmic G-protein-coupling domains. In contrast, the extracellular surface (ECS) of GPCRs is remarkably diverse and is therefore an ideal target for the discovery of subtype-selective drugs. However, little is known about the functional role of the ECS in receptor activation, or about conformational coupling of this surface to the native ligand-binding pocket. Here we use NMR spectroscopy to investigate ligand-specific conformational changes around a central structural feature in the ECS of the {beta}{sub 2} adrenergic receptor: a salt bridge linking extracellular loops 2 and 3. Small-molecule drugs that bind within the transmembrane core and exhibit different efficacies towards G-protein activation (agonist, neutral antagonist and inverse agonist) also stabilize distinct conformations of the ECS. We thereby demonstrate conformational coupling between the ECS and the orthosteric binding site, showing that drugs targeting this diverse surface could function as allosteric modulators with high subtype selectivity. Moreover, these studies provide a new insight into the dynamic behaviour of GPCRs not addressable by static, inactive-state crystal structures.

  18. Cholesterol depletion induces dynamic confinement of the G-protein coupled serotonin(1A) receptor in the plasma membrane of living cells.

    Science.gov (United States)

    Pucadyil, Thomas J; Chattopadhyay, Amitabha

    2007-03-01

    Cholesterol is an essential constituent of eukaryotic membranes and plays a crucial role in membrane organization, dynamics, function, and sorting. It is often found distributed non-randomly in domains or pools in biological and model membranes and is thought to contribute to a segregated distribution of membrane constituents. Signal transduction events mediated by seven transmembrane domain G-protein coupled receptors (GPCRs) are the primary means by which cells communicate with and respond to their external environment. We analyzed the role of cholesterol in the plasma membrane organization of the G-protein coupled serotonin(1A) receptor by fluorescence recovery after photobleaching (FRAP) measurements with varying bleach spot sizes. Our results show that lateral diffusion parameters of serotonin(1A) receptors in normal cells are consistent with models describing diffusion of molecules in a homogenous membrane. Interestingly, these characteristics are altered in cholesterol-depleted cells in a manner that is consistent with dynamic confinement of serotonin(1A) receptors in the plasma membrane. Importantly, analysis of ligand binding and downstream signaling of the serotonin(1A) receptor suggests that receptor function is affected in a significantly different manner when intact cells or isolated membranes are depleted of cholesterol. These results assume significance in the context of interpreting effects of cholesterol depletion on diffusion characteristics of membrane proteins in particular, and cholesterol-dependent cellular processes in general.

  19. G-protein-coupled receptor 81 promotes a malignant phenotype in breast cancer through angiogenic factor secretion.

    Science.gov (United States)

    Lee, Yu Jin; Shin, Kyeong Jin; Park, Soo-Ah; Park, Kyeong Su; Park, Seorim; Heo, Kyun; Seo, Young-Kyo; Noh, Dong-Young; Ryu, Sung Ho; Suh, Pann-Ghill

    2016-10-25

    G-protein-coupled receptor 81 (GPR81) functions as a receptor for lactate and plays an important role in the regulation of anti-lipolytic effects in adipocytes. However, to data, a role for GPR81 in the tumor microenvironment has not been clearly defined. Here, GPR81 expression in breast cancer patients and several breast cancer cell lines was significantly increased compared with normal mammary tissues and cells. GPR81 knockdown resulted in impaired breast cancer growth and led to apoptosis both in vitro and in vivo. Furthermore, the inhibition of GPR81 signaling suppressed angiogenesis through a phosphoinositide 3-OH kinase (PI3K)/Akt-cAMP response element binding protein (CREB) pathway, which led to decreased production of the pro-angiogenic mediator amphiregulin (AREG). Overall, these findings identify GPR81 as a tumor-promoting receptor in breast cancer progression and suggest a novel mechanism that regulates GPR81-dependent activation of the PI3K/Akt signaling axis in tumor microenvironment.

  20. DMPD: Modulation of Toll-interleukin 1 receptor mediated signaling. [Dynamic Macrophage Pathway CSML Database

    Lifescience Database Archive (English)

    Full Text Available 15662540 Modulation of Toll-interleukin 1 receptor mediated signaling. Li X, Qin J.... J Mol Med. 2005 Apr;83(4):258-66. Epub 2005 Jan 21. (.png) (.svg) (.html) (.csml) Show Modulation of Toll-i...nterleukin 1 receptor mediated signaling. PubmedID 15662540 Title Modulation of Toll-interleukin 1 receptor

  1. Extracellular signal regulated kinase 5 mediates signals triggered by the novel tumor promoter palytoxin

    International Nuclear Information System (INIS)

    Charlson, Aaron T.; Zeliadt, Nicholette A.; Wattenberg, Elizabeth V.

    2009-01-01

    Palytoxin is classified as a non-12-O-tetradecanoylphorbol-13-acetate (TPA)-type skin tumor because it does not bind to or activate protein kinase C. Palytoxin is thus a novel tool for investigating alternative signaling pathways that may affect carcinogenesis. We previously showed that palytoxin activates three major members of the mitogen activated protein kinase (MAPK) family, extracellular signal regulated kinase 1 and 2 (ERK1/2), c-Jun N-terminal kinase (JNK), and p38. Here we report that palytoxin also activates another MAPK family member, called ERK5, in HeLa cells and in keratinocytes derived from initiated mouse skin (308 cells). By contrast, TPA does not activate ERK5 in these cell lines. The major cell surface receptor for palytoxin is the Na+,K+-ATPase. Accordingly, ouabain blocked the ability of palytoxin to activate ERK5. Ouabain alone did not activate ERK5. ERK5 thus represents a divergence in the signaling pathways activated by these two agents that bind to the Na+,K+-ATPase. Cycloheximide, okadaic acid, and sodium orthovanadate did not mimic the effect of palytoxin on ERK5. These results indicate that the stimulation of ERK5 by palytoxin is not simply due to inhibition of protein synthesis or inhibition of serine/threonine or tyrosine phosphatases. Therefore, the mechanism by which palytoxin activates ERK5 differs from that by which it activates ERK1/2, JNK, and p38. Finally, studies that used pharmacological inhibitors and shRNA to block ERK5 action indicate that ERK5 contributes to palytoxin-stimulated c-Fos gene expression. These results suggest that ERK5 can act as an alternative mediator for transmitting diverse tumor promoter-stimulated signals.

  2. Characterization of the heterotrimeric G-protein family and its transmembrane regulator from capsicum (Capsicum annuum L.).

    Science.gov (United States)

    Romero-Castillo, Rafael A; Roy Choudhury, Swarup; León-Félix, Josefina; Pandey, Sona

    2015-05-01

    Throughout evolution, organisms have created numerous mechanisms to sense and respond to their environment. One such highly conserved mechanism involves regulation by heterotrimeric G-protein complex comprised of alpha (Gα), beta (Gβ) and gamma (Gγ) subunits. In plants, these proteins play important roles in signal transduction pathways related to growth and development including response to biotic and abiotic stresses and consequently affect yield. In this work, we have identified and characterized the complete heterotrimeric G-protein repertoire in the Capsicum annuum (Capsicum) genome which consists of one Gα, one Gβ and three Gγ genes. We have also identified one RGS gene in the Capsicum genome that acts as a regulator of the G-protein signaling. Biochemical activities of the proteins were confirmed by assessing the GTP-binding and GTPase activity of the recombinant Gα protein and its regulation by the GTPase acceleration activity of the RGS protein. Interaction between different subunits was established using yeast- and plant-based analyses. Gene and protein expression profiles of specific G-protein components revealed interesting spatial and temporal regulation patterns, especially during root development and during fruit development and maturation. This research thus details the characterization of the first heterotrimeric G-protein family from a domesticated, commercially important vegetable crop. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

  3. G protein in stimulation of PI hydrolysis by CCK [cholecystokinin] in isolated rat pancreatic acinar cells

    International Nuclear Information System (INIS)

    Matozaki, Takashi; Sakamoto, Choitsu; Nagao, Munehiko; Nishizaki, Hogara; Baba, Shigeaki

    1988-01-01

    To clarify the possible role of a guanine nucleotide-binding protein (G protein) in the signal transducing system activated by cholecystokinin (CCK), actions of CCK on rat pancreatic acini were compared with those of fluoride, a well-known activator of stimulatory (G s ) or inhibitory (G i ) G protein. When acini were incubated with increasing concentrations of either CCK-octapeptide (CCK8) or NaF, a maximal stimulation of amylase release from acini occurred at 100 pM CCK8 or 10 mM NaF, respectively; this secretory rate decreased as CCK8 or NaF concentration was increased. NaF caused an increase in cytoplasmic Ca 2+ concentration from the internal Ca 2+ store and stimulated accumulation of inositol phosphates in acini, as observed with CCK. Guanylimidodiphosphate activated the generation of inositol phosphates in the [ 3 H]inositol-labeled pancreatic acinar cell membrane preparation, with half-maximal and maximal stimulation at 1 and 10 μM, respectively. Furthermore, the effects of submaximal CCK concentrations on inositol phosphate accumulation in membranes were markedly potentiated in the presence of 100 μM GTP, which alone was ineffective. Combined findings of the present study strongly suggest that pancreatic CCK receptors are probably coupled to the activation of polyphosphoinositide (PI) breakdown by a G protein, which appears to be fluoride sensitive but is other than G s - or G i -like protein

  4. Pituitary adenylate cyclase 1 receptor internalization and endosomal signaling mediate the pituitary adenylate cyclase activating polypeptide-induced increase in guinea pig cardiac neuron excitability.

    Science.gov (United States)

    Merriam, Laura A; Baran, Caitlin N; Girard, Beatrice M; Hardwick, Jean C; May, Victor; Parsons, Rodney L

    2013-03-06

    After G-protein-coupled receptor activation and signaling at the plasma membrane, the receptor complex is often rapidly internalized via endocytic vesicles for trafficking into various intracellular compartments and pathways. The formation of signaling endosomes is recognized as a mechanism that produces sustained intracellular signals that may be distinct from those generated at the cell surface for cellular responses including growth, differentiation, and survival. Pituitary adenylate cyclase activating polypeptide (PACAP; Adcyap1) is a potent neurotransmitter/neurotrophic peptide and mediates its diverse cellular functions in part through internalization of its cognate G-protein-coupled PAC1 receptor (PAC1R; Adcyap1r1). In the present study, we examined whether PAC1R endocytosis participates in the regulation of neuronal excitability. Although PACAP increased excitability in 90% of guinea pig cardiac neurons, pretreatment with Pitstop 2 or dynasore to inhibit clathrin and dynamin I/II, respectively, suppressed the PACAP effect. Subsequent addition of inhibitor after the PACAP-induced increase in excitability developed gradually attenuated excitability with no changes in action potential properties. Likewise, the PACAP-induced increase in excitability was markedly decreased at ambient temperature. Receptor trafficking studies with GFP-PAC1 cell lines demonstrated the efficacy of Pitstop 2, dynasore, and low temperatures at suppressing PAC1R endocytosis. In contrast, brefeldin A pretreatments to disrupt Golgi vesicle trafficking did not blunt the PACAP effect, and PACAP/PAC1R signaling still increased neuronal cAMP production even with endocytic blockade. Our results demonstrate that PACAP/PAC1R complex endocytosis is a key step for the PACAP modulation of cardiac neuron excitability.

  5. Coupling of g proteins to reconstituted monomers and tetramers of the M2 muscarinic receptor.

    Science.gov (United States)

    Redka, Dar'ya S; Morizumi, Takefumi; Elmslie, Gwendolynne; Paranthaman, Pranavan; Shivnaraine, Rabindra V; Ellis, John; Ernst, Oliver P; Wells, James W

    2014-08-29

    G protein-coupled receptors can be reconstituted as monomers in nanodiscs and as tetramers in liposomes. When reconstituted with G proteins, both forms enable an allosteric interaction between agonists and guanylyl nucleotides. Both forms, therefore, are candidates for the complex that controls signaling at the level of the receptor. To identify the biologically relevant form, reconstituted monomers and tetramers of the purified M2 muscarinic receptor were compared with muscarinic receptors in sarcolemmal membranes for the effect of guanosine 5'-[β,γ-imido]triphosphate (GMP-PNP) on the inhibition of N-[(3)H]methylscopolamine by the agonist oxotremorine-M. With monomers, a stepwise increase in the concentration of GMP-PNP effected a lateral, rightward shift in the semilogarithmic binding profile (i.e. a progressive decrease in the apparent affinity of oxotremorine-M). With tetramers and receptors in sarcolemmal membranes, GMP-PNP effected a vertical, upward shift (i.e. an apparent redistribution of sites from a state of high affinity to one of low affinity with no change in affinity per se). The data were analyzed in terms of a mechanistic scheme based on a ligand-regulated equilibrium between uncoupled and G protein-coupled receptors (the "ternary complex model"). The model predicts a rightward shift in the presence of GMP-PNP and could not account for the effects at tetramers in vesicles or receptors in sarcolemmal membranes. Monomers present a special case of the model in which agonists and guanylyl nucleotides interact within a complex that is both constitutive and stable. The results favor oligomers of the M2 receptor over monomers as the biologically relevant state for coupling to G proteins. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

  6. Agonists for G-protein-coupled receptor 84 (GPR84) alter cellular morphology and motility but do not induce pro-inflammatory responses in microglia.

    Science.gov (United States)

    Wei, Li; Tokizane, Kyohei; Konishi, Hiroyuki; Yu, Hua-Rong; Kiyama, Hiroshi

    2017-10-03

    Several G-protein-coupled receptors (GPCRs) have been shown to be important signaling mediators between neurons and glia. In our previous screening for identification of nerve injury-associated GPCRs, G-protein-coupled receptor 84 (GPR84) mRNA showed the highest up-regulation by microglia after nerve injury. GPR84 is a pro-inflammatory receptor of macrophages in a neuropathic pain mouse model, yet its function in resident microglia in the central nervous system is poorly understood. We used endogenous, natural, and surrogate agonists for GPR84 (capric acid, embelin, and 6-OAU, respectively) and examined their effect on mouse primary cultured microglia in vitro. 6-n-Octylaminouracil (6-OAU), embelin, and capric acid rapidly induced membrane ruffling and motility in cultured microglia obtained from C57BL/6 mice, although these agonists failed to promote microglial pro-inflammatory cytokine expression. Concomitantly, 6-OAU suppressed forskolin-induced increase of cAMP in cultured microglia. Pertussis toxin, an inhibitor of Gi-coupled signaling, completely suppressed 6-OAU-induced microglial membrane ruffling and motility. In contrast, no 6-OAU-induced microglial membrane ruffling and motility was observed in microglia from DBA/2 mice, a mouse strain that does not express functional GPR84 protein due to endogenous nonsense mutation of the GPR84 gene. GPR84 mediated signaling causes microglial motility and membrane ruffling but does not promote pro-inflammatory responses. As GPR84 is a known receptor for medium-chain fatty acids, those released from damaged brain cells may be involved in the enhancement of microglial motility through GPR84 after neuronal injury.

  7. The effect of ligand efficacy on the formation and stability of a GPCR-G protein complex

    DEFF Research Database (Denmark)

    Yao, Xiao Jie; Vélez Ruiz, Gisselle; Whorton, Matthew R

    2009-01-01

    G protein-coupled receptors (GPCRs) mediate the majority of physiologic responses to hormones and neurotransmitters. However, many GPCRs exhibit varying degrees of agonist-independent G protein activation. This phenomenon is referred to as basal or constitutive activity. For many of these GPCRs...... of an agonist, the beta(2)AR and Gs can be trapped in a complex by enzymatic depletion of guanine nucleotides. Formation of the complex is enhanced by the agonist isoproterenol, and it rapidly dissociates on exposure to concentrations of GTP and GDP found in the cytoplasm. The inverse agonist ICI prevents...... formation of the beta(2)AR-Gs complex, but has little effect on preformed complexes. These results provide insights into G protein-induced conformational changes in the beta(2)AR and the structural basis for ligand efficacy....

  8. High-resolution crystal structure of an engineered human beta2-adrenergic G protein-coupled receptor

    DEFF Research Database (Denmark)

    Cherezov, Vadim; Rosenbaum, Daniel M; Hanson, Michael A

    2007-01-01

    Heterotrimeric guanine nucleotide-binding protein (G protein)-coupled receptors constitute the largest family of eukaryotic signal transduction proteins that communicate across the membrane. We report the crystal structure of a human beta2-adrenergic receptor-T4 lysozyme fusion protein bound to t...

  9. Validation of a rapid, non-radioactive method to quantify internalisation of G-protein coupled receptors

    NARCIS (Netherlands)

    Jongsma, Maikel; Florczyk, Urszula M.; Hendriks-Balk, Marieelle C.; Michel, Martin C.; Peters, Stephan L. M.; Alewijnse, Astrid E.

    2007-01-01

    Agonist exposure can cause internalisation of G-protein coupled receptors (GPCRs), which may be a part of desensitisation but also of cellular signaling. Previous methods to study internalisation have been tedious or only poorly quantitative. Therefore, we have developed and validated a quantitative

  10. Identification of NCAM-binding peptides promoting neurite outgrowth via a heterotrimeric G-protein-coupled pathway

    DEFF Research Database (Denmark)

    Hansen, Raino Kristian; Christensen, Claus; Korshunova, Irina

    2007-01-01

    the fibroblast growth factor receptor, the Src-related non-receptor tyrosine kinase Fyn, and heterotrimeric G-proteins. Interestingly, neurite outgrowth stimulated by ENFIN2 and ENFIN11 was independent of signaling through fibroblast growth factor receptor and Fyn, but could be inhibited with pertussis toxin...

  11. G protein-coupled receptor mutations and human genetic disease.

    Science.gov (United States)

    Thompson, Miles D; Hendy, Geoffrey N; Percy, Maire E; Bichet, Daniel G; Cole, David E C

    2014-01-01

    Genetic variations in G protein-coupled receptor genes (GPCRs) disrupt GPCR function in a wide variety of human genetic diseases. In vitro strategies and animal models have been used to identify the molecular pathologies underlying naturally occurring GPCR mutations. Inactive, overactive, or constitutively active receptors have been identified that result in pathology. These receptor variants may alter ligand binding, G protein coupling, receptor desensitization and receptor recycling. Receptor systems discussed include rhodopsin, thyrotropin, parathyroid hormone, melanocortin, follicle-stimulating hormone (FSH), luteinizing hormone, gonadotropin-releasing hormone (GNRHR), adrenocorticotropic hormone, vasopressin, endothelin-β, purinergic, and the G protein associated with asthma (GPRA or neuropeptide S receptor 1 (NPSR1)). The role of activating and inactivating calcium-sensing receptor (CaSR) mutations is discussed in detail with respect to familial hypocalciuric hypercalcemia (FHH) and autosomal dominant hypocalemia (ADH). The CASR mutations have been associated with epilepsy. Diseases caused by the genetic disruption of GPCR functions are discussed in the context of their potential to be selectively targeted by drugs that rescue altered receptors. Examples of drugs developed as a result of targeting GPCRs mutated in disease include: calcimimetics and calcilytics, therapeutics targeting melanocortin receptors in obesity, interventions that alter GNRHR loss from the cell surface in idiopathic hypogonadotropic hypogonadism and novel drugs that might rescue the P2RY12 receptor congenital bleeding phenotype. De-orphanization projects have identified novel disease-associated receptors, such as NPSR1 and GPR35. The identification of variants in these receptors provides genetic reagents useful in drug screens. Discussion of the variety of GPCRs that are disrupted in monogenic Mendelian disorders provides the basis for examining the significance of common

  12. Contribution of G protein-coupled estrogen receptor 1 (GPER) to 17β-estradiol-induced developmental toxicity in zebrafish.

    Science.gov (United States)

    Diamante, Graciel; Menjivar-Cervantes, Norma; Leung, Man Sin; Volz, David C; Schlenk, Daniel

    2017-05-01

    Exposure to 17β-estradiol (E2) influences the regulation of multiple signaling pathways, and E2-mediated disruption of signaling events during early development can lead to malformations such as cardiac defects. In this study, we investigated the potential role of the G-protein estrogen receptor 1 (GPER) in E2-induced developmental toxicity. Zebrafish embryos were exposed to E2 from 2h post fertilization (hpf) to 76 hpf with subsequent transcriptional measurements of heart and neural crest derivatives expressed 2 (hand2), leucine rich repeat containing 10 (lrrc10), and gper at 12, 28 and 76 hpf. Alteration in the expression of lrrc10, hand2 and gper was observed at 12 hpf and 76 hpf, but not at 28 hpf. Expression of these genes was also altered after exposure to G1 (a GPER agonist) at 76 hpf. Expression of lrrc10, hand2 and gper all coincided with the formation of cardiac edema at 76 hpf as well as other developmental abnormalities. While co-exposure of G1 with G36 (a GPER antagonist) rescued G1-induced abnormalities and altered gene expression, co-exposure of E2 with G36, or ICI 182,780 (an estrogen receptor antagonist) did not rescue E2-induced cardiac deformities or gene expression. In addition, no effects on the concentrations of downstream ER and GPER signaling molecules (cAMP or calcium) were observed in embryo homogenates after E2 treatment. These data suggest that the impacts of E2 on embryonic development at this stage are complex and may involve multiple receptor and/or signaling pathways. Copyright © 2017 Elsevier B.V. All rights reserved.

  13. G protein-coupled receptors in Anopheles gambiae.

    Science.gov (United States)

    Hill, Catherine A; Fox, A Nicole; Pitts, R Jason; Kent, Lauren B; Tan, Perciliz L; Chrystal, Mathew A; Cravchik, Anibal; Collins, Frank H; Robertson, Hugh M; Zwiebel, Laurence J

    2002-10-04

    We used bioinformatic approaches to identify a total of 276 G protein-coupled receptors (GPCRs) from the Anopheles gambiae genome. These include GPCRs that are likely to play roles in pathways affecting almost every aspect of the mosquito's life cycle. Seventy-nine candidate odorant receptors were characterized for tissue expression and, along with 76 putative gustatory receptors, for their molecular evolution relative to Drosophila melanogaster. Examples of lineage-specific gene expansions were observed as well as a single instance of unusually high sequence conservation.

  14. Molecular and functional profiling of histamine receptor-mediated calcium ion signals in different cell lines.

    Science.gov (United States)

    Meisenberg, Annika; Kaschuba, Dagmar; Balfanz, Sabine; Jordan, Nadine; Baumann, Arnd

    2015-10-01

    Calcium ions (Ca(2+)) play a pivotal role in cellular physiology. Often Ca(2+)-dependent processes are studied in commonly available cell lines. To induce Ca(2+) signals on demand, cells may need to be equipped with additional proteins. A prominent group of membrane proteins evoking Ca(2+) signals are G-protein coupled receptors (GPCRs). These proteins register external signals such as photons, odorants, and neurotransmitters and convey ligand recognition into cellular responses, one of which is Ca(2+) signaling. To avoid receptor cross-talk or cross-activation with introduced proteins, the repertoire of cell-endogenous receptors must be known. Here we examined the presence of histamine receptors in six cell lines frequently used as hosts to study cellular signaling processes. In a concentration-dependent manner, histamine caused a rise in intracellular Ca(2+) in HeLa, HEK 293, and COS-1 cells. The concentration for half-maximal activation (EC50) was in the low micromolar range. In individual cells, transient Ca(2+) signals and Ca(2+) oscillations were uncovered. The results show that (i) HeLa, HEK 293, and COS-1 cells express sufficient amounts of endogenous receptors to study cellular Ca(2+) signaling processes directly and (ii) these cell lines are suitable for calibrating Ca(2+) biosensors in situ based on histamine receptor evoked responses. Copyright © 2015 Elsevier Inc. All rights reserved.

  15. G protein-coupled receptor 39 deficiency is associated with pancreatic islet dysfunction

    DEFF Research Database (Denmark)

    Holst, Birgitte; Egerod, Kristoffer L; Jin, Chunyu

    2009-01-01

    G protein-coupled receptor (GPR)-39 is a seven-transmembrane receptor expressed mainly in endocrine and metabolic tissues that acts as a Zn(++) sensor signaling mainly through the G(q) and G(12/13) pathways. The expression of GPR39 is regulated by hepatocyte nuclear factor (HNF)-1alpha and HNF-4...... tolerance both during oral and iv glucose tolerance tests, and Gpr39(-/-) mice had decreased plasma insulin response to oral glucose. Islet architecture was normal in the Gpr39 null mice, but expression of Pdx-1 and Hnf-1alpha was reduced. Isolated, perifused islets from Gpr39 null mice secreted less...

  16. Regulation of P450-mediated permethrin resistance in Culex quinquefasciatus by the GPCR/Gαs/AC/cAMP/PKA signaling cascade.

    Science.gov (United States)

    Li, Ting; Liu, Nannan

    2017-12-01

    This study explores the role of G-protein-coupled receptor-intracellular signaling in the development of P450-mediated insecticide resistance in mosquitoes, Culex quinquefasciatus , focusing on the essential function of the GPCRs and their downstream effectors of Gs alpha subunit protein (Gαs) and adenylyl cyclase (ACs) in P450-mediated insecticide resistance of Culex mosquitoes. Our RNAi-mediated functional study showed that knockdown of Gαs caused the decreased expression of the downstream effectors of ACs and PKAs in the GPCR signaling pathway and resistance P450 genes, whereas knockdown of ACs decreased the expression of PKAs and resistance P450 genes. Knockdown of either Gαs or ACs resulted in an increased susceptibility of mosquitoes to permethrin. These results add significantly to our understanding of the molecular basis of resistance P450 gene regulation through GPCR/Gαs/AC/cAMP-PKA signaling pathways in the insecticide resistance of mosquitoes. The temporal and spatial dynamic analyses of GPCRs, Gαs, ACs, PKAs, and P450s in two insecticide resistant mosquito strains revealed that all the GPCR signaling pathway components tested, namely GPCRs, Gαs, ACs and PKAs, were most highly expressed in the brain for both resistant strains, suggesting the role played by these genes in signaling transduction and regulation. The resistance P450 genes were mainly expressed in the brain, midgut and malpighian tubules (MTs), suggesting their critical function in the central nervous system and importance for detoxification. The temporal dynamics analysis for the gene expression showed a diverse expression profile during mosquito development, indicating their initially functional importance in response to exposure to insecticides during their life stages.

  17. Comparison of MEK/ERK pathway inhibitors on the upregulation of vascular G-protein coupled receptors in rat cerebral arteries

    DEFF Research Database (Denmark)

    Sandhu, Hardip; Ansar, Saema; Edvinsson, Lars

    2010-01-01

    on translational level and increased respective contractions. The prostanoid TP receptor mediated contraction curve was left-wards shifted by organ culture. Organ culture was associated with elevated pERK1/2 in the vascular smooth muscle cells: the MEK1/2 inhibitor U0126 attenuated the endothelin ET(B) receptor......Organ culture is an in vitro method for investigating cellular mechanisms involved in upregulation of vasocontractile G-protein coupled receptors. We hypothesize that mitogen-activated-protein kinase (MEK) and/or extracellular-signal-regulated kinase (ERK) specific inhibitors will attenuate the G......), prostanoid TP receptor, and angiotensin II receptor type 1 and type 2 were investigated. Results were verified by measurement of mRNA with real time PCR and by protein immunohistochemistry. Organ culture induced transcriptional upregulation of endothelin ET(B) receptor and of serotonin 5-HT(1B) receptor...

  18. Impact of G protein-coupled receptor heteromers in endocrine systems.

    Science.gov (United States)

    Jonas, K C; Hanyaloglu, A C

    2017-07-05

    The fine-tuning of endocrine homeostasis is regulated by dynamic receptor mediated processes. The superfamily of G protein-coupled receptors (GPCRs) have diverse roles in the modulation of all endocrine axes, thus understanding the mechanisms underpinning their functionality is paramount for treatment of endocrinopathies. Evidence over the last 20 years has highlighted homo and heteromerization as a key mode of mediating GPCR functional diversity. This review will discuss the concept of GPCR heteromerization and its relevance to endocrine function, detailing in vitro and in vivo evidence, and exploring current and potential pharmacological strategies for specific targeting of GPCR heteromers in endocrine heath and disease. Crown Copyright © 2017. Published by Elsevier B.V. All rights reserved.

  19. Transmateriality: Toward an Energetics of Signal in Contemporary Mediatic Assemblages

    Directory of Open Access Journals (Sweden)

    Anna Munster

    2014-03-01

    Full Text Available This article focuses on signal as an aspect of modern technicity that precedes—or supersedes—codification. Examining DIY drone videos found on YouTube and the video art of Nam June Paik, among other sources, the article explores how each tests the flow of signal and signal processing as forms of transmateriality and transduction. the author draws particularly on the work of Gilbert Simondon and Adrian Mackenzie.

  20. Crystal Structure of a Lipid G Protein-Coupled Receptor

    Energy Technology Data Exchange (ETDEWEB)

    Hanson, Michael A; Roth, Christopher B; Jo, Euijung; Griffith, Mark T; Scott, Fiona L; Reinhart, Greg; Desale, Hans; Clemons, Bryan; Cahalan, Stuart M; Schuerer, Stephan C; Sanna, M Germana; Han, Gye Won; Kuhn, Peter; Rosen, Hugh; Stevens, Raymond C [Scripps; (Receptos)

    2012-03-01

    The lyso-phospholipid sphingosine 1-phosphate modulates lymphocyte trafficking, endothelial development and integrity, heart rate, and vascular tone and maturation by activating G protein-coupled sphingosine 1-phosphate receptors. Here, we present the crystal structure of the sphingosine 1-phosphate receptor 1 fused to T4-lysozyme (S1P1-T4L) in complex with an antagonist sphingolipid mimic. Extracellular access to the binding pocket is occluded by the amino terminus and extracellular loops of the receptor. Access is gained by ligands entering laterally between helices I and VII within the transmembrane region of the receptor. This structure, along with mutagenesis, agonist structure-activity relationship data, and modeling, provides a detailed view of the molecular recognition and requirement for hydrophobic volume that activates S1P1, resulting in the modulation of immune and stromal cell responses.

  1. Peptide drugs to target G protein-coupled receptors.

    Science.gov (United States)

    Bellmann-Sickert, Kathrin; Beck-Sickinger, Annette G

    2010-09-01

    Major indications for use of peptide-based therapeutics include endocrine functions (especially diabetes mellitus and obesity), infectious diseases, and cancer. Whereas some peptide pharmaceuticals are drugs, acting as agonists or antagonists to directly treat cancer, others (including peptide diagnostics and tumour-targeting pharmaceuticals) use peptides to 'shuttle' a chemotherapeutic agent or a tracer to the tumour and allow sensitive imaging or targeted therapy. Significant progress has been made in the last few years to overcome disadvantages in peptide design such as short half-life, fast proteolytic cleavage, and low oral bioavailability. These advances include peptide PEGylation, lipidisation or multimerisation; the introduction of peptidomimetic elements into the sequences; and innovative uptake strategies such as liposomal, capsule or subcutaneous formulations. This review focuses on peptides targeting G protein-coupled receptors that are promising drug candidates or that have recently entered the pharmaceutical market. Copyright 2010 Elsevier Ltd. All rights reserved.

  2. Recent Progress in Understanding Subtype Specific Regulation of NMDA Receptors by G Protein Coupled Receptors (GPCRs

    Directory of Open Access Journals (Sweden)

    Kai Yang

    2014-02-01

    Full Text Available G Protein Coupled Receptors (GPCRs are the largest family of receptors whose ligands constitute nearly a third of prescription drugs in the market. They are widely involved in diverse physiological functions including learning and memory. NMDA receptors (NMDARs, which belong to the ionotropic glutamate receptor family, are likewise ubiquitously expressed in the central nervous system (CNS and play a pivotal role in learning and memory. Despite its critical contribution to physiological and pathophysiological processes, few pharmacological interventions aimed directly at regulating NMDAR function have been developed to date. However, it is well established that NMDAR function is precisely regulated by cellular signalling cascades recruited downstream of G protein coupled receptor (GPCR stimulation. Accordingly, the downstream regulation of NMDARs likely represents an important determinant of outcome following treatment with neuropsychiatric agents that target selected GPCRs. Importantly, the functional consequence of such regulation on NMDAR function varies, based not only on the identity of the GPCR, but also on the cell type in which relevant receptors are expressed. Indeed, the mechanisms responsible for regulating NMDARs by GPCRs involve numerous intracellular signalling molecules and regulatory proteins that vary from one cell type to another. In the present article, we highlight recent findings from studies that have uncovered novel mechanisms by which selected GPCRs regulate NMDAR function and consequently NMDAR-dependent plasticity.

  3. Adaptive and innate immune reactions regulating mast cell activation: from receptor-mediated signaling to responses

    DEFF Research Database (Denmark)

    Tkaczyk, Christine; Jensen, Bettina M; Iwaki, Shoko

    2006-01-01

    differentially activate multiple signaling pathways within the mast cells required for the generation and/or release of inflammatory mediators. Thus, the composition of the suite of mediators released and the physiologic ramifications of these responses are dependent on the stimuli and the microenvironment...

  4. An Autocrine Proliferation Repressor Regulates Dictyostelium discoideum Proliferation and Chemorepulsion Using the G Protein-Coupled Receptor GrlH

    OpenAIRE

    Yu Tang; Yuantai Wu; Sarah E. Herlihy; Francisco J. Brito-Aleman; Jose H. Ting; Chris Janetopoulos; Richard H. Gomer; Scott D. Emr

    2018-01-01

    In eukaryotic microbes, little is known about signals that inhibit the proliferation of the cells that secrete the signal, and little is known about signals (chemorepellents) that cause cells to move away from the source of the signal. Autocrine proliferation repressor protein A (AprA) is a protein secreted by the eukaryotic microbe Dictyostelium discoideum. AprA is a chemorepellent for and inhibits the proliferation of D. discoideum. We previously found that cells sense AprA using G proteins...

  5. The β-Arrestins: Multifunctional Regulators of G Protein-coupled Receptors.

    Science.gov (United States)

    Smith, Jeffrey S; Rajagopal, Sudarshan

    2016-04-22

    The β-arrestins (βarrs) are versatile, multifunctional adapter proteins that are best known for their ability to desensitize G protein-coupled receptors (GPCRs), but also regulate a diverse array of cellular functions. To signal in such a complex fashion, βarrs adopt multiple conformations and are regulated at multiple levels to differentially activate downstream pathways. Recent structural studies have demonstrated that βarrs have a conserved structure and activation mechanism, with plasticity of their structural fold, allowing them to adopt a wide array of conformations. Novel roles for βarrs continue to be identified, demonstrating the importance of these dynamic regulators of cellular signaling. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  6. Evolution of a G protein-coupled receptor response by mutations in regulatory network interactions

    DEFF Research Database (Denmark)

    Di Roberto, Raphaël B; Chang, Belinda; Trusina, Ala

    2016-01-01

    All cellular functions depend on the concerted action of multiple proteins organized in complex networks. To understand how selection acts on protein networks, we used the yeast mating receptor Ste2, a pheromone-activated G protein-coupled receptor, as a model system. In Saccharomyces cerevisiae......, Ste2 is a hub in a network of interactions controlling both signal transduction and signal suppression. Through laboratory evolution, we obtained 21 mutant receptors sensitive to the pheromone of a related yeast species and investigated the molecular mechanisms behind this newfound sensitivity. While...... demonstrate that a new receptor-ligand pair can evolve through network-altering mutations independently of receptor-ligand binding, and suggest a potential role for such mutations in disease....

  7. Regulation of Rac1 GTPase activity by quinine through G-protein and bitter taste receptor T2R4.

    Science.gov (United States)

    Sidhu, Crystal; Jaggupilli, Appalaraju; Chelikani, Prashen; Bhullar, Rajinder P

    2017-02-01

    Rac1 belongs to the Rho family of small GTPases and regulates actin cytoskeleton reorganization. T2R4 is a bitter taste receptor belonging to the G protein-coupled receptor family of proteins. In addition to mediating bitter taste perception from the tongue, T2R4s are found in extra-oral tissues, e.g., nasal epithelium, airways, brain, testis suggesting a much broader physiological function for these receptors. Anti-malarial drug and a bitter tasting compound, quinine, is a known agonist for T2R4, whereas BCML (Nα,Nα-Bis(carboxymethyl)-L-lysine) acts as an inverse agonist. Using western blot and Ca ++ mobilization assays, the effects of quinine on Rac1 activity in HEK293T cells stably expressing T2R4/Gα 16/44 , T2R4, or Gα 16/44 and transiently transfected with HA-Rac1 were investigated. Quinine treatment caused a significant reduction in the amount of active Rac1, whereas in the presence of BCML, quinine failed to cause any significant change in active Rac1. No significant change in Rac1 activity was observed in BAPTA-AM plus quinine-treated Gα 16/44 cells, suggesting possibility of a pathway in addition to the canonical Ca ++ -dependent pathway. A noticeable role for Gα 16/44 independent of T2R4 is observed in quinine-mediated Rac1 inactivation. Further, a significant difference in quinine-induced Ca ++ response in T2R4/Gα 16/44 or T2R4 cells was observed validating the partial role of calcium and importance of Gα 16/44 . This study is the first to show an inhibitory downstream action of a T2R4 agonist on Rac1 function. Further investigation will help in better understanding the downstream signal transduction network of T2R4 and its extra-oral physiological roles.

  8. Backbone resonance assignments for G protein α(i3) subunit in the GDP-bound state.

    Science.gov (United States)

    Mase, Yoko; Yokogawa, Mariko; Osawa, Masanori; Shimada, Ichio

    2014-10-01

    Guanine-nucleotide binding proteins (G proteins) serve as molecular switches in signaling pathways, by coupling the activation of G protein-coupled receptors (GPCRs) at the cell surface to intracellular responses. In the resting state, G protein forms a heterotrimer, consisting of the G protein α subunit with GDP (Gα·GDP) and the G protein βγ subunit (Gβγ). Ligand binding to GPCRs promotes the GDP-GTP exchange on Gα, leading to the dissociation of the GTP-bound form of Gα (Gα·GTP) and Gβγ. Then, Gα·GTP and Gβγ bind to their downstream effector enzymes or ion channels and regulate their activities, leading to a variety of cellular responses. Finally, Gα hydrolyzes the bound GTP to GDP and returns to the resting state by re-associating with Gβγ. The G proteins are classified with four major families based on the amino acid sequences of Gα: i/o, s, q/11, and 12/13. Here, we established the backbone resonance assignments of human Gαi3, a member of the i/o family with a molecular weight of 41 K, in complex with GDP. The chemical shifts were compared with those of Gα(i3) in complex with a GTP-analogue, GTPγS, which we recently reported, indicating that the residues with significant chemical shift differences are mostly consistent with the regions with the structural differences between the GDP- and GTPγS-bound states, as indicated in the crystal structures. The assignments of Gα(i3)·GDP would be useful for the analyses of the dynamics of Gα(i3) and its interactions with various target molecules.

  9. Role of Detergents in Conformational Exchange of a G Protein-coupled Receptor*

    Science.gov (United States)

    Chung, Ka Young; Kim, Tae Hun; Manglik, Aashish; Alvares, Rohan; Kobilka, Brian K.; Prosser, R. Scott

    2012-01-01

    The G protein-coupled β2-adrenoreceptor (β2AR) signals through the heterotrimeric G proteins Gs and Gi and β-arrestin. As such, the energy landscape of β2AR-excited state conformers is expected to be complex. Upon tagging Cys-265 of β2AR with a trifluoromethyl probe, 19F NMR was used to assess conformations and possible equilibria between states. Here, we report key differences in β2AR conformational dynamics associated with the detergents used to stabilize the receptor. In dodecyl maltoside (DDM) micelles, the spectra are well represented by a single Lorentzian line that shifts progressively downfield with activation by appropriate ligand. The results are consistent with interconversion between two or more states on a time scale faster than the greatest difference in ligand-dependent chemical shift (i.e. >100 Hz). Given that high detergent off-rates of DDM monomers may facilitate conformational exchange between functional states of β2AR, we utilized the recently developed maltose-neopentyl glycol (MNG-3) diacyl detergent. In MNG-3 micelles, spectra indicated at least three distinct states, the relative populations of which depended on ligand, whereas no ligand-dependent shifts were observed, consistent with the slow exchange limit. Thus, detergent has a profound effect on the equilibrium kinetics between functional states. MNG-3, which has a critical micelle concentration in the nanomolar regime, exhibits an off-rate that is 4 orders of magnitude lower than that of DDM. High detergent off-rates are more likely to facilitate conformational exchange between distinct functional states associated with the G protein-coupled receptor. PMID:22893704

  10. Dysfunction of G Protein-Coupled Receptor Kinases in Alzheimer’'s Disease

    Directory of Open Access Journals (Sweden)

    William Z. Suo

    2010-01-01

    Full Text Available Although mutations and variations of several genes have been identified to be involved in Alzheimer's disease (AD, the efforts towards understanding the pathogenic mechanisms of the disease still have a long journey to go. One such effort is to identify the signal transduction deficits, for which previous studies have suggested that the central problems appear to be at the interface between G proteins and their coupled receptors. G protein-coupled receptor kinases (GRKs are a small family of serine/threonine protein kinases primarily acting at the “receptor-G protein interface””. Recent studies have indicated the possible involvement of GRK, primarily GRK2 and GRK5, dysfunction in the pathogenesis of AD. It seems that mild, soluble, β-amyloid accumulation can lead to a reduced membrane (functional and an elevated cytosolic GRK2/5. The increased cytosolic GRK2 appears to be colocalized with damaged mitochondria and neurofibrillary tangles. Moreover, the total levels of GRK2, not only in the brain, but also in peripheral blood samples, are increased in a manner inversely correlated with the patient's cognitive levels. The deficiency of GRK5, on the other hand, impairs presynaptic M2 autoreceptor desensitization, which leads to a reduced acetylcholine release, axonal/synaptic degenerative changes, and associated amnestic, mild cognitive impairment. It also promotes an evil cycle to further increase Beta-amyloid accumulation and exaggerates brain inflammation, possibly even the basal forebrain cholinergic degeneration. Therefore, continuous efforts in this direction are necessary before translating the knowledge to any therapeutic strategies.

  11. Role of detergents in conformational exchange of a G protein-coupled receptor.

    Science.gov (United States)

    Chung, Ka Young; Kim, Tae Hun; Manglik, Aashish; Alvares, Rohan; Kobilka, Brian K; Prosser, R Scott

    2012-10-19

    The G protein-coupled β(2)-adrenoreceptor (β(2)AR) signals through the heterotrimeric G proteins G(s) and G(i) and β-arrestin. As such, the energy landscape of β(2)AR-excited state conformers is expected to be complex. Upon tagging Cys-265 of β(2)AR with a trifluoromethyl probe, (19)F NMR was used to assess conformations and possible equilibria between states. Here, we report key differences in β(2)AR conformational dynamics associated with the detergents used to stabilize the receptor. In dodecyl maltoside (DDM) micelles, the spectra are well represented by a single Lorentzian line that shifts progressively downfield with activation by appropriate ligand. The results are consistent with interconversion between two or more states on a time scale faster than the greatest difference in ligand-dependent chemical shift (i.e. >100 Hz). Given that high detergent off-rates of DDM monomers may facilitate conformational exchange between functional states of β(2)AR, we utilized the recently developed maltose-neopentyl glycol (MNG-3) diacyl detergent. In MNG-3 micelles, spectra indicated at least three distinct states, the relative populations of which depended on ligand, whereas no ligand-dependent shifts were observed, consistent with the slow exchange limit. Thus, detergent has a profound effect on the equilibrium kinetics between functional states. MNG-3, which has a critical micelle concentration in the nanomolar regime, exhibits an off-rate that is 4 orders of magnitude lower than that of DDM. High detergent off-rates are more likely to facilitate conformational exchange between distinct functional states associated with the G protein-coupled receptor.

  12. An epidemic process mediated by a decaying diffusing signal

    International Nuclear Information System (INIS)

    Faria, Fernando P; Dickman, Ronald

    2012-01-01

    We study a stochastic epidemic model consisting of elements (organisms in a community or cells in tissue) with fixed positions, in which damage or disease is transmitted by diffusing agents ('signals') emitted by infected individuals. The signals decay as well as diffuse; since they are assumed to be produced in large numbers, the signal concentration is treated deterministically. The model, which includes four cellular states (susceptible, transformed, depleted, and removed), admits various interpretations: spread of an infection or infectious disease, or of damage in a tissue in which injured cells may themselves provoke further damage, and as a description of the so-called radiation-induced bystander effect, in which the signals are molecules capable of inducing cell damage and/or death in unirradiated cells. The model exhibits a continuous phase transition between spreading and nonspreading phases. We formulate two mean-field theory (MFT) descriptions of the model, one of which ignores correlations between the cellular state and the signal concentration, and another that treats such correlations in an approximate manner. Monte Carlo simulations of the spread of infection on the square lattice yield values for the critical exponents and the fractal dimension consistent with the dynamic percolation universality class

  13. The mediator complex in genomic and non-genomic signaling in cancer.

    Science.gov (United States)

    Weber, Hannah; Garabedian, Michael J

    2018-05-01

    Mediator is a conserved, multi-subunit macromolecular machine divided structurally into head, middle, and tail modules, along with a transiently associating kinase module. Mediator functions as an integrator of transcriptional regulatory activity by interacting with DNA-bound transcription factors and with RNA polymerase II (Pol II) to both activate and repress gene expression. Mediator has been shown to affect multiple steps in transcription, including chromatin looping between enhancers and promoters, pre-initiation complex formation, transcriptional elongation, and mRNA splicing. Individual Mediator subunits participate in regulation of gene expression by the estrogen and androgen receptors and are altered in a number of endocrine cancers, including breast and prostate cancer. In addition to its role in genomic signaling, MED12 has been implicated in non-genomic signaling by interacting with and activating TGF-beta receptor 2 in the cytoplasm. Recent structural studies have revealed extensive inter-domain interactions and complex architecture of the Mediator-Pol II complex, suggesting that Mediator is capable of reorganizing its conformation and composition to fit cellular needs. We propose that alterations in Mediator subunit expression that occur in various cancers could impact the organization and function of Mediator, resulting in changes in gene expression that promote malignancy. A better understanding of the role of Mediator in cancer could reveal new approaches to the diagnosis and treatment of Mediator-dependent endocrine cancers, especially in settings of therapy resistance. Copyright © 2017 Elsevier Inc. All rights reserved.

  14. Reduced beta-adrenergic receptor activation decreases G-protein expression and beta-adrenergic receptor kinase activity in porcine heart.

    OpenAIRE

    Ping, P; Gelzer-Bell, R; Roth, D A; Kiel, D; Insel, P A; Hammond, H K

    1995-01-01

    To determine whether beta-adrenergic receptor agonist activation influences guanosine 5'-triphosphate-binding protein (G-protein) expression and beta-adrenergic receptor kinase activity in the heart, we examined the effects of chronic beta 1-adrenergic receptor antagonist treatment (bisoprolol, 0.2 mg/kg per d i.v., 35 d) on components of the myocardial beta-adrenergic receptor-G-protein-adenylyl cyclase pathway in porcine myocardium. Three novel alterations in cardiac adrenergic signaling as...

  15. Notch signalling mediates reproductive constraint in the adult worker honeybee

    Science.gov (United States)

    Duncan, Elizabeth J.; Hyink, Otto; Dearden, Peter K.

    2016-01-01

    The hallmark of eusociality is the reproductive division of labour, in which one female caste reproduces, while reproduction is constrained in the subordinate caste. In adult worker honeybees (Apis mellifera) reproductive constraint is conditional: in the absence of the queen and brood, adult worker honeybees activate their ovaries and lay haploid male eggs. Here, we demonstrate that chemical inhibition of Notch signalling can overcome the repressive effect of queen pheromone and promote ovary activity in adult worker honeybees. We show that Notch signalling acts on the earliest stages of oogenesis and that the removal of the queen corresponds with a loss of Notch protein in the germarium. We conclude that the ancient and pleiotropic Notch signalling pathway has been co-opted into constraining reproduction in worker honeybees and we provide the first molecular mechanism directly linking ovary activity in adult worker bees with the presence of the queen. PMID:27485026

  16. Estradiol Membrane-Initiated Signaling in the Brain Mediates Reproduction.

    Science.gov (United States)

    Micevych, Paul E; Mermelstein, Paul G; Sinchak, Kevin

    2017-11-01

    Over the past few years our understanding of estrogen signaling in the brain has expanded rapidly. Estrogens are synthesized in the periphery and in the brain, acting on multiple receptors to regulate gene transcription, neural function, and behavior. Various estrogen-sensitive signaling pathways often operate in concert within the same cell, increasing the complexity of the system. In females, estrogen concentrations fluctuate over the estrous/menstrual cycle, dynamically modulating estrogen receptor (ER) expression, activity, and trafficking. These dynamic changes influence multiple behaviors but are particularly important for reproduction. Using the female rodent model, we review our current understanding of estradiol signaling in the regulation of sexual receptivity. Copyright © 2017 Elsevier Ltd. All rights reserved.

  17. Eukaryotic interference with homoserine lactone mediated procaryotic signalling

    DEFF Research Database (Denmark)

    Givskov, Michael Christian; de Nys, Rocky; Gram, Lone

    1996-01-01

    Acylated homoserine lactones (AHLs) plays a widespread role in intercellular communication among bacteria. The Australian macroalga Delisea pulchra produces secondary metabolites which have structural similarities to AHL molecules. We report here that these metabolites inhibited AHL-controlled pr......-controlled processes in prokaryotes. Our results suggest that the interaction between higher organisms and their surface-associated bacteria may be mediated by interference with bacterial regulatory systems....

  18. The repertoire of G protein-coupled receptors in the human parasite Schistosoma mansoni and the model organism Schmidtea mediterranea

    Directory of Open Access Journals (Sweden)

    Zamanian Mostafa

    2011-12-01

    Full Text Available Abstract Background G protein-coupled receptors (GPCRs constitute one of the largest groupings of eukaryotic proteins, and represent a particularly lucrative set of pharmaceutical targets. They play an important role in eukaryotic signal transduction and physiology, mediating cellular responses to a diverse range of extracellular stimuli. The phylum Platyhelminthes is of considerable medical and biological importance, housing major pathogens as well as established model organisms. The recent availability of genomic data for the human blood fluke Schistosoma mansoni and the model planarian Schmidtea mediterranea paves the way for the first comprehensive effort to identify and analyze GPCRs in this important phylum. Results Application of a novel transmembrane-oriented approach to receptor mining led to the discovery of 117 S. mansoni GPCRs, representing all of the major families; 105 Rhodopsin, 2 Glutamate, 3 Adhesion, 2 Secretin and 5 Frizzled. Similarly, 418 Rhodopsin, 9 Glutamate, 21 Adhesion, 1 Secretin and 11 Frizzled S. mediterranea receptors were identified. Among these, we report the identification of novel receptor groupings, including a large and highly-diverged Platyhelminth-specific Rhodopsin subfamily, a planarian-specific Adhesion-like family, and atypical Glutamate-like receptors. Phylogenetic analysis was carried out following extensive gene curation. Support vector machines (SVMs were trained and used for ligand-based classification of full-length Rhodopsin GPCRs, complementing phylogenetic and homology-based classification. Conclusions Genome-wide investigation of GPCRs in two platyhelminth genomes reveals an extensive and complex receptor signaling repertoire with many unique features. This work provides important sequence and functional leads for understanding basic flatworm receptor biology, and sheds light on a lucrative set of anthelmintic drug targets.

  19. Noncanonical thyroid hormone signaling mediates cardiometabolic effects in vivo

    DEFF Research Database (Denmark)

    Hönes, G. Sebastian; Rakov, Helena; Logan, John

    2017-01-01

    Thyroid hormone (TH) and TH receptors (TRs) α and β act by binding to TH response elements (TREs) in regulatory regions of target genes. This nuclear signaling is established as the canonical or type 1 pathway for TH action. Nevertheless, TRs also rapidly activate intracellular second-messenger s...

  20. The G protein-coupled receptors deorphanization landscape.

    Science.gov (United States)

    Laschet, Céline; Dupuis, Nadine; Hanson, Julien

    2018-07-01

    G protein-coupled receptors (GPCRs) are usually highlighted as being both the largest family of membrane proteins and the most productive source of drug targets. However, most of the GPCRs are understudied and hence cannot be used immediately for innovative therapeutic strategies. Besides, there are still around 100 orphan receptors, with no described endogenous ligand and no clearly defined function. The race to discover new ligands for these elusive receptors seems to be less intense than before. Here, we present an update of the various strategies employed to assign a function to these receptors and to discover new ligands. We focus on the recent advances in the identification of endogenous ligands with a detailed description of newly deorphanized receptors. Replication being a key parameter in these endeavors, we also discuss the latest controversies about problematic ligand-receptor pairings. In this context, we propose several recommendations in order to strengthen the reporting of new ligand-receptor pairs. Copyright © 2018 Elsevier Inc. All rights reserved.

  1. GPCRdb: an information system for G protein-coupled receptors.

    Science.gov (United States)

    Isberg, Vignir; Mordalski, Stefan; Munk, Christian; Rataj, Krzysztof; Harpsøe, Kasper; Hauser, Alexander S; Vroling, Bas; Bojarski, Andrzej J; Vriend, Gert; Gloriam, David E

    2016-01-04

    Recent developments in G protein-coupled receptor (GPCR) structural biology and pharmacology have greatly enhanced our knowledge of receptor structure-function relations, and have helped improve the scientific foundation for drug design studies. The GPCR database, GPCRdb, serves a dual role in disseminating and enabling new scientific developments by providing reference data, analysis tools and interactive diagrams. This paper highlights new features in the fifth major GPCRdb release: (i) GPCR crystal structure browsing, superposition and display of ligand interactions; (ii) direct deposition by users of point mutations and their effects on ligand binding; (iii) refined snake and helix box residue diagram looks; and (iii) phylogenetic trees with receptor classification colour schemes. Under the hood, the entire GPCRdb front- and back-ends have been re-coded within one infrastructure, ensuring a smooth browsing experience and development. GPCRdb is available at http://www.gpcrdb.org/ and it's open source code at https://bitbucket.org/gpcr/protwis. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

  2. Superfamily of G-protein coupled receptors (GPCRs – extraordinary and outstanding success of evolution

    Directory of Open Access Journals (Sweden)

    Kazimierz Kochman

    2014-10-01

    Full Text Available The G protein-coupled receptors (GPCRs are considered as very diverse and also surprisingly successful structures during the whole evolutionary process, being capable of transducing the different forms of “information” within the cell and also between cells, such as different peptides, lipids, proteins, nucleotides, nucleosides, organic odorants and photons. Complex studies as well as two-dimensional crystallization of rhodopsin, their paradigm, led to the creation of a useful model having a common central core, consisting of seven transmembrane helical domains, which undergoes appropriate structural modification during activation and signal transduction. After the complete delineation of the human genome, which is the apogee of human scientific civilization and culture, it was possible to identify more than 800 human GPCR sequences and in parallel analyze 342 unique functional nonolfactory human GPCR sequences with phylogenetic analyses. These results support, with high bootstrap values, the existence of five main families, named by the authors glutamate, rhodopsin, adhesion, frizzle/taste2, and secretin, forming the GRAFS classification system. Positions of the GPCRs in chromosomal paralogous regions indicate the importance of tetraploidizations or local gene duplication events during their creation. Some families of GPCRs show, however, very little or no similarity in the sequence of amino acid chains. They utilize an enormous number of different domains to bind ligands and to activate the appropriate G-proteins. The delicate tuning of their coupling to G proteins is further regulated by splicing, RNA editing and phosphorylation. A number of GPCRs may also form homodimers or heterodimers with structurally different GPCRs and also with membrane-bound proteins having one transmembrane domain. It should also be stressed that not all GPCRs are strictly faithful to G proteins because growing evidence indicates that they can interact directly

  3. Three α-Subunits of Heterotrimeric G Proteins and an Adenylyl Cyclase Have Distinct Roles in Fruiting Body Development in the Homothallic Fungus Sordaria macrospora

    Science.gov (United States)

    Kamerewerd, Jens; Jansson, Malin; Nowrousian, Minou; Pöggeler, Stefanie; Kück, Ulrich

    2008-01-01

    Sordaria macrospora, a self-fertile filamentous ascomycete, carries genes encoding three different α-subunits of heterotrimeric G proteins (gsa, G protein Sordaria alpha subunit). We generated knockout strains for all three gsa genes (Δgsa1, Δgsa2, and Δgsa3) as well as all combinations of double mutants. Phenotypic analysis of single and double mutants showed that the genes for Gα-subunits have distinct roles in the sexual life cycle. While single mutants show some reduction of fertility, double mutants Δgsa1Δgsa2 and Δgsa1Δgsa3 are completely sterile. To test whether the pheromone receptors PRE1 and PRE2 mediate signaling via distinct Gα-subunits, two recently generated Δpre strains were crossed with all Δgsa strains. Analyses of the corresponding double mutants revealed that compared to GSA2, GSA1 is a more predominant regulator of a signal transduction cascade downstream of the pheromone receptors and that GSA3 is involved in another signaling pathway that also contributes to fruiting body development and fertility. We further isolated the gene encoding adenylyl cyclase (AC) (sac1) for construction of a knockout strain. Analyses of the three ΔgsaΔsac1 double mutants and one Δgsa2Δgsa3Δsac1 triple mutant indicate that SAC1 acts downstream of GSA3, parallel to a GSA1–GSA2-mediated signaling pathway. In addition, the function of STE12 and PRO41, two presumptive signaling components, was investigated in diverse double mutants lacking those developmental genes in combination with the gsa genes. This analysis was further completed by expression studies of the ste12 and pro41 transcripts in wild-type and mutant strains. From the sum of all our data, we propose a model for how different Gα-subunits interact with pheromone receptors, adenylyl cyclase, and STE12 and thus cooperatively regulate sexual development in S. macrospora. PMID:18723884

  4. Acidic tumor microenvironment and pH-sensing G protein-coupled receptors.

    Science.gov (United States)

    Justus, Calvin R; Dong, Lixue; Yang, Li V

    2013-12-05

    The tumor microenvironment is acidic due to glycolytic cancer cell metabolism, hypoxia, and deficient blood perfusion. It is proposed that acidosis in the tumor microenvironment is an important stress factor and selection force for cancer cell somatic evolution. Acidic pH has pleiotropic effects on the proliferation, migration, invasion, metastasis, and therapeutic response of cancer cells and the function of immune cells, vascular cells, and other stromal cells. However, the molecular mechanisms by which cancer cells and stromal cells sense and respond to acidic pH in the tumor microenvironment are poorly understood. In this article the role of a family of pH-sensing G protein-coupled receptors (GPCRs) in tumor biology is reviewed. Recent studies show that the pH-sensing GPCRs, including GPR4, GPR65 (TDAG8), GPR68 (OGR1), and GPR132 (G2A), regulate cancer cell metastasis and proliferation, immune cell function, inflammation, and blood vessel formation. Activation of the proton-sensing GPCRs by acidosis transduces multiple downstream G protein signaling pathways. Since GPCRs are major drug targets, small molecule modulators of the pH-sensing GPCRs are being actively developed and evaluated. Research on the pH-sensing GPCRs will continue to provide important insights into the molecular interaction between tumor and its acidic microenvironment and may identify new targets for cancer therapy and chemoprevention.

  5. β2-Adrenergic receptors and G-protein-coupled receptor kinase 2 in rabbit pleural mesothelium.

    Science.gov (United States)

    Sironi, Chiara; Bodega, Francesca; Armilli, Marta; Porta, Cristina; Zocchi, Luciano; Agostoni, Emilio

    2010-09-30

    Former studies on net rate of liquid absorption from small Ringer or 1% albumin-Ringer hydrothoraces in rabbits indicated that Na+ transport and solute-coupled liquid absorption by mesothelium is increased by pleural liquid dilution, and stimulation of β2-adrenoreceptors (β2AR). In this research we tried to provide molecular evidence for β2AR in visceral and parietal mesothelium of rabbit pleura. Moreover, because prolonged stimulation of β2AR may lead to desensitization mediated by G-protein-coupled receptor kinase 2 (GRK2), we also checked whether GRK2 is expressed in pleural mesothelium. To this end we performed immunoblot assays on total protein extracts from scraped visceral and parietal mesothelium, and from cultured pleural mesothelial cells of rabbits. All three samples showed β2AR and GRK2 specific bands. Copyright 2010 Elsevier B.V. All rights reserved.

  6. Discovery and Characterization of a Novel Small-Molecule Agonist for Medium-Chain Free Fatty Acid Receptor G Protein-Coupled Receptor 84.

    Science.gov (United States)

    Zhang, Qing; Yang, Hui; Li, Jing; Xie, Xin

    2016-05-01

    G protein-coupled receptor 84 (GPR84) is a free fatty acid receptor activated by medium-chain free fatty acids with 9-14 carbons. It is expressed mainly in the immune-related tissues, such as spleen, bone marrow, and peripheral blood leukocytes. GPR84 plays significant roles in inflammatory processes and may represent a novel drug target for the treatment of immune-mediated diseases. However, the lack of potent and specific ligands for GPR84 hindered the study of its functions and the development of potential clinical applications. Here, we report the screen of 160,000 small-molecule compounds with a calcium mobilization assay using a human embryonic kidney 293 cell line stably expressing GPR84 and Gα16, and the identification of 2-(hexylthio)pyrimidine-4,6-diol (ZQ-16) as a potent and selective agonist of GPR84 with a novel structure. ZQ-16 activates several GPR84-mediated signaling pathways, including calcium mobilization, inhibition of cAMP accumulation, phosphorylation of extracellular signal-regulated protein kinase 1/2, receptor desensitization and internalization, and receptor-β-arrestin interaction. This compound may be a useful tool to study the functions of GPR84 and a potential candidate for further structural optimization. Copyright © 2016 by The American Society for Pharmacology and Experimental Therapeutics.

  7. Physiological stress mediates the honesty of social signals.

    Directory of Open Access Journals (Sweden)

    Gary R Bortolotti

    Full Text Available Extravagant ornaments used as social signals evolved to advertise their bearers' quality. The Immunocompetence Handicap Hypothesis proposes that testosterone-dependent ornaments reliably signal health and parasite resistance; however, empirical studies have shown mixed support. Alternatively, immune function and parasite resistance may be indirectly or directly related to glucocorticoid stress hormones. We propose that an understanding of the interplay between the individual and its environment, particularly how they cope with stressors, is crucial for understanding the honesty of social signals.We analyzed corticosterone deposited in growing feathers as an integrated measure of hypothalamic-pituitary-adrenal activity in a wild territorial bird, the red grouse Lagopus lagopus scoticus. We manipulated two key, interrelated components, parasites and testosterone, which influence both ornamentation and fitness. Birds were initially purged of parasites, and later challenged with parasites or not, while at the same time being given testosterone or control implants, using a factorial experimental design. At the treatment level, testosterone enhanced ornamentation, while parasites reduced it, but only in males not implanted with testosterone. Among individuals, the degree to which both parasites and testosterone had an effect was strongly dependent on the amount of corticosterone in the feather grown during the experiment. The more stressors birds had experienced (i.e., higher corticosterone, the more parasites developed, and the less testosterone enhanced ornamentation.With this unique focus on the individual, and a novel, integrative, measure of response to stressors, we show that ornamentation is ultimately a product of the cumulative physiological response to environmental challenges. These findings lead toward a more realistic concept of honesty in signaling as well as a broader discussion of the concept of stress.

  8. The ins and outs of lysophosphatidic acid signaling

    NARCIS (Netherlands)

    Moolenaar, Wouter H; van Meeteren, Laurens A; Giepmans, Ben N G

    Lysophosphatidic acid (LPA) is a lipid mediator with a wide variety of biological actions, particularly as an inducer of cell proliferation, migration and survival. LPA binds to specific G-protein-coupled receptors and thereby activates multiple signal transduction pathways, including those

  9. Event generation and production of signal inputs for the search of dark matter mediator signal at a future hadron collider

    CERN Document Server

    Chalise, Darshan

    2017-01-01

    The interaction between Dark Matter particles and Standard Model particles is possible through a force mediated by a Dark Matter(DM) - Standard Model(SM) mediator. If that mediator decays through a dijet event, the reconstructed invariant mass of the jets will peak at a specific value, in contrast to the smooth QCD background. This analysis is a preliminary work towards the understanding of how changes in detector conditions at the Future Circular Collider affect the sensitivity of the mediator signal. MadGraph 5 was used to produce events with 30 TeV DM mediator and Heppy was used to produce flat n-tuples for ROOT analysis. MadAnalysis 5 was then used to produce histograms of MadGraph events and PyRoot was used to analyze Heppy output. Histograms of invariant mass of the jets after event production through MadGraph as well as after Heppy analysis showed a peak at 30 TeV. This verified the production of a 30 TeV mediator during event production.

  10. Plant cell surface receptor-mediated signaling - a common theme amid diversity.

    Science.gov (United States)

    He, Yunxia; Zhou, Jinggeng; Shan, Libo; Meng, Xiangzong

    2018-01-29

    Sessile plants employ a diverse array of plasma membrane-bound receptors to perceive endogenous and exogenous signals for regulation of plant growth, development and immunity. These cell surface receptors include receptor-like kinases (RLKs) and receptor-like proteins (RLPs) that harbor different extracellular domains for perception of distinct ligands. Several RLK and RLP signaling pathways converge at the somatic embryogenesis receptor kinases (SERKs), which function as shared co-receptors. A repertoire of receptor-like cytoplasmic kinases (RLCKs) associate with the receptor complexes to relay intracellular signaling. Downstream of the receptor complexes, mitogen-activated protein kinase (MAPK) cascades are among the key signaling modules at which the signals converge, and these cascades regulate diverse cellular and physiological responses through phosphorylation of different downstream substrates. In this Review, we summarize the emerging common theme that underlies cell surface receptor-mediated signaling pathways in Arabidopsis thaliana : the dynamic association of RLKs and RLPs with specific co-receptors and RLCKs for signal transduction. We further discuss how signaling specificities are maintained through modules at which signals converge, with a focus on SERK-mediated receptor signaling. © 2018. Published by The Company of Biologists Ltd.

  11. Cigarette smoke regulates VEGFR2-mediated survival signaling in rat lungs

    Directory of Open Access Journals (Sweden)

    Stevenson Christopher S

    2010-02-01

    Full Text Available Abstract Background Vascular endothelial growth factor (VEGF and VEGF receptor 2 (VEGFR2-mediated survival signaling is critical to endothelial cell survival, maintenance of the vasculature and alveolar structure and regeneration of lung tissue. Reduced VEGF and VEGFR2 expression in emphysematous lungs has been linked to increased endothelial cell death and vascular regression. Previously, we have shown that CS down-regulated the VEGFR2 and its downstream signaling in mouse lungs. However, the VEGFR2-mediated survival signaling in response to oxidants/cigarette smoke (CS is not known. We hypothesized that CS exposure leads to disruption of VEGFR2-mediated endothelial survival signaling in rat lungs. Methods Adult male Sprague-Dawley rats were exposed CS for 3 days, 8 weeks and 6 months to investigate the effect of CS on VEGFR2-mediated survival signaling by measuring the Akt/PI3-kinase/eNOS downstream signaling in rat lungs. Results and Discussion We show that CS disrupts VEGFR2/PI3-kinase association leading to decreased Akt and eNOS phosphorylation. This may further alter the phosphorylation of the pro-apoptotic protein Bad and increase the Bad/Bcl-xl association. However, this was not associated with a significant lung cell death as evidenced by active caspase-3 levels. These data suggest that although CS altered the VEGFR2-mediated survival signaling in the rat lungs, but it was not sufficient to cause lung cell death. Conclusion The rat lungs exposed to CS in acute, sub-chronic and chronic levels may be representative of smokers where survival signaling is altered but was not associated with lung cell death whereas emphysema is known to be associated with lung cell apoptosis.

  12. Curcumin mediates anticancer effects by modulating multiple cell signaling pathways.

    Science.gov (United States)

    Kunnumakkara, Ajaikumar B; Bordoloi, Devivasha; Harsha, Choudhary; Banik, Kishore; Gupta, Subash C; Aggarwal, Bharat B

    2017-08-01

    Curcumin, a component of a spice native to India, was first isolated in 1815 by Vogel and Pelletier from the rhizomes of Curcuma longa (turmeric) and, subsequently, the chemical structure of curcumin as diferuloylmethane was reported by Milobedzka et al. [(1910) 43., 2163-2170]. Since then, this polyphenol has been shown to exhibit antioxidant, anti-inflammatory, anticancer, antiviral, antibacterial, and antifungal activities. The current review primarily focuses on the anticancer potential of curcumin through the modulation of multiple cell signaling pathways. Curcumin modulates diverse transcription factors, inflammatory cytokines, enzymes, kinases, growth factors, receptors, and various other proteins with an affinity ranging from the pM to the mM range. Furthermore, curcumin effectively regulates tumor cell growth via modulation of numerous cell signaling pathways and potentiates the effect of chemotherapeutic agents and radiation against cancer. Curcumin can interact with most of the targets that are modulated by FDA-approved drugs for cancer therapy. The focus of this review is to discuss the molecular basis for the anticancer activities of curcumin based on preclinical and clinical findings. © 2017 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.

  13. IP3 production in the hypersensitive response of lemon seedlings against Alternaria alternata involves active protein tyrosine kinases but not a G-protein

    Directory of Open Access Journals (Sweden)

    XIMENA ORTEGA

    2005-01-01

    Full Text Available IP3 increase and de novo synthesis of scoparone are produced in the hypersensitive response (HR of lemon seedlings against the fungus Alternaria alternata. To elucidate whether a G-protein and/or a protein tyrosine kinase (PTK are involved in signal transduction leading to the production of such a defensive response, we studied the HR in this plant system after treatment with G-protein activators alone and PTK inhibitors in the presence of fungal conidia. No changes in the level of IP3 were detected in response to the treatment with the G-protein activators cholera toxin or mastoparan, although the HR was observed in response to these compounds as determined by the scoparone synthesis. On the contrary, the PTK inhibitors lavendustin A and 2,5-dihidroxy methyl cinnamate (DHMC not only prevented the IP3 changes observed in response to the fungal inoculation of lemon seedlings but also blocked the development of the HR. These results suggest that the IP3 changes observed in response to A. alternata require a PTK activity and are the result of a G-protein independent Phospholipase C activity, even though the activation of a G-protein can also lead to the development of a HR. Therefore, it appears that more than one signaling pathway may be activated for the development of HR in lemon seedlings: one involving a G-protein and the other involving a PTK-dependent PLC.

  14. TSLP signaling pathway map: a platform for analysis of TSLP-mediated signaling.

    Science.gov (United States)

    Zhong, Jun; Sharma, Jyoti; Raju, Rajesh; Palapetta, Shyam Mohan; Prasad, T S Keshava; Huang, Tai-Chung; Yoda, Akinori; Tyner, Jeffrey W; van Bodegom, Diederik; Weinstock, David M; Ziegler, Steven F; Pandey, Akhilesh

    2014-01-01

    Thymic stromal lymphopoietin (TSLP) is a four-helix bundle cytokine that plays a critical role in the regulation of immune responses and in the differentiation of hematopoietic cells. TSLP signals through a heterodimeric receptor complex consisting of an interleukin-7 receptor α chain and a unique TSLP receptor (TSLPR) [also known as cytokine receptor-like factor 2 (CRLF2)]. Cellular targets of TSLP include dendritic cells, B cells, mast cells, regulatory T (Treg) cells and CD4+ and CD8+ T cells. The TSLP/TSLPR axis can activate multiple signaling transduction pathways including the JAK/STAT pathway and the PI-3 kinase pathway. Aberrant TSLP/TSLPR signaling has been associated with a variety of human diseases including asthma, atopic dermatitis, nasal polyposis, inflammatory bowel disease, eosinophilic eosophagitis and, most recently, acute lymphoblastic leukemia. A centralized resource of the TSLP signaling pathway cataloging signaling events is not yet available. In this study, we present a literature-annotated resource of reactions in the TSLP signaling pathway. This pathway map is publicly available through NetPath (http://www.netpath.org/), an open access signal transduction pathway resource developed previously by our group. This map includes 236 molecules and 252 reactions that are involved in TSLP/TSLPR signaling pathway. We expect that the TSLP signaling pathway map will provide a rich resource to study the biology of this important cytokine as well as to identify novel therapeutic targets for diseases associated with dysregulated TSLP/TSLPR signaling. Database URL: http://www.netpath.org/pathways?path_id=NetPath_24.

  15. Concurrent inhibition of kit- and FcepsilonRI-mediated signaling: coordinated suppression of mast cell activation

    DEFF Research Database (Denmark)

    Jensen, Bettina M; Beaven, Michael A; Iwaki, Shoko

    2008-01-01

    Although primarily required for the growth, differentiation, and survival of mast cells, Kit ligand (stem cell factor) is also required for optimal antigen-mediated mast cell activation. Therefore, concurrent inhibition of Kit- and FcepsilonRI-mediated signaling would be an attractive approach...... characterized Kit inhibitor imatinib mesylate (imatinib). In contrast to imatinib, however, hypothemycin also effectively inhibited FcepsilonRI-mediated degranulation and cytokine production in addition to the potentiation of these responses via Kit. The effect of hypothemycin on Kit-mediated responses could...... be explained by its inhibition of Kit kinase activity, whereas the inhibitory effects on FcepsilonRI-dependent signaling were at the level of Btk activation. Because hypothemycin also significantly reduced the mouse passive cutaneous anaphylaxis response in vivo, these data provide proof of principle...

  16. Evidence of G-protein-coupled receptor and substrate transporter heteromerization at a single molecule level.

    Science.gov (United States)

    Fischer, Jana; Kleinau, Gunnar; Rutz, Claudia; Zwanziger, Denise; Khajavi, Noushafarin; Müller, Anne; Rehders, Maren; Brix, Klaudia; Worth, Catherine L; Führer, Dagmar; Krude, Heiko; Wiesner, Burkhard; Schülein, Ralf; Biebermann, Heike

    2018-06-01

    G-protein-coupled receptors (GPCRs) can constitute complexes with non-GPCR integral membrane proteins, while such interaction has not been demonstrated at a single molecule level so far. We here investigated the potential interaction between the thyrotropin receptor (TSHR) and the monocarboxylate transporter 8 (MCT8), a member of the major facilitator superfamily (MFS), using fluorescence cross-correlation spectroscopy (FCCS). Both the proteins are expressed endogenously on the basolateral plasma membrane of the thyrocytes and are involved in stimulation of thyroid hormone production and release. Indeed, we demonstrate strong interaction between both the proteins which causes a suppressed activation of G q/11 by TSH-stimulated TSHR. Thus, we provide not only evidence for a novel interaction between the TSHR and MCT8, but could also prove this interaction on a single molecule level. Moreover, this interaction forces biased signaling at the TSHR. These results are of general interest for both the GPCR and the MFS research fields.

  17. Regulation of Epithelial Morphogenesis by the G-Protein Coupled Receptor Mist and its Ligand Fog*

    Science.gov (United States)

    Manning, Alyssa J.; Peters, Kimberly A.; Peifer, Mark; Rogers, Stephen L.

    2014-01-01

    Epithelial morphogenesis is essential for shaping organs and tissues and for establishment of the three embryonic germ layers during gastrulation. Studies of gastrulation in Drosophila have provided insight into how epithelial morphogenesis is governed by developmental patterning mechanisms. We developed an assay to recapitulate morphogenetic shape changes in individual cultured cells, and used RNAi-based screening to identify Mist, a Drosophila G protein-coupled receptor (GPCR) that transduces signals from the secreted ligand Folded gastrulation (Fog) in cultured cells. Mist functioned in Fog-dependent embryonic morphogenesis, and the transcription factor Snail regulated expression of mist in zygotes. Our data revealed how a cell fate transcriptional program acts through a ligand-GPCR pair to stimulate epithelial morphogenetic shape changes. PMID:24222713

  18. Staphylococcal Superantigens Spark Host-Mediated Danger Signals

    Directory of Open Access Journals (Sweden)

    Terry eKrakauer

    2016-02-01

    Full Text Available Staphylococcal enterotoxin B (SEB of Staphylococcus aureus, and related superantigenic toxins produced by myriad microbes, are potent stimulators of the immune system causing a variety of human diseases from transient food poisoning to lethal toxic shock. These protein toxins bind directly to specific V regions of T-cell receptors (TCR and major histocompatibility complex (MHC class II on antigen-presenting cells, resulting in hyperactivation of T lymphocytes and monocytes / macrophages. Activated host cells produce excessive amounts of proinflammatory cytokines and chemokines, especially tumor necrosis factor α, interleukin 1 (IL-1, IL-2, interferon γ (IFNγ, and macrophage chemoattractant protein 1 causing clinical symptoms of fever, hypotension, and shock. Because of superantigen-induced T cells skewed towards TH1 helper cells, and the induction of proinflammatory cytokines, superantigens can exacerbate autoimmune diseases. Upon TCR / MHC ligation, pathways induced by superantigens include the mitogen-activated protein kinase cascades and cytokine receptor signaling, resulting in activation of NFκB and the phosphoinositide 3-kinase / mammalian target of rapamycin pathways. Various mouse models exist to study SEB-induced shock including those with potentiating agents, transgenic mice and an SEB-only model. However, therapeutics to treat toxic shock remain elusive as host response genes central to pathogenesis of superantigens have only been identified recently. Gene profiling of a murine model for SEB-induced shock reveals novel molecules upregulated in multiple organs not previously associated with SEB-induced responses. The pivotal genes include intracellular DNA / RNA sensors, apoptosis / DNA damage-related molecules, immunoproteasome components, as well as anti-viral and IFN-stimulated genes. The host-wide induction of these, and other, anti-microbial defense genes provide evidence that SEB elicits danger signals resulting in multi

  19. Bmp signaling mediates endoderm pouch morphogenesis by regulating Fgf signaling in zebrafish

    Science.gov (United States)

    Swartz, Mary E.; McCarthy, Neil; Norrie, Jacqueline L.; Eberhart, Johann K.

    2016-01-01

    The endodermal pouches are a series of reiterated structures that segment the pharyngeal arches and help pattern the vertebrate face. Multiple pathways regulate the complex process of endodermal development, including the Bone morphogenetic protein (Bmp) pathway. However, the role of Bmp signaling in pouch morphogenesis is poorly understood. Using genetic and chemical inhibitor approaches, we show that pouch morphogenesis requires Bmp signaling from 10-18 h post-fertilization, immediately following gastrulation. Blocking Bmp signaling during this window results in morphological defects to the pouches and craniofacial skeleton. Using genetic chimeras we show that Bmp signals directly to the endoderm for proper morphogenesis. Time-lapse imaging and analysis of reporter transgenics show that Bmp signaling is necessary for pouch outpocketing via the Fibroblast growth factor (Fgf) pathway. Double loss-of-function analyses demonstrate that Bmp and Fgf signaling interact synergistically in craniofacial development. Collectively, our analyses shed light on the tissue and signaling interactions that regulate development of the vertebrate face. PMID:27122171

  20. Allergen-Induced Dermatitis Causes Alterations in Cutaneous Retinoid-Mediated Signaling in Mice

    Science.gov (United States)

    Gericke, Janine; Ittensohn, Jan; Mihály, Johanna; Dubrac, Sandrine; Rühl, Ralph

    2013-01-01

    Nuclear receptor-mediated signaling via RARs and PPARδ is involved in the regulation of skin homeostasis. Moreover, activation of both RAR and PPARδ was shown to alter skin inflammation. Endogenous all-trans retinoic acid (ATRA) can activate both receptors depending on specific transport proteins: Fabp5 initiates PPARδ signaling whereas Crabp2 promotes RAR signaling. Repetitive topical applications of ovalbumin (OVA) in combination with intraperitoneal injections of OVA or only intraperitoneal OVA applications were used to induce allergic dermatitis. In our mouse model, expression of IL-4, and Hbegf increased whereas expression of involucrin, Abca12 and Spink5 decreased in inflamed skin, demonstrating altered immune response and epidermal barrier homeostasis. Comprehensive gene expression analysis showed alterations of the cutaneous retinoid metabolism and retinoid-mediated signaling in allergic skin immune response. Notably, ATRA synthesis was increased as indicated by the elevated expression of retinaldehyde dehydrogenases and increased levels of ATRA. Consequently, the expression pattern of genes downstream to RAR was altered. Furthermore, the increased ratio of Fabp5 vs. Crabp2 may indicate an up-regulation of the PPARδ pathway in allergen-induced dermatitis in addition to the altered RAR signaling. Thus, our findings suggest that ATRA levels, RAR-mediated signaling and signaling involved in PPARδ pathways are mainly increased in allergen-induced dermatitis and may contribute to the development and/or maintenance of allergic skin diseases. PMID:23977003

  1. Ligand-induced dynamics of heterotrimeric G protein-coupled receptor-like kinase complexes.

    Directory of Open Access Journals (Sweden)

    Meral Tunc-Ozdemir

    Full Text Available Arabidopsis, 7-transmembrane Regulator of G signaling protein 1 (AtRGS1 modulates canonical G protein signaling by promoting the inactive state of heterotrimeric G protein complex on the plasma membrane. It is known that plant leucine-rich repeat receptor-like kinases (LRR RLKs phosphorylate AtRGS1 in vitro but little is known about the in vivo interaction, molecular dynamics, or the cellular consequences of this interaction.Therefore, a subset of the known RLKs that phosphorylate AtRGS1 were selected for elucidation, namely, BAK1, BIR1, FLS2. Several microscopies for both static and dynamic protein-protein interactions were used to follow in vivo interactions between the RLKs and AtRGS1 after the presentation of the Pathogen-associated Molecular Pattern, Flagellin 22 (Flg22. These microscopies included Förster Resonance Energy Transfer, Bimolecular Fluoresence Complementation, and Cross Number and Brightness Fluorescence Correlation Spectroscopy. In addition, reactive oxygen species and calcium changes in living cells were quantitated using luminometry and R-GECO1 microscopy.The LRR RLKs BAK1 and BIR1, interact with AtRGS1 at the plasma membrane. The RLK ligand flg22 sets BAK1 in motion toward AtRGS1 and BIR1 away, both returning to the baseline orientations by 10 minutes. The C-terminal tail of AtRGS1 is important for the interaction with BAK1 and for the tempo of the AtRGS1/BIR1 dynamics. This window of time corresponds to the flg22-induced transient production of reactive oxygen species and calcium release which are both attenuated in the rgs1 and the bak1 null mutants.A temporal model of these interactions is proposed. flg22 binding induces nearly instantaneous dimerization between FLS2 and BAK1. Phosphorylated BAK1 interacts with and enables AtRGS1 to move away from BIR1 and AtRGS1 becomes phosphorylated leading to its endocytosis thus leading to de-repression by permitting AtGPA1 to exchange GDP for GTP. Finally, the G protein complex

  2. Role of heterotrimeric G protein and calcium in cardiomyocyte hypertrophy induced by IGF-1.

    Science.gov (United States)

    Carrasco, Loreto; Cea, Paola; Rocco, Paola; Peña-Oyarzún, Daniel; Rivera-Mejias, Pablo; Sotomayor-Flores, Cristian; Quiroga, Clara; Criollo, Alfredo; Ibarra, Cristian; Chiong, Mario; Lavandero, Sergio

    2014-04-01

    In the heart, insulin-like growth factor-1 (IGF-1) is a peptide with pro-hypertrophic and anti-apoptotic actions. The pro-hypertrophic properties of IGF-1 have been attributed to the extracellular regulated kinase (ERK) pathway. Recently, we reported that IGF-1 also increases intracellular Ca(2+) levels through a pertussis toxin (PTX)-sensitive G protein. Here we investigate whether this Ca(2+) signal is involved in IGF-1-induced cardiomyocyte hypertrophy. Our results show that the IGF-1-induced increase in Ca(2+) level is abolished by the IGF-1 receptor tyrosine kinase inhibitor AG538, PTX and the peptide inhibitor of Gβγ signaling, βARKct. Increases in the activities of Ca(2+) -dependent enzymes calcineurin, calmodulin kinase II (CaMKII), and protein kinase Cα (PKCα) were observed at 5 min after IGF-1 exposure. AG538, PTX, βARKct, and the dominant negative PKCα prevented the IGF-1-dependent phosphorylation of ERK1/2. Participation of calcineurin and CaMKII in ERK phosphorylation was discounted. IGF-1-induced cardiomyocyte hypertrophy, determined by cell size and β-myosin heavy chain (β-MHC), was prevented by AG538, PTX, βARKct, dominant negative PKCα, and the MEK1/2 inhibitor PD98059. Inhibition of calcineurin with CAIN did not abolish IGF-1-induced cardiac hypertrophy. We conclude that IGF-1 induces hypertrophy in cultured cardiomyocytes by activation of the receptor tyrosine kinase activity/βγ-subunits of a PTX-sensitive G protein/Ca(2+) /PKCα/ERK pathway without the participation of calcineurin. © 2013 Wiley Periodicals, Inc.

  3. Twenty years of the G protein-coupled estrogen receptor GPER: Historical and personal perspectives.

    Science.gov (United States)

    Barton, Matthias; Filardo, Edward J; Lolait, Stephen J; Thomas, Peter; Maggiolini, Marcello; Prossnitz, Eric R

    2018-02-01

    Estrogens play a critical role in many aspects of physiology, particularly female reproductive function, but also in pathophysiology, and are associated with protection from numerous diseases in premenopausal women. Steroids and the effects of estrogen have been known for ∼90 years, with the first evidence for a receptor for estrogen presented ∼50 years ago. The original ancestral steroid receptor, extending back into evolution more than 500 million years, was likely an estrogen receptor, whereas G protein-coupled receptors (GPCRs) trace their origins back into history more than one billion years. The classical estrogen receptors (ERα and ERβ) are ligand-activated transcription factors that confer estrogen sensitivity upon many genes. It was soon apparent that these, or novel receptors may also be responsible for the "rapid"/"non-genomic" membrane-associated effects of estrogen. The identification of an orphan GPCR (GPR30, published in 1996) opened a new field of research with the description in 2000 that GPR30 expression is required for rapid estrogen signaling. In 2005-2006, the field was greatly stimulated by two studies that described the binding of estrogen to GPR30-expressing cell membranes, followed by the identification of a GPR30-selective agonist (that lacked binding and activity towards ERα and ERβ). Renamed GPER (G protein-coupled estrogen receptor) by IUPHAR in 2007, the total number of articles in PubMed related to this receptor recently surpassed 1000. In this article, the authors present personal perspectives on how they became involved in the discovery and/or advancement of GPER research. These areas include non-genomic effects on vascular tone, receptor cloning, molecular and cellular biology, signal transduction mechanisms and pharmacology of GPER, highlighting the roles of GPER and GPER-selective compounds in diseases such as obesity, diabetes, and cancer and the obligatory role of GPER in propagating cardiovascular aging, arterial

  4. Constitutive stimulatory G protein activity in limb mesenchyme impairs bone growth.

    Science.gov (United States)

    Karaca, Anara; Malladi, Vijayram Reddy; Zhu, Yan; Tafaj, Olta; Paltrinieri, Elena; Wu, Joy Y; He, Qing; Bastepe, Murat

    2018-05-01

    GNAS mutations leading to constitutively active stimulatory G protein alpha-subunit (Gsα) cause different tumors, fibrous dysplasia of bone, and McCune-Albright syndrome, which are typically not associated with short stature. Enhanced signaling of the parathyroid hormone/parathyroid hormone-related peptide receptor, which couples to multiple G proteins including Gsα, leads to short bones with delayed endochondral ossification. It has remained unknown whether constitutive Gsα activity also impairs bone growth. Here we generated mice expressing a constitutively active Gsα mutant (Gsα-R201H) conditionally upon Cre recombinase (cGsα R201H mice). Gsα-R201H was expressed in cultured bone marrow stromal cells from cGsα R201H mice upon adenoviral-Cre transduction. When crossed with mice in which Cre is expressed in a tamoxifen-regulatable fashion (CAGGCre-ER™), tamoxifen injection resulted in mosaic expression of the transgene in double mutant offspring. We then crossed the cGsα R201H mice with Prx1-Cre mice, in which Cre is expressed in early limb-bud mesenchyme. The double mutant offspring displayed short limbs at birth, with narrow hypertrophic chondrocyte zones in growth plates and delayed formation of secondary ossification center. Consistent with enhanced Gsα signaling, bone marrow stromal cells from these mice demonstrated increased levels of c-fos mRNA. Our findings indicate that constitutive Gsα activity during limb development disrupts endochondral ossification and bone growth. Given that Gsα haploinsufficiency also leads to short bones, as in patients with Albright's hereditary osteodystrophy, these results suggest that a tight control of Gsα activity is essential for normal growth plate physiology. Copyright © 2018 Elsevier Inc. All rights reserved.

  5. Adaptor Protein Complex-2 (AP-2) and Epsin-1 Mediate Protease-activated Receptor-1 Internalization via Phosphorylation- and Ubiquitination-dependent Sorting Signals*

    Science.gov (United States)

    Chen, Buxin; Dores, Michael R.; Grimsey, Neil; Canto, Isabel; Barker, Breann L.; Trejo, JoAnn

    2011-01-01

    Signaling by protease-activated receptor-1 (PAR1), a G protein-coupled receptor (GPCR) for thrombin, is regulated by desensitization and internalization. PAR1 desensitization is mediated by β-arrestins, like most classic GPCRs. In contrast, internalization of PAR1 occurs through a clathrin- and dynamin-dependent pathway independent of β-arrestins. PAR1 displays two modes of internalization. Constitutive internalization of unactivated PAR1 is mediated by the clathrin adaptor protein complex-2 (AP-2), where the μ2-adaptin subunit binds directly to a tyrosine-based motif localized within the receptor C-tail domain. However, AP-2 depletion only partially inhibits agonist-induced internalization of PAR1, suggesting a function for other clathrin adaptors in this process. Here, we now report that AP-2 and epsin-1 are both critical mediators of agonist-stimulated PAR1 internalization. We show that ubiquitination of PAR1 and the ubiquitin-interacting motifs of epsin-1 are required for epsin-1-dependent internalization of activated PAR1. In addition, activation of PAR1 promotes epsin-1 de-ubiquitination, which may increase its endocytic adaptor activity to facilitate receptor internalization. AP-2 also regulates activated PAR1 internalization via recognition of distal C-tail phosphorylation sites rather than the canonical tyrosine-based motif. Thus, AP-2 and epsin-1 are both required to promote efficient internalization of activated PAR1 and recognize discrete receptor sorting signals. This study defines a new pathway for internalization of mammalian GPCRs. PMID:21965661

  6. G protein-coupled receptor 56 regulates mechanical overload-induced muscle hypertrophy.

    Science.gov (United States)

    White, James P; Wrann, Christiane D; Rao, Rajesh R; Nair, Sreekumaran K; Jedrychowski, Mark P; You, Jae-Sung; Martínez-Redondo, Vicente; Gygi, Steven P; Ruas, Jorge L; Hornberger, Troy A; Wu, Zhidan; Glass, David J; Piao, Xianhua; Spiegelman, Bruce M

    2014-11-04

    Peroxisome proliferator-activated receptor gamma coactivator 1-alpha 4 (PGC-1α4) is a protein isoform derived by alternative splicing of the PGC1α mRNA and has been shown to promote muscle hypertrophy. We show here that G protein-coupled receptor 56 (GPR56) is a transcriptional target of PGC-1α4 and is induced in humans by resistance exercise. Furthermore, the anabolic effects of PGC-1α4 in cultured murine muscle cells are dependent on GPR56 signaling, because knockdown of GPR56 attenuates PGC-1α4-induced muscle hypertrophy in vitro. Forced expression of GPR56 results in myotube hypertrophy through the expression of insulin-like growth factor 1, which is dependent on Gα12/13 signaling. A murine model of overload-induced muscle hypertrophy is associated with increased expression of both GPR56 and its ligand collagen type III, whereas genetic ablation of GPR56 expression attenuates overload-induced muscle hypertrophy and associated anabolic signaling. These data illustrate a signaling pathway through GPR56 which regulates muscle hypertrophy associated with resistance/loading-type exercise.

  7. A ligand channel through the G protein coupled receptor opsin.

    Directory of Open Access Journals (Sweden)

    Peter W Hildebrand

    Full Text Available The G protein coupled receptor rhodopsin contains a pocket within its seven-transmembrane helix (TM structure, which bears the inactivating 11-cis-retinal bound by a protonated Schiff-base to Lys296 in TM7. Light-induced 11-cis-/all-trans-isomerization leads to the Schiff-base deprotonated active Meta II intermediate. With Meta II decay, the Schiff-base bond is hydrolyzed, all-trans-retinal is released from the pocket, and the apoprotein opsin reloaded with new 11-cis-retinal. The crystal structure of opsin in its active Ops* conformation provides the basis for computational modeling of retinal release and uptake. The ligand-free 7TM bundle of opsin opens into the hydrophobic membrane layer through openings A (between TM1 and 7, and B (between TM5 and 6, respectively. Using skeleton search and molecular docking, we find a continuous channel through the protein that connects these two openings and comprises in its central part the retinal binding pocket. The channel traverses the receptor over a distance of ca. 70 A and is between 11.6 and 3.2 A wide. Both openings are lined with aromatic residues, while the central part is highly polar. Four constrictions within the channel are so narrow that they must stretch to allow passage of the retinal beta-ionone-ring. Constrictions are at openings A and B, respectively, and at Trp265 and Lys296 within the retinal pocket. The lysine enforces a 90 degrees elbow-like kink in the channel which limits retinal passage. With a favorable Lys side chain conformation, 11-cis-retinal can take the turn, whereas passage of the all-trans isomer would require more global conformational changes. We discuss possible scenarios for the uptake of 11-cis- and release of all-trans-retinal. If the uptake gate of 11-cis-retinal is assigned to opening B, all-trans is likely to leave through the same gate. The unidirectional passage proposed previously requires uptake of 11-cis-retinal through A and release of photolyzed all

  8. Elevated carbon dioxide blunts mammalian cAMP signaling dependent on inositol 1,4,5-triphosphate receptor-mediated Ca2+ release.

    Science.gov (United States)

    Cook, Zara C; Gray, Michael A; Cann, Martin J

    2012-07-27

    Elevated CO(2) is generally detrimental to animal cells, suggesting an interaction with core processes in cell biology. We demonstrate that elevated CO(2) blunts G protein-activated cAMP signaling. The effect of CO(2) is independent of changes in intracellular and extracellular pH, independent of the mechanism used to activate the cAMP signaling pathway, and is independent of cell context. A combination of pharmacological and genetic tools demonstrated that the effect of elevated CO(2) on cAMP levels required the activity of the IP(3) receptor. Consistent with these findings, CO(2) caused an increase in steady state cytoplasmic Ca(2+) concentrations not observed in the absence of the IP(3) receptor or under nonspecific acidotic conditions. We examined the well characterized cAMP-dependent inhibition of the isoform 3 Na(+)/H(+) antiporter (NHE3) to demonstrate a functional relevance for CO(2)-mediated reductions in cellular cAMP. Consistent with the cellular biochemistry, elevated CO(2) abrogated the inhibitory effect of cAMP on NHE3 function via an IP(3) receptor-dependent mechanism.

  9. Elevated Carbon Dioxide Blunts Mammalian cAMP Signaling Dependent on Inositol 1,4,5-Triphosphate Receptor-mediated Ca2+ Release*

    Science.gov (United States)

    Cook, Zara C.; Gray, Michael A.; Cann, Martin J.

    2012-01-01

    Elevated CO2 is generally detrimental to animal cells, suggesting an interaction with core processes in cell biology. We demonstrate that elevated CO2 blunts G protein-activated cAMP signaling. The effect of CO2 is independent of changes in intracellular and extracellular pH, independent of the mechanism used to activate the cAMP signaling pathway, and is independent of cell context. A combination of pharmacological and genetic tools demonstrated that the effect of elevated CO2 on cAMP levels required the activity of the IP3 receptor. Consistent with these findings, CO2 caused an increase in steady state cytoplasmic Ca2+ concentrations not observed in the absence of the IP3 receptor or under nonspecific acidotic conditions. We examined the well characterized cAMP-dependent inhibition of the isoform 3 Na+/H+ antiporter (NHE3) to demonstrate a functional relevance for CO2-mediated reductions in cellular cAMP. Consistent with the cellular biochemistry, elevated CO2 abrogated the inhibitory effect of cAMP on NHE3 function via an IP3 receptor-dependent mechanism. PMID:22654111

  10. Lunatic fringe-mediated Notch signaling regulates adult hippocampal neural stem cell maintenance.

    Science.gov (United States)

    Semerci, Fatih; Choi, William Tin-Shing; Bajic, Aleksandar; Thakkar, Aarohi; Encinas, Juan Manuel; Depreux, Frederic; Segil, Neil; Groves, Andrew K; Maletic-Savatic, Mirjana

    2017-07-12

    Hippocampal neural stem cells (NSCs) integrate inputs from multiple sources to balance quiescence and activation. Notch signaling plays a key role during this process. Here, we report that Lunatic fringe ( Lfng), a key modifier of the Notch receptor, is selectively expressed in NSCs. Further, Lfng in NSCs and Notch ligands Delta1 and Jagged1, expressed by their progeny, together influence NSC recruitment, cell cycle duration, and terminal fate. We propose a new model in which Lfng-mediated Notch signaling enables direct communication between a NSC and its descendants, so that progeny can send feedback signals to the 'mother' cell to modify its cell cycle status. Lfng-mediated Notch signaling appears to be a key factor governing NSC quiescence, division, and fate.

  11. Plasmodesmata-mediated intercellular signaling during plant growth and development

    Directory of Open Access Journals (Sweden)

    Shri Ram eYadav

    2014-02-01

    Full Text Available Plasmodesmata (PD are cytoplasmic channels that connect neighboring cells for cell-to-cell communication. PD structure and function vary temporally and spatially to allow formation of symplastic domains during different stages of plant development. Reversible deposition of callose at PD plays an important role in controlling molecular trafficking through PD by regulating their size exclusion limit (SEL. Previously, we reported several semi-dominant mutants for CALLOSE SYNTHASE 3 (CALS3 gene, which overproduce callose at PD in Arabidopsis. By combining two of these mutations in a LexA-VP16-ER (XVE-based estradiol inducible vector system, a tool known as the icals3m system was developed to temporally obstruct the symplastic connections in a specified spatial domain. The system has been successfully tested and used, in combination with other methods, to investigate the route for mobile signals such as the SHR protein, microRNA165/6, and cytokinins in Arabidopsis roots, and also to understand the role of symplastic domain formation during lateral root development. We envision that this tool may also be useful for identifying tissue-specific symplastic regulatory networks and to analyze symplastic movement of metabolites.

  12. Plasmodesmata-mediated intercellular signaling during plant growth and development.

    Science.gov (United States)

    Yadav, Shri R; Yan, Dawei; Sevilem, Iris; Helariutta, Ykä

    2014-01-01

    Plasmodesmata (PD) are cytoplasmic channels that connect neighboring cells for cell-to-cell communication. PD structure and function vary temporally and spatially to allow formation of symplastic domains during different stages of plant development. Reversible deposition of callose at PD plays an important role in controlling molecular trafficking through PD by regulating their size exclusion limit. Previously, we reported several semi-dominant mutants for CALLOSE SYNTHASE 3 (CALS3) gene, which overproduce callose at PD in Arabidopsis. By combining two of these mutations in a LexA-VP16-ER (XVE)-based estradiol inducible vector system, a tool known as the "icals3m system" was developed to temporally obstruct the symplastic connections in a specified spatial domain. The system has been successfully tested and used, in combination with other methods, to investigate the route for mobile signals such as the SHR protein, microRNA165/6, and cytokinins in Arabidopsis roots, and also to understand the role of symplastic domain formation during lateral root development. We envision that this tool may also be useful for identifying tissue-specific symplastic regulatory networks and to analyze symplastic movement of metabolites.

  13. Lysophospholipid Growth Factors and Their G Protein-Coupled Receptors in Immunity, Coronary Artery Disease, and Cancer

    Directory of Open Access Journals (Sweden)

    Edward J. Goetzl

    2002-01-01

    Full Text Available The physiological lysophospholipids (LPLs, exemplified by lysophosphatidic acid (LPA and sphingosine 1-phosphate (S1P, are omnific mediators of normal cellular proliferation, survival, and functions. Although both LPA and S1P attain micromolar concentrations in many biological fluids, numerous aspects of their biosynthesis, transport, and metabolic degradation are unknown. Eight members of a new subfamily of G protein-coupled LPA/S1P receptors, originally termed Edg Rs, bind either LPA or S1P with high affinity and transduce a series of growth-related and/or cytoskeleton-based functional responses. The most critical areas of LPL biology and pathobiology are neural development and neurodegeneration, immunity, atherosclerosis and myocardial injury, and cancer. Data from analyses of T cells established two basic points: (1 the plasticity and adaptability of expression of LPA/S1P Rs by some cells as a function of activation, and (2 the role of opposing signals from two different receptors for the same ligand as a mechanism for fine control of effects of LPLs. In the heart, LPLs may promote coronary atherosclerosis, but are effectively cytoprotective for hypoxic cardiac myocytes and those exposed to oxygen free radicals. The findings of production of LPA by some types of tumor cells, overexpression of selected sets of LPA receptors by the same tumor cells, and augmentation of the effects of protein growth factors by LPA have suggested pathogenetic roles for the LPLs in cancer. The breadth of physiologic and pathologic activities of LPLs emphasizes the importance of developing bioavailable nonlipid agonists and antagonists of the LPA/S1P receptors for diverse therapeutic applications.

  14. Involvement of the G-protein-coupled receptor 4 in RANKL expression by osteoblasts in an acidic environment

    Energy Technology Data Exchange (ETDEWEB)

    Okito, Asuka [Department of Cellular Physiological Chemistry, Tokyo Medical and Dental University, Tokyo (Japan); Department of Orthodontic Science, Tokyo Medical and Dental University, Tokyo (Japan); Nakahama, Ken-ichi, E-mail: nakacell@tmd.ac.jp [Department of Cellular Physiological Chemistry, Tokyo Medical and Dental University, Tokyo (Japan); Akiyama, Masako [Department of Cellular Physiological Chemistry, Tokyo Medical and Dental University, Tokyo (Japan); Ono, Takashi [Department of Orthodontic Science, Tokyo Medical and Dental University, Tokyo (Japan); Morita, Ikuo [Department of Cellular Physiological Chemistry, Tokyo Medical and Dental University, Tokyo (Japan)

    2015-03-06

    Osteoclast activity is enhanced in acidic environments following systemic or local inflammation. However, the regulatory mechanism of receptor activator of NF-κB ligand (RANKL) expression in osteoblasts under acidic conditions is not fully understood. In the present paper, we detected the mRNA expression of the G-protein-coupled receptor (GPR) proton sensors GPR4 and GPR65 (T-cell death-associated gene 8, TDAG8), in osteoblasts. RANKL expression and the cyclic AMP (cAMP) level in osteoblasts were up-regulated under acidic culture conditions. Acidosis-induced up-regulation of RANKL was abolished by the protein kinase A inhibitor H89. To clarify the role of GPR4 in RANKL expression, GPR4 gain and loss of function experiments were performed. Gene knockdown and forced expression of GPR4 caused reduction and induction of RANKL expression, respectively. These results suggested that, at least in part, RANKL expression by osteoblasts in an acidic environment was mediated by cAMP/PKA signaling resulting from GPR4 activation. A comprehensive microarray analysis of gene expression of osteoblasts revealed that, under acidic conditions, the phenotype of osteoblasts was that of an osteoclast supporting cell rather than that of a mineralizing cell. These findings will contribute to a molecular understanding of bone disruption in an acidic environment. - Highlights: • RANKL expression was increased in osteoblasts under acidosis via cAMP/PKA pathway. • GRP4 knockdown resulted in decrease of RANKL expression. • GRP4 overexpression resulted in increase of RANKL expression. • Osteoblast mineralization was reduced under acidic condition.

  15. Protein kinase D1 signaling in angiogenic gene expression and VEGF-mediated angiogenesis

    Directory of Open Access Journals (Sweden)

    Bin eRen MD, Phd, FAHA

    2016-05-01

    Full Text Available Protein kinase D 1 (PKD-1 is a signaling kinase important in fundamental cell functions including migration, proliferation and differentiation. PKD-1 is also a key regulator of gene expression and angiogenesis that is essential for cardiovascular development and tumor progression. Further understanding molecular aspects of PKD-1 signaling in the regulation of angiogenesis may have translational implications in obesity, cardiovascular disease and cancer. The author will summarize and provide the insights into molecular mechanisms by which PKD-1 regulates transcriptional expression of angiogenic genes, focusing on the transcriptional regulation of CD36 by PKD-1-FoxO1 signaling axis along with the potential implications of this axis in arterial differentiation and morphogenesis. He will also discuss a new concept of dynamic balance between proangiogenic and antiangiogenic signaling in determining angiogenic switch, and stress how PKD-1 signaling regulates VEGF signaling-mediated angiogenesis.

  16. Volatile-Mediated within-Plant Signaling in Hybrid Aspen: Required for Systemic Responses.

    Science.gov (United States)

    Li, Tao; Blande, James D

    2017-04-01

    Plant volatiles play crucial roles in signaling between plants and their associated community members, but their role in within-plant signaling remains largely unexplored, particularly under field conditions. Using a system comprising the hybrid aspen (Populus tremula x tremuloides) and the specialized herbivorous leaf beetle (Phratora laticollis) and, combining field, greenhouse and laboratory experiments, we examined whether local damage triggered systemic responses in undamaged branches that lack vascular connection to the damaged branches, and to what extent this was caused by airborne volatile signals versus internal signals. An experiment tracing dye through the vasculature of saplings revealed no downward movement of the dye from upper to lower branches, suggesting a lack of vascular connectivity among branches. However, we found under both field and laboratory conditions that herbivore feeding on upper branches elicited volatile emissions by undamaged lower branches. Greenhouse experiments manipulating air contact between damaged and undamaged branches showed that systemic induction of volatiles was almost eliminated when air contact was interrupted. Our findings clearly demonstrate that herbivore-induced volatiles overcome vascular constraints and mediate within-plant signaling. Further, we found that volatile signaling led to induction of different classes of volatiles under field and environment controlled conditions, with a weaker response observed in the field. This difference not only reflects the dose- and time-dependent nature of volatile signaling, but also points out that future studies should focus more on field observations to better understand the ecological role of volatile-mediated within-plant signaling.

  17. Identification of a novel Gnao-mediated alternate olfactory signaling pathway in murine OSNs

    Directory of Open Access Journals (Sweden)

    Paul eScholz

    2016-03-01

    Full Text Available It is generally agreed that in olfactory sensory neurons (OSNs, the binding of odorant molecules to their specific olfactory receptor (OR triggers a cAMP-dependent signaling cascade, activating cyclic-nucleotide gated (CNG channels. However, considerable controversy dating back more than 20 years has surrounded the question of whether alternate signaling plays a role in mammalian olfactory transduction. In this study, we demonstrate a specific alternate signaling pathway in Olfr73-expressing OSNs. Methylisoeugenol (MIEG and at least one other known weak Olfr73 agonist (Raspberry Ketone trigger a signaling cascade independent from the canonical pathway, leading to the depolarization of the cell. Interestingly, this pathway is mediated by Gnao activation, leading to Cl- efflux; however, the activation of adenylyl cyclase III (ACIII, the recruitment of Ca2+ from extra-or intracellular stores, and phosphatidylinositol 3-kinase-dependent signaling (PI signaling are not involved. Furthermore, we demonstrated that our newly identified pathway coexists with the canonical olfactory cAMP pathway in the same OSN and can be triggered by the same OR in a ligand-selective manner. We suggest that this pathway might reflect a mechanism for odor recognition predominantly used in early developmental stages before olfactory cAMP signaling is fully developed. Taken together, our findings support the existence of at least one odor-induced alternate signal transduction pathway in native OSNs mediated by Olfr73 in a ligand-selective manner.

  18. Trichoderma G protein-coupled receptors: functional characterisation of a cAMP receptor-like protein from Trichoderma atroviride.

    Science.gov (United States)

    Brunner, Kurt; Omann, Markus; Pucher, Marion E; Delic, Marizela; Lehner, Sylvia M; Domnanich, Patrick; Kratochwill, Klaus; Druzhinina, Irina; Denk, Dagmar; Zeilinger, Susanne

    2008-12-01

    Galpha subunits act to regulate vegetative growth, conidiation, and the mycoparasitic response in Trichoderma atroviride. To extend our knowledge on G protein signalling, we analysed G protein-coupled receptors (GPCRs). As the genome sequence of T. atroviride is not publicly available yet, we carried out an in silico exploration of the genome database of the close relative T. reesei. Twenty genes encoding putative GPCRs distributed over eight classes and additional 35 proteins similar to the Magnaporthe grisea PTH11 receptor were identified. Subsequently, four T. atroviride GPCR-encoding genes were isolated and affiliated to the cAMP receptor-like family by phylogenetic and topological analyses. All four genes showed lowest expression on glycerol and highest mRNA levels upon carbon starvation. Transcription of gpr3 and gpr4 responded to exogenously added cAMP and the shift from liquid to solid media. gpr3 mRNA levels also responded to the presence of fungal hyphae or cellulose membranes. Further characterisation of mutants bearing a gpr1-silencing construct revealed that Gpr1 is essential for vegetative growth, conidiation and conidial germination. Four genes encoding the first GPCRs described in Trichoderma were isolated and their expression characterized. At least one of these GPCRs is important for several cellular processes, supporting the fundamental role of G protein signalling in this fungus.

  19. Δ9-THC-caused synaptic and memory impairments are mediated through COX-2 signaling

    Science.gov (United States)

    Yang, Hongwei; Tang, Ya-ping; Sun, Hao; Song, Yunping; Chen, Chu

    2013-01-01

    SUMMARY Marijuana has been used for thousands of years as a treatment for medical conditions. However, untoward side effects limit its medical value. Here we show that synaptic and cognitive impairments following repeated exposure to Δ9-tetrahydrocannabinol (Δ9-THC) are associated with the induction of cyclooxygenase-2 (COX-2), an inducible enzyme that converts arachidonic acid to prostanoids, in the brain. COX-2 induction by Δ9-THC is mediated via CB1 receptor-coupled G-protein βγ subunits. Pharmacological or genetic inhibition of COX-2 blocks down-regulation and internalization of glutamate receptor subunits and alterations of the dendritic spine density of hippocampal neurons induced by repeated Δ9-THC exposures. Ablation of COX-2 also eliminates Δ9-THC-impaired hippocampal long-term synaptic plasticity, spatial, and fear memories. Importantly, the beneficial effects of decreasing β-amyloid plaques and neurodegeneration by Δ9-THC in Alzheimer’s disease animals are retained in the presence of COX-2 inhibition. These results suggest that the applicability of medical marijuana would be broadened by concurrent inhibition of COX-2. PMID:24267894

  20. Δ9-THC-caused synaptic and memory impairments are mediated through COX-2 signaling.

    Science.gov (United States)

    Chen, Rongqing; Zhang, Jian; Fan, Ni; Teng, Zhao-Qian; Wu, Yan; Yang, Hongwei; Tang, Ya-Ping; Sun, Hao; Song, Yunping; Chen, Chu

    2013-11-21

    Marijuana has been used for thousands of years as a treatment for medical conditions. However, untoward side effects limit its medical value. Here, we show that synaptic and cognitive impairments following repeated exposure to Δ(9)-tetrahydrocannabinol (Δ(9)-THC) are associated with the induction of cyclooxygenase-2 (COX-2), an inducible enzyme that converts arachidonic acid to prostanoids in the brain. COX-2 induction by Δ(9)-THC is mediated via CB1 receptor-coupled G protein βγ subunits. Pharmacological or genetic inhibition of COX-2 blocks downregulation and internalization of glutamate receptor subunits and alterations of the dendritic spine density of hippocampal neurons induced by repeated Δ(9)-THC exposures. Ablation of COX-2 also eliminates Δ(9)-THC-impaired hippocampal long-term synaptic plasticity, working, and fear memories. Importantly, the beneficial effects of decreasing β-amyloid plaques and neurodegeneration by Δ(9)-THC in Alzheimer's disease animals are retained in the presence of COX-2 inhibition. These results suggest that the applicability of medical marijuana would be broadened by concurrent inhibition of COX-2. Copyright © 2013 Elsevier Inc. All rights reserved.

  1. Metabolite-Sensing G Protein-Coupled Receptors-Facilitators of Diet-Related Immune Regulation.

    Science.gov (United States)

    Tan, Jian K; McKenzie, Craig; Mariño, Eliana; Macia, Laurence; Mackay, Charles R

    2017-04-26

    Nutrition and the gut microbiome regulate many systems, including the immune, metabolic, and nervous systems. We propose that the host responds to deficiency (or sufficiency) of dietary and bacterial metabolites in a dynamic way, to optimize responses and survival. A family of G protein-coupled receptors (GPCRs) termed the metabolite-sensing GPCRs bind to various metabolites and transmit signals that are important for proper immune and metabolic functions. Members of this family include GPR43, GPR41, GPR109A, GPR120, GPR40, GPR84, GPR35, and GPR91. In addition, bile acid receptors such as GPR131 (TGR5) and proton-sensing receptors such as GPR65 show similar features. A consistent feature of this family of GPCRs is that they provide anti-inflammatory signals; many also regulate metabolism and gut homeostasis. These receptors represent one of the main mechanisms whereby the gut microbiome affects vertebrate physiology, and they also provide a link between the immune and metabolic systems. Insufficient signaling through one or more of these metabolite-sensing GPCRs likely contributes to human diseases such as asthma, food allergies, type 1 and type 2 diabetes, hepatic steatosis, cardiovascular disease, and inflammatory bowel diseases.

  2. Dynamic regulation of GDP binding to G proteins revealed by magnetic field-dependent NMR relaxation analyses.

    Science.gov (United States)

    Toyama, Yuki; Kano, Hanaho; Mase, Yoko; Yokogawa, Mariko; Osawa, Masanori; Shimada, Ichio

    2017-02-22

    Heterotrimeric guanine-nucleotide-binding proteins (G proteins) serve as molecular switches in signalling pathways, by coupling the activation of cell surface receptors to intracellular responses. Mutations in the G protein α-subunit (Gα) that accelerate guanosine diphosphate (GDP) dissociation cause hyperactivation of the downstream effector proteins, leading to oncogenesis. However, the structural mechanism of the accelerated GDP dissociation has remained unclear. Here, we use magnetic field-dependent nuclear magnetic resonance relaxation analyses to investigate the structural and dynamic properties of GDP bound Gα on a microsecond timescale. We show that Gα rapidly exchanges between a ground-state conformation, which tightly binds to GDP and an excited conformation with reduced GDP affinity. The oncogenic D150N mutation accelerates GDP dissociation by shifting the equilibrium towards the excited conformation.

  3. G protein-coupled receptor 84, a microglia-associated protein expressed in neuroinflammatory conditions.

    Science.gov (United States)

    Bouchard, Caroline; Pagé, Julie; Bédard, Andréanne; Tremblay, Pierrot; Vallières, Luc

    2007-06-01

    G protein-coupled receptor 84 (GPR84) is a recently discovered member of the seven transmembrane receptor superfamily whose function and regulation are unknown. Here, we report that in mice suffering from endotoxemia, microglia express GPR84 in a strong and sustained manner. This property is shared by subpopulations of peripheral macrophages and, to a much lesser extent, monocytes. The induction of GPR84 expression by endotoxin is mediated, at least in part, by proinflammatory cytokines, notably tumor necrosis factor (TNF) and interleukin-1 (IL-1), because mice lacking either one or both of these molecules have fewer GPR84-expressing cells in their cerebral cortex than wild-type mice during the early phase of endotoxemia. Moreover, when injected intracerebrally or added to microglial cultures, recombinant TNF stimulates GPR84 expression through a dexamethasone-insensitive mechanism. Finally, we show that microglia produce GPR84 not only during endotoxemia, but also during experimental autoimmune encephalomyelitis (EAE), a model of multiple sclerosis. In conclusion, this study reports the identification of a new sensitive marker of microglial activation, which may play an important regulatory role in neuroimmunological processes, acting downstream to the effects of proinflammatory mediators.

  4. Characterization of murine melanocortin receptors mediating adipocyte lipolysis and examination of signalling pathways involved

    DEFF Research Database (Denmark)

    Møller, Cathrine Laustrup; Raun, Kirsten; Jacobsen, Marianne Lambert

    2011-01-01

    hormone (a-MSH) generated from proopiomelanocortin (POMC), as well as synthetic MSH analogues to stimulate lipolysis in murine 3T3-L1 adipocytes it is shown that MC2R and MC5R are lipolytic mediators in differentiated 3T3-L1 adipocytes. Involvement of cAMP, phosphorylated extracellular signal...

  5. The Bucket System – A computer mediated signaling system for group improvisation

    DEFF Research Database (Denmark)

    Dahlstedt, Palle; Nilsson, Per Anders; Robair, Gino

    2015-01-01

    The Bucket System is a new system for computer-mediated ensemble improvisation, designed by improvisers for improvisers. Coming from a tradition of structured free ensemble improvisation practices (comprovisation), influenced by post-WW2 experimental music practices, it is a signaling system...

  6. Essential roles of Gab1 tyrosine phosphorylation in growth factor-mediated signaling and angiogenesis.

    Science.gov (United States)

    Wang, Weiye; Xu, Suowen; Yin, Meimei; Jin, Zheng Gen

    2015-02-15

    Growth factors and their downstream receptor tyrosine kinases (RTKs) mediate a number of biological processes controlling cell function. Adaptor (docking) proteins, which consist exclusively of domains and motifs that mediate molecular interactions, link receptor activation to downstream effectors. Recent studies have revealed that Grb2-associated-binders (Gab) family members (including Gab1, Gab2, and Gab3), when phosphorylated on tyrosine residues, provide binding sites for multiple effector proteins, such as Src homology-2 (SH2)-containing protein tyrosine phosphatase 2 (SHP2) and phosphatidylinositol 3-kinase (PI3K) regulatory subunit p85, thereby playing important roles in transducing RTKs-mediated signals into pathways with diversified biological functions. Here, we provide an up-to-date overview on the domain structure and biological functions of Gab1, the most intensively studied Gab family protein, in growth factor signaling and biological functions, with a special focus on angiogenesis. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

  7. Rearrangement of a polar core provides a conserved mechanism for constitutive activation of class B G protein-coupled receptors

    Science.gov (United States)

    Yin, Yanting; de Waal, Parker W.; He, Yuanzheng; Zhao, Li-Hua; Yang, Dehua; Cai, Xiaoqing; Jiang, Yi; Melcher, Karsten; Wang, Ming-Wei; Xu, H. Eric

    2017-01-01

    The glucagon receptor (GCGR) belongs to the secretin-like (class B) family of G protein-coupled receptors (GPCRs) and is activated by the peptide hormone glucagon. The structures of an activated class B GPCR have remained unsolved, preventing a mechanistic understanding of how these receptors are activated. Using a combination of structural modeling and mutagenesis studies, we present here two modes of ligand-independent activation of GCGR. First, we identified a GCGR-specific hydrophobic lock comprising Met-338 and Phe-345 within the IC3 loop and transmembrane helix 6 (TM6) and found that this lock stabilizes the TM6 helix in the inactive conformation. Disruption of this hydrophobic lock led to constitutive G protein and arrestin signaling. Second, we discovered a polar core comprising conserved residues in TM2, TM3, TM6, and TM7, and mutations that disrupt this polar core led to constitutive GCGR activity. On the basis of these results, we propose a mechanistic model of GCGR activation in which TM6 is held in an inactive conformation by the conserved polar core and the hydrophobic lock. Mutations that disrupt these inhibitory elements allow TM6 to swing outward to adopt an active TM6 conformation similar to that of the canonical β2-adrenergic receptor complexed with G protein and to that of rhodopsin complexed with arrestin. Importantly, mutations in the corresponding polar core of several other members of class B GPCRs, including PTH1R, PAC1R, VIP1R, and CRFR1, also induce constitutive G protein signaling, suggesting that the rearrangement of the polar core is a conserved mechanism for class B GPCR activation. PMID:28356352

  8. Pivotal role of extended linker 2 in the activation of Gα by G protein-coupled receptor.

    Science.gov (United States)

    Huang, Jianyun; Sun, Yutong; Zhang, J Jillian; Huang, Xin-Yun

    2015-01-02

    G protein-coupled receptors (GPCRs) relay extracellular signals mainly to heterotrimeric G-proteins (Gαβγ) and they are the most successful drug targets. The mechanisms of G-protein activation by GPCRs are not well understood. Previous studies have revealed a signal relay route from a GPCR via the C-terminal α5-helix of Gα to the guanine nucleotide-binding pocket. Recent structural and biophysical studies uncover a role for the opening or rotating of the α-helical domain of Gα during the activation of Gα by a GPCR. Here we show that β-adrenergic receptors activate eight Gαs mutant proteins (from a screen of 66 Gαs mutants) that are unable to bind Gβγ subunits in cells. Five of these eight mutants are in the αF/Linker 2/β2 hinge region (extended Linker 2) that connects the Ras-like GTPase domain and the α-helical domain of Gαs. This extended Linker 2 is the target site of a natural product inhibitor of Gq. Our data show that the extended Linker 2 is critical for Gα activation by GPCRs. We propose that a GPCR via its intracellular loop 2 directly interacts with the β2/β3 loop of Gα to communicate to Linker 2, resulting in the opening and closing of the α-helical domain and the release of GDP during G-protein activation. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  9. Rearrangement of a polar core provides a conserved mechanism for constitutive activation of class B G protein-coupled receptors.

    Science.gov (United States)

    Yin, Yanting; de Waal, Parker W; He, Yuanzheng; Zhao, Li-Hua; Yang, Dehua; Cai, Xiaoqing; Jiang, Yi; Melcher, Karsten; Wang, Ming-Wei; Xu, H Eric

    2017-06-16

    The glucagon receptor (GCGR) belongs to the secretin-like (class B) family of G protein-coupled receptors (GPCRs) and is activated by the peptide hormone glucagon. The structures of an activated class B GPCR have remained unsolved, preventing a mechanistic understanding of how these receptors are activated. Using a combination of structural modeling and mutagenesis studies, we present here two modes of ligand-independent activation of GCGR. First, we identified a GCGR-specific hydrophobic lock comprising Met-338 and Phe-345 within the IC3 loop and transmembrane helix 6 (TM6) and found that this lock stabilizes the TM6 helix in the inactive conformation. Disruption of this hydrophobic lock led to constitutive G protein and arrestin signaling. Second, we discovered a polar core comprising conserved residues in TM2, TM3, TM6, and TM7, and mutations that disrupt this polar core led to constitutive GCGR activity. On the basis of these results, we propose a mechanistic model of GCGR activation in which TM6 is held in an inactive conformation by the conserved polar core and the hydrophobic lock. Mutations that disrupt these inhibitory elements allow TM6 to swing outward to adopt an active TM6 conformation similar to that of the canonical β 2 -adrenergic receptor complexed with G protein and to that of rhodopsin complexed with arrestin. Importantly, mutations in the corresponding polar core of several other members of class B GPCRs, including PTH1R, PAC1R, VIP1R, and CRFR1, also induce constitutive G protein signaling, suggesting that the rearrangement of the polar core is a conserved mechanism for class B GPCR activation. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  10. Regional differences in mu and kappa opioid receptor G-protein activation in brain in male and female prairie voles.

    Science.gov (United States)

    Martin, T J; Sexton, T; Kim, S A; Severino, A L; Peters, C M; Young, L J; Childers, S R

    2015-12-17

    Prairie voles are unusual mammals in that, like humans, they are capable of forming socially monogamous pair bonds, display biparental care, and engage in alloparental behaviors. Both mu and kappa opioid receptors are involved in behaviors that either establish and maintain, or result from pair bond formation in these animals. Mu and kappa opioid receptors both utilize inhibitory G-proteins in signal transduction mechanisms, however the efficacy by which these receptor subtypes stimulate G-protein signaling across the prairie vole neuraxis is not known. Utilizing [(35)S]GTPγS autoradiography, we characterized the efficacy of G-protein stimulation in coronal sections throughout male and female prairie vole brains by [D-Ala2,NMe-Phe4,Gly-ol5]-enkephalin (DAMGO) and U50,488H, selective mu and kappa opioid agonists, respectively. DAMGO stimulation was highest in the forebrain, similar to that found with other rodent species. U-50,488H produced greater stimulation in prairie voles than is typically seen in mice and rats, particularly in select forebrain areas. DAMGO produced higher stimulation in the core versus the shell of the nucleus accumbens (NAc) in females, while the distribution of U-50,488H stimulation was the opposite. There were no gender differences for U50,488H stimulation of G-protein activity across the regions examined, while DAMGO stimulation was greater in sections from females compared to those from males for NAc core, entopeduncular nucleus, and hippocampus. These data suggest that the kappa opioid system may be more sensitive to manipulation in prairie voles compared to mice and rats, and that female prairie voles may be more sensitive to mu agonists in select brain regions than males. Copyright © 2015 IBRO. Published by Elsevier Ltd. All rights reserved.

  11. Rac1 modulates G-protein-coupled receptor-induced bronchial smooth muscle contraction.

    Science.gov (United States)

    Sakai, Hiroyasu; Kai, Yuki; Sato, Ken; Ikebe, Mitsuo; Chiba, Yohihiko

    2018-01-05

    Increasing evidence suggests a functional role of RhoA/Rho-kinase signalling as a mechanism for smooth muscle contraction; however, little is known regarding the roles of Rac1 and other members of the Rho protein family. This study aimed to examine whether Rac1 modulates bronchial smooth muscle contraction. Ring preparations of bronchi isolated from rats were suspended in an organ bath, and isometric contraction of circular smooth muscle was measured. Immunoblotting was used to examine myosin light chain phosphorylation in bronchial smooth muscle. Our results demonstrated that muscle contractions induced by carbachol (CCh) and endothelin-1 (ET-1) were inhibited by EHT1864, a selective Rac1 inhibitor, and NSC23766, a selective inhibitor of Rac1-specific guanine nucleotide exchange factors. Similarly, myosin light chain and myosin phosphatase target subunit 1 (MYPT1) at Thr853 phosphorylation induced by contractile agonist were inhibited with Rac1 inhibition. However, contractions induced by high K + , calyculin A (a potent protein phosphatase inhibitor) and K + /PDBu were not inhibited by these Rac1 inhibitors. Interestingly, NaF (a G-protein activator)-induced contractions were inhibited by EHT1864 but not by NSC23766. We next examined the effects of a trans-acting activator of transcription protein transduction domain (PTD) fusion protein with Rac1 (PTD-Rac1) on muscle contraction. The constitutively active form of PTD-Rac1 directly induced force development and contractions were abolished by EHT1864. These results suggest that Rac1, activated by G protein-coupled receptor agonists, such as CCh and ET-1, may induce myosin light chain and MYPT phosphorylation and modulate the contraction of bronchial smooth muscle. Copyright © 2017 Elsevier B.V. All rights reserved.

  12. Molecular evolution of a chordate specific family of G protein-coupled receptors

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    Leese Florian

    2011-08-01

    Full Text Available Abstract Background Chordate evolution is a history of innovations that is marked by physical and behavioral specializations, which led to the development of a variety of forms from a single ancestral group. Among other important characteristics, vertebrates obtained a well developed brain, anterior sensory structures, a closed circulatory system and gills or lungs as blood oxygenation systems. The duplication of pre-existing genes had profound evolutionary implications for the developmental complexity in vertebrates, since mutations modifying the function of a duplicated protein can lead to novel functions, improving the evolutionary success. Results We analyzed here the evolution of the GPRC5 family of G protein-coupled receptors by comprehensive similarity searches and found that the receptors are only present in chordates and that the size of the receptor family expanded, likely due to genome duplication events in the early history of vertebrate evolution. We propose that a single GPRC5 receptor coding gene originated in a stem chordate ancestor and gave rise by duplication events to a gene family comprising three receptor types (GPRC5A-C in vertebrates, and a fourth homologue present only in mammals (GPRC5D. Additional duplications of GPRC5B and GPRC5C sequences occurred in teleost fishes. The finding that the expression patterns of the receptors are evolutionarily conserved indicates an important biological function of these receptors. Moreover, we found that expression of GPRC5B is regulated by vitamin A in vivo, confirming previous findings that linked receptor expression to retinoic acid levels in tumor cell lines and strengthening the link between the receptor expression and the development of a complex nervous system in chordates, known to be dependent on retinoic acid signaling. Conclusions GPRC5 receptors, a class of G protein-coupled receptors with unique sequence characteristics, may represent a molecular novelty that helped non

  13. G Protein-Coupled Receptor 87 (GPR87 Promotes Cell Proliferation in Human Bladder Cancer Cells

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    Xia Zhang

    2015-10-01

    Full Text Available G protein-coupled receptor 87 (GPR87 is a newly deorphanized member of the cell surface molecule G protein-coupled receptor family. GPR signaling was shown to play a role in promotion of cell growth and survival, metastasis, and drug resistance. The overexpression of GPR87 has also been reported in many malignant tumors including bladder cancer. The aim of the present study is to examine the effect of silencing GPR87 expression with a replication-deficient recombinant adenoviral vector expressing short hairpin RNA targeting GPR87 (Ad-shGPR87 and to explore the underlying molecular mechanisms in bladder cancer cells. Six GPR87-expressing human bladder cancer cells, HT1197, HT1376, J82, RT112, TCCSUP and UMUC3, were used. Infection with Ad-shGPR87 effectively downregulated the GPR87 expression, and significantly reduced the percentage of viable cells in 4 of 6 cell lines as detected by an MTT assay. Significant inhibition on cell proliferation with Ad-shGPR87 was observed in the wild-type p53 bladder cancer cell lines (HT1197, RT112, TCCSUP and UMUC3, but not in the mutant p53 cells (HT1376 and J82. As represented by a wild-type p53 RT112 cell, Ad-shGPR87 infection significantly enhanced p53 and p21 expression and caused caspase-dependent apoptosis. Furthermore, the treatment with Ad-shGPR87 exerted a significant antitumor effect against the GPR87-expressing RT112 xenografts. GPR87 appeared to be a promising target for gene therapy, and Ad-shGPR87 had strong antitumor effects, specifically anti-proliferative and pro-apoptotic effects, against GPR87-expressing human bladder cancer cells.

  14. Notch Signaling Mediates Skeletal Muscle Atrophy in Cancer Cachexia Caused by Osteosarcoma

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    Xiaodong Mu

    2016-01-01

    Full Text Available Skeletal muscle atrophy in cancer cachexia is mediated by the interaction between muscle stem cells and various tumor factors. Although Notch signaling has been known as a key regulator of both cancer development and muscle stem cell activity, the potential involvement of Notch signaling in cancer cachexia and concomitant muscle atrophy has yet to be elucidated. The murine K7M2 osteosarcoma cell line was used to generate an orthotopic model of sarcoma-associated cachexia, and the role of Notch signaling was evaluated. Skeletal muscle atrophy was observed in the sarcoma-bearing mice, and Notch signaling was highly active in both tumor tissues and the atrophic skeletal muscles. Systemic inhibition of Notch signaling reduced muscle atrophy. In vitro coculture of osteosarcoma cells with muscle-derived stem cells (MDSCs isolated from normal mice resulted in decreased myogenic potential of MDSCs, while the application of Notch inhibitor was able to rescue this repressed myogenic potential. We further observed that Notch-activating factors reside in the exosomes of osteosarcoma cells, which activate Notch signaling in MDSCs and subsequently repress myogenesis. Our results revealed that signaling between tumor and muscle via the Notch pathway may play an important role in mediating the skeletal muscle atrophy seen in cancer cachexia.

  15. Functionally Selective Signaling for Morphine and Fentanyl Antinociception and Tolerance Mediated by the Rat Periaqueductal Gray

    Science.gov (United States)

    Morgan, Michael M.; Reid, Rachel A.; Saville, Kimber A.

    2014-01-01

    Functionally selective signaling appears to contribute to the variability in mechanisms that underlie tolerance to the antinociceptive effects of opioids. The present study tested this hypothesis by examining the contribution of G protein-coupled receptor kinase (GRK)/Protein kinase C (PKC) and C-Jun N-terminal kinase (JNK) activation on both the expression and development of tolerance to morphine and fentanyl microinjected into the ventrolateral periaqueductal gray of the rat. Microinjection of morphine or fentanyl into the periaqueductal gray produced a dose-dependent increase in hot plate latency. Microinjection of the non-specific GRK/PKC inhibitor Ro 32-0432 into the periaqueductal gray to block mu-opioid receptor phosphorylation enhanced the antinociceptive effect of morphine but had no effect on fentanyl antinociception. Microinjection of the JNK inhibitor SP600125 had no effect on morphine or fentanyl antinociception, but blocked the expression of tolerance to repeated morphine microinjections. In contrast, a microinjection of Ro 32-0432 blocked the expression of fentanyl, but not morphine tolerance. Repeated microinjections of Ro 32-0432 blocked the development of morphine tolerance and inhibited fentanyl antinociception whether rats were tolerant or not. Repeated microinjections of SP600125 into the periaqueductal gray blocked the development of tolerance to both morphine and fentanyl microinjections. These data demonstrate that the signaling molecules that contribute to tolerance vary depending on the opioid and methodology used to assess tolerance (expression vs. development of tolerance). This signaling difference is especially clear for the expression of tolerance in which JNK contributes to morphine tolerance and GRK/PKC contributes to fentanyl tolerance. PMID:25503060

  16. Identification of potential pathway mediation targets in Toll-like receptor signaling.

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    Fan Li

    2009-02-01

    Full Text Available Recent advances in reconstruction and analytical methods for signaling networks have spurred the development of large-scale models that incorporate fully functional and biologically relevant features. An extended reconstruction of the human Toll-like receptor signaling network is presented herein. This reconstruction contains an extensive complement of kinases, phosphatases, and other associated proteins that mediate the signaling cascade along with a delineation of their associated chemical reactions. A computational framework based on the methods of large-scale convex analysis was developed and applied to this network to characterize input-output relationships. The input-output relationships enabled significant modularization of the network into ten pathways. The analysis identified potential candidates for inhibitory mediation of TLR signaling with respect to their specificity and potency. Subsequently, we were able to identify eight novel inhibition targets through constraint-based modeling methods. The results of this study are expected to yield meaningful avenues for further research in the task of mediating the Toll-like receptor signaling network and its effects.

  17. Hetero-oligomeric Complex between the G Protein-coupled Estrogen Receptor 1 and the Plasma Membrane Ca2+-ATPase 4b.

    Science.gov (United States)

    Tran, Quang-Kim; VerMeer, Mark; Burgard, Michelle A; Hassan, Ali B; Giles, Jennifer

    2015-05-22

    The new G protein-coupled estrogen receptor 1 (GPER/GPR30) plays important roles in many organ systems. The plasma membrane Ca(2+)-ATPase (PMCA) is essential for removal of cytoplasmic Ca(2+) and for shaping the time courses of Ca(2+)-dependent activities. Here, we show that PMCA and GPER/GPR30 physically interact and functionally influence each other. In primary endothelial cells, GPER/GPR30 agonist G-1 decreases PMCA-mediated Ca(2+) extrusion by promoting PMCA tyrosine phosphorylation. GPER/GPR30 overexpression decreases PMCA activity, and G-1 further potentiates this effect. GPER/GPR30 knockdown increases PMCA activity, whereas PMCA knockdown substantially reduces GPER/GPR30-mediated phosphorylation of the extracellular signal-related kinase (ERK1/2). GPER/GPR30 co-immunoprecipitates with PMCA with or without treatment with 17β-estradiol, thapsigargin, or G-1. Heterologously expressed GPER/GPR30 in HEK 293 cells co-localizes with PMCA4b, the main endothelial PMCA isoform. Endothelial cells robustly express the PDZ post-synaptic density protein (PSD)-95, whose knockdown reduces the association between GPER/GPR30 and PMCA. Additionally, the association between PMCA4b and GPER/GPR30 is substantially reduced by truncation of either or both of their C-terminal PDZ-binding motifs. Functionally, inhibition of PMCA activity is significantly reduced by truncation of GPER/GPR30's C-terminal PDZ-binding motif. These data strongly indicate that GPER/GPR30 and PMCA4b form a hetero-oligomeric complex in part via the anchoring action of PSD-95, in which they constitutively affect each other's function. Activation of GPER/GPR30 further inhibits PMCA activity through tyrosine phosphorylation of the pump. These interactions represent cross-talk between Ca(2+) signaling and GPER/GPR30-mediated activities. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  18. Homer2 protein regulates plasma membrane Ca²⁺-ATPase-mediated Ca²⁺ signaling in mouse parotid gland acinar cells.

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    Yang, Yu-Mi; Lee, Jiae; Jo, Hae; Park, Soonhong; Chang, Inik; Muallem, Shmuel; Shin, Dong Min

    2014-09-05

    Homer proteins are scaffold molecules with a domain structure consisting of an N-terminal Ena/VASP homology 1 protein-binding domain and a C-terminal leucine zipper/coiled-coil domain. The Ena/VASP homology 1 domain recognizes proline-rich motifs and binds multiple Ca(2+)-signaling proteins, including G protein-coupled receptors, inositol 1,4,5-triphosphate receptors, ryanodine receptors, and transient receptor potential channels. However, their role in Ca(2+) signaling in nonexcitable cells is not well understood. In this study, we investigated the role of Homer2 on Ca(2+) signaling in parotid gland acinar cells using Homer2-deficient (Homer2(-/-)) mice. Homer2 is localized at the apical pole in acinar cells. Deletion of Homer2 did not affect inositol 1,4,5-triphosphate receptor localization or channel activity and did not affect the expression and activity of sarco/endoplasmic reticulum Ca(2+)-ATPase pumps. In contrast, Homer2 deletion markedly increased expression of plasma membrane Ca(2+)-ATPase (PMCA) pumps, in particular PMCA4, at the apical pole. Accordingly, Homer2 deficiency increased Ca(2+) extrusion by acinar cells. These findings were supported by co-immunoprecipitation of Homer2 and PMCA in wild-type parotid cells and transfected human embryonic kidney 293 (HEK293) cells. We identified a Homer-binding PPXXF-like motif in the N terminus of PMCA that is required for interaction with Homer2. Mutation of the PPXXF-like motif did not affect the interaction of PMCA with Homer1 but inhibited its interaction with Homer2 and increased Ca(2+) clearance by PMCA. These findings reveal an important regulation of PMCA by Homer2 that has a central role on PMCA-mediated Ca(2+) signaling in parotid acinar cells. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

  19. PDL1 Signals through Conserved Sequence Motifs to Overcome Interferon-Mediated Cytotoxicity

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    Maria Gato-Cañas

    2017-08-01

    Full Text Available PDL1 blockade produces remarkable clinical responses, thought to occur by T cell reactivation through prevention of PDL1-PD1 T cell inhibitory interactions. Here, we find that PDL1 cell-intrinsic signaling protects cancer cells from interferon (IFN cytotoxicity and accelerates tumor progression. PDL1 inhibited IFN signal transduction through a conserved class of sequence motifs that mediate crosstalk with IFN signaling. Abrogation of PDL1 expression or antibody-mediated PDL1 blockade strongly sensitized cancer cells to IFN cytotoxicity through a STAT3/caspase-7-dependent pathway. Moreover, somatic mutations found in human carcinomas within these PDL1 sequence motifs disrupted motif regulation, resulting in PDL1 molecules with enhanced protective activities from type I and type II IFN cytotoxicity. Overall, our results reveal a mode of action of PDL1 in cancer cells as a first line of defense against IFN cytotoxicity.

  20. Single-cell analysis reveals a link between CD3- and CD59-mediated signaling pathways in Jurkat T cells

    International Nuclear Information System (INIS)

    Lipp, A. M.

    2012-01-01

    Elevation of intracellular free calcium concentration ([Ca2+]i) is a key signal during T cell activation and is commonly used as a read-out parameter for stimulation of T cell signaling. Upon T cell stimulation a variety of calcium signals is produced by individual cells of the T cell population and the type of calcium signal strongly influences cell fate decisions. The heterogeneous nature of T cells is masked in ensemble measurements, which highlights the need for single-cell measurements. In this study we used single-cell calcium measurements in Jurkat cells to investigate signaling pathways, which are triggered by different proteins, namely CD3 and CD59. By application of an automated cluster algorithm the presented assay provides unbiased analysis of a large data set of individual calcium time traces generated by the whole cell population. By using this method we could demonstrate that the Jurkat population generates heterogeneous calcium signals in a stimulus-dependent manner. Furthermore, our data revealed the existence of a link between CD3- and CD59-mediated signaling pathways. Single-cell calcium measurements in Jurkat cells expressing different levels of the T cell receptor (TCR) complex indicated that CD59-mediated calcium signaling is critically dependent on TCR surface expression levels. In addition, triggering CD59-mediated calcium signaling resulted in down-regulation of TCR surface expression levels, which is known to happen upon direct TCR triggering too. Moreover, by using siRNA-mediated protein knock-downs and protein knock-out Jurkat mutants we could show that CD3- and CD59-mediated calcium signaling require identical key proteins. We therefore explored by which mechanism CD59-mediated signaling couples into TCR-mediated signaling. Fluorescence recovery after photobleaching (FRAP) experiments and live-cell protein-protein interaction assays provided no evidence of a direct physical interaction between CD3- and CD59-mediated signaling pathways

  1. [G-protein potentiates the activation of TNF-alpha on calcium-activated potassium channel in ECV304].

    Science.gov (United States)

    Lin, L; Zheng, Y; Qu, J; Bao, G

    2000-06-01

    Observe the effect of tumor necrosis factor-alpha (TNF-alpha) on calcium-activated potassium channel in ECV304 and the possible involvement of G-protein mediation in the action of TNF-alpha. Using the cell-attached configuration of patch clamp technique. (1) the activity of high-conductance calcium-activated potassium channel (BKca) was recorded. Its conductance is (202.54 +/- 16.62) pS; (2) the activity of BKca was potentiated by 200 U/ml TNF-alpha; (3) G-protein would intensify this TNF-alpha activation. TNF-alpha acted on vascular endothelial cell ECV304 could rapidly activate the activity of BKca. Opening of BKca resulted in membrane hyper-polarization which could increase electro-chemical gradient for the resting Ca2+ influx and open leakage calcium channel, thus resting cytoplasmic free Ca2+ concentration could be elevated. G-protein may exert an important regulation in this process.

  2. NPR-9, a Galanin-Like G-Protein Coupled Receptor, and GLR-1 Regulate Interneuronal Circuitry Underlying Multisensory Integration of Environmental Cues in Caenorhabditis elegans.

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    Jason C Campbell

    2016-05-01

    Full Text Available C. elegans inhabit environments that require detection of diverse stimuli to modulate locomotion in order to avoid unfavourable conditions. In a mammalian context, a failure to appropriately integrate environmental signals can lead to Parkinson's, Alzheimer's, and epilepsy. Provided that the circuitry underlying mammalian sensory integration can be prohibitively complex, we analyzed nematode behavioral responses in differing environmental contexts to evaluate the regulation of context dependent circuit reconfiguration and sensorimotor control. Our work has added to the complexity of a known parallel circuit, mediated by interneurons AVA and AIB, that integrates sensory cues and is responsible for the initiation of backwards locomotion. Our analysis of the galanin-like G-protein coupled receptor NPR-9 in C. elegans revealed that upregulation of galanin signaling impedes the integration of sensory evoked neuronal signals. Although the expression pattern of npr-9 is limited to AIB, upregulation of the receptor appears to impede AIB and AVA circuits to broadly prevent backwards locomotion, i.e. reversals, suggesting that these two pathways functionally interact. Galanin signaling similarly plays a broadly inhibitory role in mammalian models. Moreover, our identification of a mutant, which rarely initiates backwards movement, allowed us to interrogate locomotory mechanisms underlying chemotaxis. In support of the pirouette model of chemotaxis, organisms that did not exhibit reversal behavior were unable to navigate towards an attractant peak. We also assessed ionotropic glutamate receptor GLR-1 cell-specifically within AIB and determined that GLR-1 fine-tunes AIB activity to modify locomotion following reversal events. Our research highlights that signal integration underlying the initiation and fine-tuning of backwards locomotion is AIB and NPR-9 dependent, and has demonstrated the suitability of C. elegans for analysis of multisensory integration

  3. Mutations Affecting G-Protein Subunit α11 in Hypercalcemia and Hypocalcemia

    Science.gov (United States)

    Babinsky, Valerie N.; Head, Rosie A.; Cranston, Treena; Rust, Nigel; Hobbs, Maurine R.; Heath, Hunter; Thakker, Rajesh V.

    2013-01-01

    BACKGROUND Familial hypocalciuric hypercalcemia is a genetically heterogeneous disorder with three variants: types 1, 2, and 3. Type 1 is due to loss-of-function mutations of the calcium-sensing receptor, a guanine nucleotide–binding protein (G-protein)–coupled receptor that signals through the G-protein subunit α11 (Gα11). Type 3 is associated with adaptor-related protein complex 2, sigma 1 subunit (AP2S1) mutations, which result in altered calcium-sensing receptor endocytosis. We hypothesized that type 2 is due to mutations effecting Gα11 loss of function, since Gα11 is involved in calcium-sensing receptor signaling, and its gene (GNA11) and the type 2 locus are colocalized on chromosome 19p13.3. We also postulated that mutations effecting Gα11 gain of function, like the mutations effecting calcium-sensing receptor gain of function that cause autosomal dominant hypocalcemia type 1, may lead to hypocalcemia. METHODS We performed GNA11 mutational analysis in a kindred with familial hypocalciuric hypercalcemia type 2 and in nine unrelated patients with familial hypocalciuric hypercalcemia who did not have mutations in the gene encoding the calcium-sensing receptor (CASR) or AP2S1. We also performed this analysis in eight unrelated patients with hypocalcemia who did not have CASR mutations. In addition, we studied the effects of GNA11 mutations on Gα11 protein structure and calcium-sensing receptor signaling in human embryonic kidney 293 (HEK293) cells. RESULTS The kindred with familial hypocalciuric hypercalcemia type 2 had an in-frame deletion of a conserved Gα11 isoleucine (Ile200del), and one of the nine unrelated patients with familial hypocalciuric hypercalcemia had a missense GNA11 mutation (Leu135Gln). Missense GNA11 mutations (Arg181Gln and Phe341Leu) were detected in two unrelated patients with hypocalcemia; they were therefore identified as having autosomal dominant hypocalcemia type 2. All four GNA11 mutations predicted disrupted protein

  4. Sodium modulates opioid receptors through a membrane component different from G-proteins. Demonstration by target size analysis

    International Nuclear Information System (INIS)

    Ott, S.; Costa, T.; Herz, A.

    1988-01-01

    The target size for opioid receptor binding was studied after manipulations known to affect the interactions between receptor and GTP-binding regulatory proteins (G-proteins). Addition of GTP or its analogs to the binding reaction, exposure of intact cells to pertussis toxin prior to irradiation, or treatment of irradiated membranes with N-ethylmaleimide did not change the target size (approximately equal to 100 kDa) for opioid receptors in NG 108-15 cells and rat brain. These data suggest that the 100-kDa species does not include an active subunit of a G-protein or alternatively that GTP does not promote the dissociation of the receptor-G-protein complex. The presence of Na+ (100 mM) in the radioligand binding assay induced a biphasic decay curve for agonist binding and a flattening of the monoexponential decay curve for a partial agonist. In both cases the effect was explained by an irradiation-induced loss of the low affinity state of the opioid receptor produced by the addition of Na+. This suggests that an allosteric inhibitor that mediates the effect of sodium on the receptor is destroyed at low doses of irradiation, leaving receptors which are no longer regulated by sodium. The effect of Na+ on target size was slightly increased by the simultaneous addition of GTP but was not altered by pertussis toxin treatment. Thus, the sodium unit is distinct from G-proteins and may represent a new component of the opioid receptor complex. Assuming a simple bimolecular model of one Na+ unit/receptor, the size of this inhibitor can be measured as 168 kDa

  5. Key mediators of intracellular amino acids signaling to mTORC1 activation.

    Science.gov (United States)

    Duan, Yehui; Li, Fengna; Tan, Kunrong; Liu, Hongnan; Li, Yinghui; Liu, Yingying; Kong, Xiangfeng; Tang, Yulong; Wu, Guoyao; Yin, Yulong

    2015-05-01

    Mammalian target of rapamycin complex 1 (mTORC1) is activated by amino acids to promote cell growth via protein synthesis. Specifically, Ras-related guanosine triphosphatases (Rag GTPases) are activated by amino acids, and then translocate mTORC1 to the surface of late endosomes and lysosomes. Ras homolog enriched in brain (Rheb) resides on this surface and directly activates mTORC1. Apart from the presence of intracellular amino acids, Rag GTPases and Rheb, other mediators involved in intracellular amino acid signaling to mTORC1 activation include human vacuolar sorting protein-34 (hVps34) and mitogen-activating protein kinase kinase kinase kinase-3 (MAP4K3). Those molecular links between mTORC1 and its mediators form a complicate signaling network that controls cellular growth, proliferation, and metabolism. Moreover, it is speculated that amino acid signaling to mTORC1 may start from the lysosomal lumen. In this review, we discussed the function of these mediators in mTORC1 pathway and how these mediators are regulated by amino acids in details.

  6. Tre1, a G protein-coupled receptor, directs transepithelial migration of Drosophila germ cells.

    Directory of Open Access Journals (Sweden)

    Prabhat S Kunwar

    2003-12-01

    Full Text Available In most organisms, germ cells are formed distant from the somatic part of the gonad and thus have to migrate along and through a variety of tissues to reach the gonad. Transepithelial migration through the posterior midgut (PMG is the first active step during Drosophila germ cell migration. Here we report the identification of a novel G protein-coupled receptor (GPCR, Tre1, that is essential for this migration step. Maternal tre1 RNA is localized to germ cells, and tre1 is required cell autonomously in germ cells. In tre1 mutant embryos, most germ cells do not exit the PMG. The few germ cells that do leave the midgut early migrate normally to the gonad, suggesting that this gene is specifically required for transepithelial migration and that mutant germ cells are still able to recognize other guidance cues. Additionally, inhibiting small Rho GTPases in germ cells affects transepithelial migration, suggesting that Tre1 signals through Rho1. We propose that Tre1 acts in a manner similar to chemokine receptors required during transepithelial migration of leukocytes, implying an evolutionarily conserved mechanism of transepithelial migration. Recently, the chemokine receptor CXCR4 was shown to direct migration in vertebrate germ cells. Thus, germ cells may more generally use GPCR signaling to navigate the embryo toward their target.

  7. Extracellular acidification activates ovarian cancer G-protein-coupled receptor 1 and GPR4 homologs of zebra fish

    Energy Technology Data Exchange (ETDEWEB)

    Mochimaru, Yuta [Laboratory of Cell Signaling Regulation, Department of Life Sciences, School of Agriculture, Meiji University, Kawasaki 214-8571 (Japan); Azuma, Morio [Laboratory of Regulatory Biology, Graduate School of Science and Engineering, University of Toyama, 3190 Gofuku, Toyama 930-8555 (Japan); Oshima, Natsuki; Ichijo, Yuta; Satou, Kazuhiro [Laboratory of Cell Signaling Regulation, Department of Life Sciences, School of Agriculture, Meiji University, Kawasaki 214-8571 (Japan); Matsuda, Kouhei [Laboratory of Regulatory Biology, Graduate School of Science and Engineering, University of Toyama, 3190 Gofuku, Toyama 930-8555 (Japan); Asaoka, Yoichi; Nishina, Hiroshi [Department of Developmental and Regenerative Biology, Medical Research Institute, Tokyo Medical and Dental University, Tokyo 113-8510 (Japan); Nakakura, Takashi [Department of Anatomy, Graduate School of Medicine, Teikyo University, 2-11-1 Kaga Itabashi-Ku, Tokyo 173-8605 (Japan); Mogi, Chihiro; Sato, Koichi; Okajima, Fumikazu [Laboratory of Signal Transduction, Institute for Molecular and Cellular Regulation, Gunma University, Maebashi 371-8512 (Japan); Tomura, Hideaki, E-mail: tomurah@meiji.ac.jp [Laboratory of Cell Signaling Regulation, Department of Life Sciences, School of Agriculture, Meiji University, Kawasaki 214-8571 (Japan)

    2015-02-20

    Mammalian ovarian G-protein-coupled receptor 1 (OGR1) and GPR4 are identified as a proton-sensing G-protein-coupled receptor coupling to multiple intracellular signaling pathways. In the present study, we examined whether zebra fish OGR1 and GPR4 homologs (zOGR1 and zGPR4) could sense protons and activate the multiple intracellular signaling pathways and, if so, whether the similar positions of histidine residue, which is critical for sensing protons in mammalian OGR and GPR4, also play a role to sense protons and activate the multiple signaling pathways in the zebra fish receptors. We found that extracellular acidic pH stimulated CRE-, SRE-, and NFAT-promoter activities in zOGR1 overexpressed cells and stimulated CRE- and SRE- but not NFAT-promoter activities in zGPR4 overexpressed cells. The substitution of histidine residues at the 12th, 15th, 162th, and 264th positions from the N-terminal of zOGR1 with phenylalanine attenuated the proton-induced SRE-promoter activities. The mutation of the histidine residue at the 78th but not the 84th position from the N-terminal of zGPR4 to phenylalanine attenuated the proton-induced SRE-promoter activities. These results suggest that zOGR1 and zGPR4 are also proton-sensing G-protein-coupled receptors, and the receptor activation mechanisms may be similar to those of the mammalian receptors. - Highlights: • Zebra fish OGR1 and GPR4 homologs (zOGR1, zGPR4) are proton-sensing receptors. • The signaling pathways activated by zOGR1 and zGPR4 are different. • Histidine residues critical for sensing protons are conserved.

  8. Allosteric modulation of G-protein coupled receptors

    DEFF Research Database (Denmark)

    Jensen, Anders A.; Spalding, Tracy A

    2004-01-01

    are believed to activate (agonists) or inhibit (competitive antagonists) receptor signalling by binding the receptor at the same site as the endogenous agonist, the orthosteric site. In contrast, allosteric ligands modulate receptor function by binding to different regions in the receptor, allosteric sites....... In recent years, combinatorial chemistry and high throughput screening have helped identify several allosteric GPCR modulators with novel structures, several of which already have become valuable pharmacological tools and may be candidates for clinical testing in the near future. This mini review outlines...... the current status and perspectives of allosteric modulation of GPCR function with emphasis on the pharmacology of endogenous and synthesised modulators, their receptor interactions and the therapeutic prospects of allosteric ligands compared to orthosteric ligands....

  9. Varicose and cheerio collaborate with pebble to mediate semaphorin-1a reverse signaling in Drosophila.

    Science.gov (United States)

    Jeong, Sangyun; Yang, Da-Som; Hong, Young Gi; Mitchell, Sarah P; Brown, Matthew P; Kolodkin, Alex L

    2017-09-26

    The transmembrane semaphorin Sema-1a acts as both a ligand and a receptor to regulate axon-axon repulsion during neural development. Pebble (Pbl), a Rho guanine nucleotide exchange factor, mediates Sema-1a reverse signaling through association with the N-terminal region of the Sema-1a intracellular domain (ICD), resulting in cytoskeletal reorganization. Here, we uncover two additional Sema-1a interacting proteins, varicose (Vari) and cheerio (Cher), each with neuronal functions required for motor axon pathfinding. Vari is a member of the membrane-associated guanylate kinase (MAGUK) family of proteins, members of which can serve as scaffolds to organize signaling complexes. Cher is related to actin filament cross-linking proteins that regulate actin cytoskeleton dynamics. The PDZ domain binding motif found in the most C-terminal region of the Sema-1a ICD is necessary for interaction with Vari, but not Cher, indicative of distinct binding modalities. Pbl/Sema-1a-mediated repulsive guidance is potentiated by both vari and cher Genetic analyses further suggest that scaffolding functions of Vari and Cher play an important role in Pbl-mediated Sema-1a reverse signaling. These results define intracellular components critical for signal transduction from the Sema-1a receptor to the cytoskeleton and provide insight into mechanisms underlying semaphorin-induced localized changes in cytoskeletal organization.

  10. Pannexin 1 channels mediate 'find-me' signal release and membrane permeability during apoptosis.

    Science.gov (United States)

    Chekeni, Faraaz B; Elliott, Michael R; Sandilos, Joanna K; Walk, Scott F; Kinchen, Jason M; Lazarowski, Eduardo R; Armstrong, Allison J; Penuela, Silvia; Laird, Dale W; Salvesen, Guy S; Isakson, Brant E; Bayliss, Douglas A; Ravichandran, Kodi S

    2010-10-14

    Apoptotic cells release 'find-me' signals at the earliest stages of death to recruit phagocytes. The nucleotides ATP and UTP represent one class of find-me signals, but their mechanism of release is not known. Here, we identify the plasma membrane channel pannexin 1 (PANX1) as a mediator of find-me signal/nucleotide release from apoptotic cells. Pharmacological inhibition and siRNA-mediated knockdown of PANX1 led to decreased nucleotide release and monocyte recruitment by apoptotic cells. Conversely, PANX1 overexpression enhanced nucleotide release from apoptotic cells and phagocyte recruitment. Patch-clamp recordings showed that PANX1 was basally inactive, and that induction of PANX1 currents occurred only during apoptosis. Mechanistically, PANX1 itself was a target of effector caspases (caspases 3 and 7), and a specific caspase-cleavage site within PANX1 was essential for PANX1 function during apoptosis. Expression of truncated PANX1 (at the putative caspase cleavage site) resulted in a constitutively open channel. PANX1 was also important for the 'selective' plasma membrane permeability of early apoptotic cells to specific dyes. Collectively, these data identify PANX1 as a plasma membrane channel mediating the regulated release of find-me signals and selective plasma membrane permeability during apoptosis, and a new mechanism of PANX1 activation by caspases.

  11. BMPRIA mediated signaling is essential for temporomandibular joint development in mice.

    Directory of Open Access Journals (Sweden)

    Shuping Gu

    Full Text Available The central importance of BMP signaling in the development and homeostasis of synovial joint of appendicular skeleton has been well documented, but its role in the development of temporomandibular joint (TMJ, also classified as a synovial joint, remains completely unknown. In this study, we investigated the function of BMPRIA mediated signaling in TMJ development in mice by transgenic loss-of- and gain-of-function approaches. We found that BMPRIA is expressed in the cranial neural crest (CNC-derived developing condyle and glenoid fossa, major components of TMJ, as well as the interzone mesenchymal cells. Wnt1-Cre mediated tissue specific inactivation of BmprIa in CNC lineage led to defective TMJ development, including failure of articular disc separation from a hypoplastic condyle, persistence of interzone cells, and failed formation of a functional fibrocartilage layer on the articular surface of the glenoid fossa and condyle, which could be at least partially attributed to the down-regulation of Ihh in the developing condyle and inhibition of apoptosis in the interzone. On the other hand, augmented BMPRIA signaling by Wnt1-Cre driven expression of a constitutively active form of BmprIa (caBmprIa inhibited osteogenesis of the glenoid fossa and converted the condylar primordium from secondary cartilage to primary cartilage associated with ectopic activation of Smad-dependent pathway but inhibition of JNK pathway, leading to TMJ agenesis. Our results present unambiguous evidence for an essential role of finely tuned BMPRIA mediated signaling in TMJ development.

  12. Avian metapneumovirus SH gene end and G protein mutations influence the level of protection of live-vaccine candidates.

    Science.gov (United States)

    Naylor, Clive J; Ling, Roger; Edworthy, Nicole; Savage, Carol E; Easton, Andrew J

    2007-06-01

    A prototype avian metapneumovirus (AMPV) vaccine (P20) was previously shown to give variable outcomes in experimental trials. Following plaque purification, three of 12 viruses obtained from P20 failed to induce protection against virulent challenge, whilst the remainder retained their protective capacity. The genome sequences of two protective viruses were identical to the P20 consensus, whereas two non-protective viruses differed only in the SH gene transcription termination signal. Northern blotting showed that the alterations in the SH gene-end region of the non-protective viruses led to enhanced levels of dicistronic mRNA produced by transcriptional readthrough. A synthetic minigenome was used to demonstrate that the altered SH gene-end region reduced the level of protein expression from a downstream gene. The genomes of the remaining eight plaque-purified viruses were sequenced in the region where the P20 consensus sequence differed from the virulent progenitor. The seven protective clones were identical, whereas the non-protective virus retained the virulent progenitor sequence at two positions and contained extensive alterations in its attachment (G) protein sequence associated with a reduced or altered expression pattern of G protein on Western blots. The data indicate that the efficacy of a putative protective vaccine strain is affected by mutations altering the balance of G protein expression.

  13. G-protein-coupled inward rectifier potassium current contributes to ventricular repolarization

    DEFF Research Database (Denmark)

    Liang, Bo; Nissen, Jakob D; Laursen, Morten

    2014-01-01

    The purpose of this study was to investigate the functional role of G-protein-coupled inward rectifier potassium (GIRK) channels in the cardiac ventricle.......The purpose of this study was to investigate the functional role of G-protein-coupled inward rectifier potassium (GIRK) channels in the cardiac ventricle....

  14. Role of CD137 signaling in dengue virus-mediated apoptosis

    International Nuclear Information System (INIS)

    Nagila, Amar; Netsawang, Janjuree; Srisawat, Chatchawan; Noisakran, Sansanee; Morchang, Atthapan; Yasamut, Umpa; Puttikhunt, Chunya; Kasinrerk, Watchara

    2011-01-01

    Highlights: → For the first time the role of CD137 in dengue virus (DENV) infection. → Induction of DENV-mediated apoptosis by CD137 signaling. → Sensitization to CD137-mediated apoptosis by dengue virus capsid protein (DENV C). → Nuclear localization of DENV C is required for CD137-mediated apoptosis. -- Abstract: Hepatic dysfunction is a well recognized feature of dengue virus (DENV) infection. However, molecular mechanisms of hepatic injury are still poorly understood. A complex interaction between DENV and the host immune response contributes to DENV-mediated tissue injury. DENV capsid protein (DENV C) physically interacts with the human death domain-associated protein Daxx. A double substitution mutation in DENV C (R85A/K86A) abrogates Daxx interaction, nuclear localization and apoptosis. Therefore we compared the expression of cell death genes between HepG2 cells expressing DENV C and DENV C (R85A/K86A) using a real-time PCR array. Expression of CD137, which is a member of the tumor necrosis factor receptor family, increased significantly in HepG2 cells expressing DENV C compared to HepG2 cells expressing DENV C (R85A/K86A). In addition, CD137-mediated apoptotic activity in HepG2 cells expressing DENV C was significantly increased by anti-CD137 antibody compared to that of HepG2 cells expressing DENV C (R85A/K86A). In DENV-infected HepG2 cells, CD137 mRNA and CD137 positive cells significantly increased and CD137-mediated apoptotic activity was increased by anti-CD137 antibody. This work is the first to demonstrate the contribution of CD137 signaling to DENV-mediated apoptosis.

  15. Role of CD137 signaling in dengue virus-mediated apoptosis

    Energy Technology Data Exchange (ETDEWEB)

    Nagila, Amar [Medical Molecular Biology Unit, Office for Research and Development, Faculty of Medicine, Siriraj Hospital, Mahidol University, Bangkok (Thailand); Department of Biochemistry, Faculty of Medicine, Siriraj Hospital, Mahidol University, Bangkok (Thailand); Netsawang, Janjuree [Faculty of Medical Technology, Rangsit University, Bangkok (Thailand); Srisawat, Chatchawan [Department of Biochemistry, Faculty of Medicine, Siriraj Hospital, Mahidol University, Bangkok (Thailand); Noisakran, Sansanee [Dengue Hemorrhagic Fever Research Unit, Office for Research and Development, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok (Thailand); Medical Biotechnology Unit, National Center for Genetic Engineering and Biotechnology, National Science and Technology Development Agency, Bangkok (Thailand); Morchang, Atthapan; Yasamut, Umpa [Medical Molecular Biology Unit, Office for Research and Development, Faculty of Medicine, Siriraj Hospital, Mahidol University, Bangkok (Thailand); Department of Immunology, Faculty of Medicine, Siriraj Hospital, Mahidol University, Bangkok (Thailand); Puttikhunt, Chunya [Dengue Hemorrhagic Fever Research Unit, Office for Research and Development, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok (Thailand); Medical Biotechnology Unit, National Center for Genetic Engineering and Biotechnology, National Science and Technology Development Agency, Bangkok (Thailand); Kasinrerk, Watchara [Division of Clinical Immunology, Department of Medical Technology, Faculty of Associated Medical Sciences, Chiang Mai University, Chiang Mai (Thailand); Biomedical Technology Research Center, National Center for Genetic Engineering and Biotechnology, National Science and Technology Development Agency at Chiang Mai University, Chiang Mai (Thailand); and others

    2011-07-08

    Highlights: {yields} For the first time the role of CD137 in dengue virus (DENV) infection. {yields} Induction of DENV-mediated apoptosis by CD137 signaling. {yields} Sensitization to CD137-mediated apoptosis by dengue virus capsid protein (DENV C). {yields} Nuclear localization of DENV C is required for CD137-mediated apoptosis. -- Abstract: Hepatic dysfunction is a well recognized feature of dengue virus (DENV) infection. However, molecular mechanisms of hepatic injury are still poorly understood. A complex interaction between DENV and the host immune response contributes to DENV-mediated tissue injury. DENV capsid protein (DENV C) physically interacts with the human death domain-associated protein Daxx. A double substitution mutation in DENV C (R85A/K86A) abrogates Daxx interaction, nuclear localization and apoptosis. Therefore we compared the expression of cell death genes between HepG2 cells expressing DENV C and DENV C (R85A/K86A) using a real-time PCR array. Expression of CD137, which is a member of the tumor necrosis factor receptor family, increased significantly in HepG2 cells expressing DENV C compared to HepG2 cells expressing DENV C (R85A/K86A). In addition, CD137-mediated apoptotic activity in HepG2 cells expressing DENV C was significantly increased by anti-CD137 antibody compared to that of HepG2 cells expressing DENV C (R85A/K86A). In DENV-infected HepG2 cells, CD137 mRNA and CD137 positive cells significantly increased and CD137-mediated apoptotic activity was increased by anti-CD137 antibody. This work is the first to demonstrate the contribution of CD137 signaling to DENV-mediated apoptosis.

  16. Methods for the Analysis of Protein Phosphorylation-Mediated Cellular Signaling Networks

    Science.gov (United States)

    White, Forest M.; Wolf-Yadlin, Alejandro

    2016-06-01

    Protein phosphorylation-mediated cellular signaling networks regulate almost all aspects of cell biology, including the responses to cellular stimulation and environmental alterations. These networks are highly complex and comprise hundreds of proteins and potentially thousands of phosphorylation sites. Multiple analytical methods have been developed over the past several decades to identify proteins and protein phosphorylation sites regulating cellular signaling, and to quantify the dynamic response of these sites to different cellular stimulation. Here we provide an overview of these methods, including the fundamental principles governing each method, their relative strengths and weaknesses, and some examples of how each method has been applied to the analysis of complex signaling networks. When applied correctly, each of these techniques can provide insight into the topology, dynamics, and regulation of protein phosphorylation signaling networks.

  17. SPATA2-Mediated Binding of CYLD to HOIP Enables CYLD Recruitment to Signaling Complexes

    Directory of Open Access Journals (Sweden)

    Sebastian Kupka

    2016-08-01

    Full Text Available Recruitment of the deubiquitinase CYLD to signaling complexes is mediated by its interaction with HOIP, the catalytically active component of the linear ubiquitin chain assembly complex (LUBAC. Here, we identify SPATA2 as a constitutive direct binding partner of HOIP that bridges the interaction between CYLD and HOIP. SPATA2 recruitment to TNFR1- and NOD2-signaling complexes is dependent on HOIP, and loss of SPATA2 abolishes CYLD recruitment. Deficiency in SPATA2 exerts limited effects on gene activation pathways but diminishes necroptosis induced by tumor necrosis factor (TNF, resembling loss of CYLD. In summary, we describe SPATA2 as a previously unrecognized factor in LUBAC-dependent signaling pathways that serves as an adaptor between HOIP and CYLD, thereby enabling recruitment of CYLD to signaling complexes.

  18. A comparative analysis of the heterotrimeric G-protein Gα, Gβ and Gγ subunits in the wheat pathogen Stagonospora nodorum

    Directory of Open Access Journals (Sweden)

    Gummer Joel P A

    2012-07-01

    Full Text Available Abstract Background It has been well established that the Gα subunit of the heterotrimeric G-protein in the wheat pathogen Stagonospora nodorum is required for a variety of phenotypes including pathogenicity, melanisation and asexual differentiation. The roles though of the Gγ and Gβ subunits though were unclear. The objective of this study was to identify and understand the role of these subunits and assess their requirement for pathogenicity and development. Results G-protein Gγ and Gβ subunits, named Gga1 and Gba1 respectively, were identified in the Stagonospora nodorum genome by comparative analysis with known fungal orthologues. A reverse genetics technique was used to study the role of these and revealed that the mutant strains displayed altered in vitro growth including a differential response to a variety of exogenous carbon sources. Pathogenicity assays showed that Stagonospora nodorum strains lacking Gba1 were essentially non-pathogenic whilst Gga1-impaired strains displayed significantly slower growth in planta. Subsequent sporulation assays showed that like the previously described Gα subunit mutants, both Gba1 and Gga1 were required for asexual sporulation with neither mutant strain being able to differentiate either pycnidia nor pycnidiospores under normal growth conditions. Continued incubation at 4°C was found to complement the mutation in each of the G-protein subunits with nearly wild-type levels of pycnidia recovered. Conclusion This study provides further evidence on the significance of cAMP-dependent signal transduction for many aspects of fungal development and pathogenicity. The observation that cold temperatures can complement the G-protein sporulation defect now provides an ideal tool by which asexual differentiation can now be dissected.

  19. Transcription Profiles Reveal Sugar and Hormone Signaling Pathways Mediating Flower Induction in Apple (Malus domestica Borkh.).

    Science.gov (United States)

    Xing, Li-Bo; Zhang, Dong; Li, You-Mei; Shen, Ya-Wen; Zhao, Cai-Ping; Ma, Juan-Juan; An, Na; Han, Ming-Yu

    2015-10-01

    Flower induction in apple (Malus domestica Borkh.) is regulated by complex gene networks that involve multiple signal pathways to ensure flower bud formation in the next year, but the molecular determinants of apple flower induction are still unknown. In this research, transcriptomic profiles from differentiating buds allowed us to identify genes potentially involved in signaling pathways that mediate the regulatory mechanisms of flower induction. A hypothetical model for this regulatory mechanism was obtained by analysis of the available transcriptomic data, suggesting that sugar-, hormone- and flowering-related genes, as well as those involved in cell-cycle induction, participated in the apple flower induction process. Sugar levels and metabolism-related gene expression profiles revealed that sucrose is the initiation signal in flower induction. Complex hormone regulatory networks involved in cytokinin (CK), abscisic acid (ABA) and gibberellic acid pathways also induce apple flower formation. CK plays a key role in the regulation of cell formation and differentiation, and in affecting flowering-related gene expression levels during these processes. Meanwhile, ABA levels and ABA-related gene expression levels gradually increased, as did those of sugar metabolism-related genes, in developing buds, indicating that ABA signals regulate apple flower induction by participating in the sugar-mediated flowering pathway. Furthermore, changes in sugar and starch deposition levels in buds can be affected by ABA content and the expression of the genes involved in the ABA signaling pathway. Thus, multiple pathways, which are mainly mediated by crosstalk between sugar and hormone signals, regulate the molecular network involved in bud growth and flower induction in apple trees. © The Author 2015. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists.

  20. Progesterone receptors (PR) mediate STAT actions: PR and prolactin receptor signaling crosstalk in breast cancer models.

    Science.gov (United States)

    Leehy, Katherine A; Truong, Thu H; Mauro, Laura J; Lange, Carol A

    2018-02-01

    Estrogen is the major mitogenic stimulus of mammary gland development during puberty wherein ER signaling acts to induce abundant PR expression. PR signaling, in contrast, is the primary driver of mammary epithelial cell proliferation in adulthood. The high circulating levels of progesterone during pregnancy signal through PR, inducing expression of the prolactin receptor (PRLR). Cooperation between PR and prolactin (PRL) signaling, via regulation of downstream components in the PRL signaling pathway including JAKs and STATs, facilitates the alveolar morphogenesis observed during pregnancy. Indeed, these pathways are fully integrated via activation of shared signaling pathways (i.e. JAKs, MAPKs) as well as by the convergence of PRs and STATs at target genes relevant to both mammary gland biology and breast cancer progression (i.e. proliferation, stem cell outgrowth, tissue cell type heterogeneity). Thus, rather than a single mediator such as ER, transcription factor cascades (ER>PR>STATs) are responsible for rapid proliferative and developmental programming in the normal mammary gland. It is not surprising that these same mediators typify uncontrolled proliferation in a majority of breast cancers, where ER and PR are most often co-expressed and may cooperate to drive malignant tumor progression. This review will primarily focus on the integration of PR and PRL signaling in breast cancer models and the importance of this cross-talk in cancer progression in the context of mammographic density. Components of these PR/PRL signaling pathways could offer alternative drug targets and logical complements to anti-ER or anti-estrogen-based endocrine therapies. Copyright © 2017 Elsevier Ltd. All rights reserved.

  1. Importance of Mediator complex in the regulation and integration of diverse signaling pathways in plants.

    Science.gov (United States)

    Samanta, Subhasis; Thakur, Jitendra K

    2015-01-01

    Basic transcriptional machinery in eukaryotes is assisted by a number of cofactors, which either increase or decrease the rate of transcription. Mediator complex is one such cofactor, and recently has drawn a lot of interest because of its integrative power to converge different signaling pathways before channeling the transcription instructions to the RNA polymerase II machinery. Like yeast and metazoans, plants do possess the Mediator complex across the kingdom, and its isolation and subunit analyses have been reported from the model plant, Arabidopsis. Genetic, and molecular analyses have unraveled important regulatory roles of Mediator subunits at every stage of plant life cycle starting from flowering to embryo and organ development, to even size determination. It also contributes immensely to the survival of plants against different environmental vagaries by the timely activation of its resistance mechanisms. Here, we have provided an overview of plant Mediator complex starting from its discovery to regulation of stoichiometry of its subunits. We have also reviewed involvement of different Mediator subunits in different processes and pathways including defense response pathways evoked by diverse biotic cues. Wherever possible, attempts have been made to provide mechanistic insight of Mediator's involvement in these processes.

  2. Charge-signal multiplication mediated by urea wires inside Y-shaped carbon nanotubes

    International Nuclear Information System (INIS)

    Lv, Mei; Liu, Zengrong; He, Bing; Xiu, Peng; Tu, Yusong

    2014-01-01

    In previous studies, we reported molecular dynamics (MD) simulations showing that single-file water wires confined inside Y-shaped single-walled carbon nanotubes (Y-SWNTs) held strong and robust capability to convert and multiply charge signals [Y. S. Tu, P. Xiu, R. Z. Wan, J. Hu, R. H. Zhou, and H. P. Fang, Proc. Natl. Acad. Sci. U.S.A. 106, 18120 (2009); Y. Tu, H. Lu, Y. Zhang, T. Huynh, and R. Zhou, J. Chem. Phys. 138, 015104 (2013)]. It is fascinating to see whether the signal multiplication can be realized by other kinds of polar molecules with larger dipole moments (which make the experimental realization easier). In this article, we use MD simulations to study the urea-mediated signal conversion and multiplication with Y-SWNTs. We observe that when a Y-SWNT with an external charge of magnitude 1.0 e (the model of a signal at the single-electron level) is solvated in 1 M urea solutions, urea can induce drying of the Y-SWNT and fill its interiors in single-file, forming Y-shaped urea wires. The external charge can effectively control the dipole orientation of the urea wire inside the main channel (i.e., the signal can be readily converted), and this signal can further be multiplied into 2 (or more) output signals by modulating dipole orientations of urea wires in bifurcated branch channels of the Y-SWNT. This remarkable signal transduction capability arises from the strong dipole-induced ordering of urea wires under extreme confinement. We also discuss the advantage of urea as compared with water in the signal multiplication, as well as the robustness and biological implications of our findings. This study provides the possibility for multiplying signals by using urea molecules (or other polar organic molecules) with Y-shaped nanochannels and might also help understand the mechanism behind signal conduction in both physical and biological systems

  3. The viral G protein-coupled receptor ORF74 hijacks β-arrestins for endocytic trafficking in response to human chemokines

    NARCIS (Netherlands)

    De Munnik, Sabrina M.; Kooistra, Albert J.; Van Offenbeek, Jody; Nijmeijer, Saskia; de Graaf, C.; Smit, Martine J.; Leurs, Rob; Vischer, Henry F.

    2015-01-01

    Kaposi's sarcoma-associated herpesvirus-infected cells express the virally encoded G protein-coupled receptor ORF74. Although ORF74 is constitutively active, it binds human CXC chemokines that modulate this basal activity. ORF74-induced signaling has been demonstrated to underlie the development of

  4. G Protein Coupled Receptor Kinase 3 Regulates Breast Cancer Migration, Invasion, and Metastasis

    Science.gov (United States)

    Billard, Matthew J.; Fitzhugh, David J.; Parker, Joel S.; Brozowski, Jaime M.; McGinnis, Marcus W.; Timoshchenko, Roman G.; Serafin, D. Stephen; Lininger, Ruth; Klauber-Demore, Nancy; Sahagian, Gary; Truong, Young K.; Sassano, Maria F.; Serody, Jonathan S.; Tarrant, Teresa K.

    2016-01-01

    Triple negative breast cancer (TNBC) is a heterogeneous disease that has a poor prognosis and limited treatment options. Chemokine receptor interactions are important modulators of breast cancer metastasis; however, it is now recognized that quantitative surface expression of one important chemokine receptor, CXCR4, may not directly correlate with metastasis and that its functional activity in breast cancer may better inform tumor pathogenicity. G protein coupled receptor kinase 3 (GRK3) is a negative regulator of CXCR4 activity, and we show that GRK expression correlates with tumorigenicity, molecular subtype, and metastatic potential in human tumor microarray analysis. Using established human breast cancer cell lines and an immunocompetent in vivo mouse model, we further demonstrate that alterations in GRK3 expression levels in tumor cells directly affect migration and invasion in vitro and the establishment of distant metastasis in vivo. The effects of GRK3 modulation appear to be specific to chemokine-mediated migration behaviors without influencing tumor cell proliferation or survival. These data demonstrate that GRK3 dysregulation may play an important part in TNBC metastasis. PMID:27049755

  5. G Protein Coupled Receptor Kinase 3 Regulates Breast Cancer Migration, Invasion, and Metastasis.

    Directory of Open Access Journals (Sweden)

    Matthew J Billard

    Full Text Available Triple negative breast cancer (TNBC is a heterogeneous disease that has a poor prognosis and limited treatment options. Chemokine receptor interactions are important modulators of breast cancer metastasis; however, it is now recognized that quantitative surface expression of one important chemokine receptor, CXCR4, may not directly correlate with metastasis and that its functional activity in breast cancer may better inform tumor pathogenicity. G protein coupled receptor kinase 3 (GRK3 is a negative regulator of CXCR4 activity, and we show that GRK expression correlates with tumorigenicity, molecular subtype, and metastatic potential in human tumor microarray analysis. Using established human breast cancer cell lines and an immunocompetent in vivo mouse model, we further demonstrate that alterations in GRK3 expression levels in tumor cells directly affect migration and invasion in vitro and the establishment of distant metastasis in vivo. The effects of GRK3 modulation appear to be specific to chemokine-mediated migration behaviors without influencing tumor cell proliferation or survival. These data demonstrate that GRK3 dysregulation may play an important part in TNBC metastasis.

  6. Expression and functional roles of G-protein-coupled estrogen receptor (GPER) in human eosinophils.

    Science.gov (United States)

    Tamaki, Mami; Konno, Yasunori; Kobayashi, Yoshiki; Takeda, Masahide; Itoga, Masamichi; Moritoki, Yuki; Oyamada, Hajime; Kayaba, Hiroyuki; Chihara, Junichi; Ueki, Shigeharu

    2014-07-01

    Sexual dimorphism in asthma links the estrogen and allergic immune responses. The function of estrogen was classically believed to be mediated through its nuclear receptors, i.e., estrogen receptors (ERs). However, recent studies established the important roles of G-protein-coupled estrogen receptor (GPER/GPR30) as a novel membrane receptor for estrogen. To date, the role of GPER in allergic inflammation is poorly understood. The purpose of this study was to examine whether GPER might affect the functions of eosinophils, which play an important role in the pathogenesis of asthma. Here, we demonstrated that GPER was expressed in purified human peripheral blood eosinophils both at the mRNA and protein levels. Although GPER agonist G-1 did not induce eosinophil chemotaxis or chemokinesis, preincubation with G-1 enhanced eotaxin (CCL11)-directed eosinophil chemotaxis. G-1 inhibited eosinophil spontaneous apoptosis and caspase-3 activities. The anti-apoptotic effect was not affected by the cAMP-phospodiesterase inhibitor rolipram or phosphoinositide 3-kinase inhibitors. In contrast to resting eosinophils, G-1 induced apoptosis and increased caspase-3 activities when eosinophils were co-stimulated with IL-5. No effect of G-1 was observed on eosinophil degranulation in terms of release of eosinophil-derived neurotoxin (EDN). The current study indicates the functional capacities of GPER on human eosinophils and also provides the previously unrecognized mechanisms of interaction between estrogen and allergic inflammation. Copyright © 2014 Elsevier B.V. All rights reserved.

  7. Synchronized integrin engagement and chemokine activation is crucial in neutrophil extracellular trap-mediated sterile inflammation

    NARCIS (Netherlands)

    Rossaint, Jan; Herter, Jan M.; van Aken, Hugo; Napirei, Markus; Döring, Yvonne; Weber, Christian; Soehnlein, Oliver; Zarbock, Alexander

    2014-01-01

    There is emerging evidence that neutrophil extracellular traps (NETs) play important roles in inflammatory processes. Here we report that neutrophils have to be simultaneously activated by integrin-mediated outside-in- and G-protein-coupled receptor (GPCR) signaling to induce NET formation in acute

  8. Diet-induced obesity mediated by the JNK/DIO2 signal transduction pathway

    Science.gov (United States)

    Vernia, Santiago; Cavanagh-Kyros, Julie; Barrett, Tamera; Jung, Dae Young; Kim, Jason K.; Davis, Roger J.

    2013-01-01

    The cJun N-terminal kinase (JNK) signaling pathway is a key mediator of metabolic stress responses caused by consuming a high-fat diet, including the development of obesity. To test the role of JNK, we examined diet-induced obesity in mice with targeted ablation of Jnk genes in the anterior pituitary gland. These mice exhibited an increase in the pituitary expression of thyroid-stimulating hormone (TSH), an increase in the blood concentration of thyroid hormone (T4), increased energy expenditure, and markedly reduced obesity compared with control mice. The increased amount of pituitary TSH was caused by reduced expression of type 2 iodothyronine deiodinase (Dio2), a gene that is required for T4-mediated negative feedback regulation of TSH expression. These data establish a molecular mechanism that accounts for the regulation of energy expenditure and the development of obesity by the JNK signaling pathway. PMID:24186979

  9. Wnt5a signaling is a substantial constituent in bone morphogenetic protein-2-mediated osteoblastogenesis