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Sample records for camp receptor protein

  1. cAMP biosensors applied in molecular pharmacological studies of G protein-coupled receptors

    DEFF Research Database (Denmark)

    Mathiesen, Jesper Mosolff; Vedel, Line; Bräuner-Osborne, Hans

    2013-01-01

    end-point assays for quantifying GPCR-mediated changes in intracellular cAMP levels exist. More recently, fluorescence resonance energy transfer (FRET)-based cAMP biosensors that can quantify intracellular cAMP levels in real time have been developed. These FRET-based cAMP biosensors have been used...... primarily in single cell FRET microscopy to monitor and visualize changes in cAMP upon GPCR activation. Here, a similar cAMP biosensor with a more efficient mCerulean/mCitrine FRET pair is described for use in the 384-well plate format. After cloning and expression in HEK293 cells, the biosensor...... is characterized in the 384-well plate format and used for measuring the signaling of the G(s)-coupled ß(2)-adrenergic receptor. The procedures described may be applied for other FRET-based biosensors in terms of characterization and conversion to the 384-well plate format....

  2. Identification of the subunit of cAMP receptor protein (CRP) that functionally interacts with CytR in CRP-CytR-mediated transcriptional repression

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    Meibom, K L; Kallipolitis, B H; Ebright, R H

    2000-01-01

    At promoters of the Escherichia coli CytR regulon, the cAMP receptor protein (CRP) interacts with the repressor CytR to form transcriptionally inactive CRP-CytR-promoter or (CRP)(2)-CytR-promoter complexes. Here, using "oriented heterodimer" analysis, we show that only one subunit of the CRP dime...

  3. Improving acetate tolerance of Escherichia coli by rewiring its global regulator cAMP receptor protein (CRP.

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    Huiqing Chong

    Full Text Available The presence of acetate exceeding 5 g/L is a major concern during E. coli fermentation due to its inhibitory effect on cell growth, thereby limiting high-density cell culture and recombinant protein production. Hence, engineered E. coli strains with enhanced acetate tolerance would be valuable for these bioprocesses. In this work, the acetate tolerance of E. coli was much improved by rewiring its global regulator cAMP receptor protein (CRP, which is reported to regulate 444 genes. Error-prone PCR method was employed to modify crp and the mutagenesis libraries (~3×10(6 were subjected to M9 minimal medium supplemented with 5-10 g/L sodium acetate for selection. Mutant A2 (D138Y was isolated and its growth rate in 15 g/L sodium acetate was found to be 0.083 h(-1, much higher than that of the control (0.016 h(-1. Real-time PCR analysis via OpenArray(® system revealed that over 400 CRP-regulated genes were differentially expressed in A2 with or without acetate stress, including those involved in the TCA cycle, phosphotransferase system, etc. Eight genes were chosen for overexpression and the overexpression of uxaB was found to lead to E. coli acetate sensitivity.

  4. Computational prediction of cAMP receptor protein (CRP binding sites in cyanobacterial genomes

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    Su Zhengchang

    2009-01-01

    Full Text Available Abstract Background Cyclic AMP receptor protein (CRP, also known as catabolite gene activator protein (CAP, is an important transcriptional regulator widely distributed in many bacteria. The biological processes under the regulation of CRP are highly diverse among different groups of bacterial species. Elucidation of CRP regulons in cyanobacteria will further our understanding of the physiology and ecology of this important group of microorganisms. Previously, CRP has been experimentally studied in only two cyanobacterial strains: Synechocystis sp. PCC 6803 and Anabaena sp. PCC 7120; therefore, a systematic genome-scale study of the potential CRP target genes and binding sites in cyanobacterial genomes is urgently needed. Results We have predicted and analyzed the CRP binding sites and regulons in 12 sequenced cyanobacterial genomes using a highly effective cis-regulatory binding site scanning algorithm. Our results show that cyanobacterial CRP binding sites are very similar to those in E. coli; however, the regulons are very different from that of E. coli. Furthermore, CRP regulons in different cyanobacterial species/ecotypes are also highly diversified, ranging from photosynthesis, carbon fixation and nitrogen assimilation, to chemotaxis and signal transduction. In addition, our prediction indicates that crp genes in modern cyanobacteria are likely inherited from a common ancestral gene in their last common ancestor, and have adapted various cellular functions in different environments, while some cyanobacteria lost their crp genes as well as CRP binding sites during the course of evolution. Conclusion The CRP regulons in cyanobacteria are highly diversified, probably as a result of divergent evolution to adapt to various ecological niches. Cyanobacterial CRPs may function as lineage-specific regulators participating in various cellular processes, and are important in some lineages. However, they are dispensable in some other lineages. The

  5. Enhancing E. coli tolerance towards oxidative stress via engineering its global regulator cAMP receptor protein (CRP.

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    Souvik Basak

    Full Text Available Oxidative damage to microbial hosts often occurs under stressful conditions during bioprocessing. Classical strain engineering approaches are usually both time-consuming and labor intensive. Here, we aim to improve E. coli performance under oxidative stress via engineering its global regulator cAMP receptor protein (CRP, which can directly or indirectly regulate redox-sensing regulators SoxR and OxyR, and other ~400 genes in E. coli. Error-prone PCR technique was employed to introduce modifications to CRP, and three mutants (OM1~OM3 were identified with improved tolerance via H(2O(2 enrichment selection. The best mutant OM3 could grow in 12 mM H(2O(2 with the growth rate of 0.6 h(-1, whereas the growth of wild type was completely inhibited at this H(2O(2 concentration. OM3 also elicited enhanced thermotolerance at 48°C as well as resistance against cumene hydroperoxide. The investigation about intracellular reactive oxygen species (ROS, which determines cell viability, indicated that the accumulation of ROS in OM3 was always lower than in WT with or without H(2O(2 treatment. Genome-wide DNA microarray analysis has shown not only CRP-regulated genes have demonstrated great transcriptional level changes (up to 8.9-fold, but also RpoS- and OxyR-regulated genes (up to 7.7-fold. qRT-PCR data and enzyme activity assay suggested that catalase (katE could be a major antioxidant enzyme in OM3 instead of alkyl hydroperoxide reductase or superoxide dismutase. To our knowledge, this is the first work on improving E. coli oxidative stress resistance by reframing its transcription machinery through its native global regulator. The positive outcome of this approach may suggest that engineering CRP can be successfully implemented as an efficient strain engineering alternative for E. coli.

  6. Improving ethanol tolerance of Escherichia coli by rewiring its global regulator cAMP receptor protein (CRP.

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    Huiqing Chong

    Full Text Available A major challenge in bioethanol fermentation is the low tolerance of the microbial host towards the end product bioethanol. Here we report to improve the ethanol tolerance of E. coli from the transcriptional level by engineering its global transcription factor cAMP receptor protein (CRP, which is known to regulate over 400 genes in E. coli. Three ethanol tolerant CRP mutants (E1- E3 were identified from error-prone PCR libraries. The best ethanol-tolerant strain E2 (M59T had the growth rate of 0.08 h(-1 in 62 g/L ethanol, higher than that of the control at 0.06 h(-1. The M59T mutation was then integrated into the genome to create variant iE2. When exposed to 150 g/l ethanol, the survival of iE2 after 15 min was about 12%, while that of BW25113 was <0.01%. Quantitative real-time reverse transcription PCR analysis (RT-PCR on 444 CRP-regulated genes using OpenArray® technology revealed that 203 genes were differentially expressed in iE2 in the absence of ethanol, whereas 92 displayed differential expression when facing ethanol stress. These genes belong to various functional groups, including central intermediary metabolism (aceE, acnA, sdhD, sucA, iron ion transport (entH, entD, fecA, fecB, and general stress response (osmY, rpoS. Six up-regulated and twelve down-regulated common genes were found in both iE2 and E2 under ethanol stress, whereas over one hundred common genes showed differential expression in the absence of ethanol. Based on the RT-PCR results, entA, marA or bhsA was knocked out in iE2 and the resulting strains became more sensitive towards ethanol.

  7. Expression of orphan G-protein coupled receptor GPR174 in CHO cells induced morphological changes and proliferation delay via increasing intracellular cAMP

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    Sugita, Kazuya; Yamamura, Chiaki; Tabata, Ken-ichi [Laboratory of Pharmacoinformatics, Graduate School of Ritsumeikan University, Kusatsu, Shiga 525-8577 (Japan); Fujita, Norihisa, E-mail: nori@ph.ritsumei.ac.jp [Laboratory of Pharmacoinformatics, Graduate School of Ritsumeikan University, Kusatsu, Shiga 525-8577 (Japan); School of Pharmacy, Ristumeikan University, Kusatsu, Shiga 525-8577 (Japan)

    2013-01-04

    Highlights: Black-Right-Pointing-Pointer Expression of GPR174 in CHO cells induces morphological changes and proliferation delay. Black-Right-Pointing-Pointer These are due to increase in intracellular cAMP concentration. Black-Right-Pointing-Pointer Lysophosphatidylserine was identified to stimulate GPR174 leading to activate ACase. Black-Right-Pointing-Pointer The potencies of fatty acid moiety on LysoPS were oleoyl Greater-Than-Or-Slanted-Equal-To stearoyl > palmitoyl. Black-Right-Pointing-Pointer We propose that GPR174 is a lysophosphatidylserine receptor. -- Abstract: We established cell lines that stably express orphan GPCR GPR174 using CHO cells, and studied physiological and pharmacological features of the receptor. GPR174-expressing cells showed cell-cell adhesion with localization of actin filaments to cell membrane, and revealed significant delay of cell proliferation. Since the morphological changes of GPR174-cells were very similar to mock CHO cells treated with cholera toxin, we measured the concentration of intracellular cAMP. The results showed the concentration was significantly elevated in GPR174-cells. By measuring intracellular cAMP concentration in GPR174-cells, we screened lipids and nucleotides to identify ligands for GPR174. We found that lysophosphatidylserine (LysoPS) stimulated increase in intracellular cAMP in a dose-dependent manner. Moreover, phosphorylation of Erk was elevated by LysoPS in GPR174 cells. These LysoPS responses were inhibited by NF449, an inhibitor of G{alpha}{sub s} protein. These results suggested that GPR174 was a putative LysoPS receptor conjugating with G{alpha}{sub s}, and its expression induced morphological changes in CHO cells by constitutively activating adenylyl cycles accompanied with cell conjunctions and delay of proliferation.

  8. Sphingosylphosphorylcholine antagonizes proton-sensing ovarian cancer G-protein-coupled receptor 1 (OGR1)-mediated inositol phosphate production and cAMP accumulation.

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    Mogi, Chihiro; Tomura, Hideaki; Tobo, Masayuki; Wang, Ju-Qiang; Damirin, Alatangaole; Kon, Junko; Komachi, Mayumi; Hashimoto, Kinji; Sato, Koichi; Okajima, Fumikazu

    2005-10-01

    Ovarian cancer G-protein-coupled receptor 1 (OGR1), previously proposed as a receptor for sphingosylphosphorylcholine (SPC), has recently been identified as a proton-sensing or extracellular pH-responsive G-protein-coupled receptor stimulating inositol phosphate production, reflecting the activation of phospholipase C. In the present study, we found that acidic pH stimulated cAMP accumulation, reflecting the activation of adenylyl cyclase, in addition to inositol phosphate production in OGR1-expressing cells. The cAMP response was hardly affected by the inhibition of phospholipase C. SPC inhibited the acidification-induced actions in a pH-dependent manner, while no OGR1-dependent agonistic action of SPC was observed. Thus, the dose-response curves of the proton-induced actions were shifted to the right in the presence of SPC regardless of stereoisoform. The antagonistic property was also observed for psychosine and glucosylsphingosine. In conclusion, OGR1 stimulation may lead to the activation of adenylyl cyclase in addition to phospholipase C in response to extracellular acidification but not to SPC. However, SPC and related lysolipids antagonize the proton-induced and OGR1-mediated actions.

  9. Regulation of cAMP Responsive Element Binding Protein 3-Like 1 (Creb3l1 Expression by Orphan Nuclear Receptor Nr4a1

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    Michael P. Greenwood

    2017-12-01

    Full Text Available Cyclic AMP (cAMP inducible transcription factor cAMP responsive element binding protein 3 like 1 (Creb3l1 is strongly activated in the hypothalamus in response to hyperosmotic cues such as dehydration (DH. We have recently shown that Creb3l1 expression is upregulated by cAMP pathways in vitro, however the exact mechanisms are not known. Here we show that increasing Creb3l1 transcription by raising cAMP levels in mouse pituitary AtT20 cells automatically initiates cleavage of Creb3l1, leading to a greater abundance of the transcriptionally active N-terminal portion. Inhibiting protein synthesis indicated that de novo protein synthesis of an intermediary transcription factor was required for Creb3l1 induction. Strategic mining of our microarray data from dehydrated rodent hypothalamus revealed four candidates, reduced to two by analysis of acute hyperosmotic-induced transcriptional activation profiles in the hypothalamus, and one, orphan nuclear receptor Nr4a1, by direct shRNA mediated silencing in AtT20 cells. We show that activation of Creb3l1 transcription by Nr4a1 involves interaction with a single NBRE site in the promoter region. The ability to activate Creb3l1 transcription by this pathway in vitro is dictated by the level of methylation of a CpG island within the proximal promoter/5′UTR of this gene. We thus identify a novel cAMP-Nr4a1-Creb3l1 transcriptional pathway in AtT20 cells and also, our evidence would suggest, in the hypothalamus.

  10. Extracellular receptor kinase and cAMP response element binding protein activation in the neonatal rat heart after perinatal cocaine exposure.

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    Sun, Lena S; Quamina, Aaron

    2004-12-01

    Prenatal exposure to cocaine has been shown to induce an increase in the myocardial expression and activation of the cAMP response binding protein (CREB), a transcriptional factor that has been shown to regulate gene expression. Several different kinases, including protein kinase A, calcium calmodulin kinase II, and mitogen-activated protein kinase can induce phosphorylation of CREB at serine 133, a necessary step for CREB activation. We examined whether the mitogen-activated protein kinase-extracellular receptor kinase (ERK) pathway may be involved in mediating the serine 133 CREB phosphorylation in cardiac nuclei after perinatal cocaine exposure. Pregnant rats were treated daily with saline or cocaine at 60 mg/kg (C60) by intragastric administration during the entire gestational period, and treatment was continued in the nursing dams after delivery until the time of the study. Nuclear extracts were isolated from hearts of 1-d- and 7-d-old neonatal rats. We performed immunoblotting experiments using an antibody that recognized CREB with phosphorylation specifically at the serine 133 site and an antibody that recognized both the phosphorylated and the unphosphorylated forms of CREB, as well as antibodies for total ERK, phospho-ERK, total ribosomal S6 kinase 1 (RSK1), RSK2, and phospho-RSK. We assessed the interaction of RSK with CREB or CREB-binding protein by performing co-immunoprecipitation experiments. We found that perinatal cocaine exposure increased both phospho-ERK and phospho-RSK expression, indicative of an increased activity of these two enzymes. Furthermore, we demonstrated that phospho-RSK was immunoprecipitated with CREB in all neonatal cardiac nuclei and that the greatest interaction was found in day 7 hearts after perinatal cocaine exposure. Our results thus illustrate that the ERK-RSK pathway was active in the postnatal rat heart at 1 and 7 d of age and that this pathway may mediate the increase in myocardial CREB activation after perinatal cocaine

  11. The activation of G protein-coupled estrogen receptor induces relaxation via cAMP as well as potentiates contraction via EGFR transactivation in porcine coronary arteries.

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    Xuan Yu

    Full Text Available Estrogen exerts protective effects against cardiovascular diseases in premenopausal women, but is associated with an increased risk of both coronary heart disease and stroke in older postmenopausal women. Studies have shown that activation of the G-protein-coupled estrogen receptor 1 (GPER can cause either relaxation or contraction of arteries. It is highly likely that these dual actions of GPER may contribute to the seemingly paradoxical effects of estrogen in regulating coronary artery function. The objective of this study was to test the hypothesis that activation of GPER enhances agonist-stimulated porcine coronary artery contraction via epidermal growth factor receptor (EGFR transactivation and its downstream extracellular signal-regulated kinases (ERK1/2 pathway. Isometric tension studies and western blot were performed to determine the effect of GPER activation on coronary artery contraction. Our findings demonstrated that G-1 caused concentration-dependent relaxation of ET-1-induced contraction, while pretreatment of arterial rings with G-1 significantly enhanced ET-1-induced contraction. GPER antagonist, G-36, significantly inhibited both the G-1-induced relaxation effect and G-1-enhanced ET-1 contraction. Gallein, a Gβγ inhibitor, significantly increased G-1-induced relaxation, yet inhibited G-1-enhanced ET-1-mediated contraction. Similarly, inhibition of EGFR with AG1478 or inhibition of Src with phosphatase 2 further increased G-1-induced relaxation responses in coronary arteries, but decreased G-1-enhanced ET-1-induced contraction. Western blot experiments in porcine coronary artery smooth muscle cells (PCASMC showed that G-1 increased tyrosine phosphorylation of EGFR, which was inhibited by AG-1478. Furthermore, enzyme-linked immunosorbent assays showed that the level of heparin-binding EGF (HB-EGF released by ET-1 treatment increased two-fold; whereas pre-incubation with G-1 further increased ET-1-induced HB-EGF release to four

  12. Endogenous 5-HT2C Receptors Phosphorylate the cAMP Response Element Binding Protein via Protein Kinase C-Promoted Activation of Extracellular-Regulated Kinases-1/2 in Hypothalamic mHypoA-2/10 Cells.

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    Lauffer, Lisa; Glas, Evi; Gudermann, Thomas; Breit, Andreas

    2016-07-01

    Serotonin 5-HT2C receptors (5-HT2CR) activate Gq proteins and are expressed in the central nervous system (CNS). 5-HT2CR regulate emotion, feeding, reward, or cognition and may serve as promising drug targets to treat psychiatric disorders or obesity. Owing to technical difficulties in isolating cells from the CNS and the lack of suitable cell lines endogenously expressing 5-HT2CR, our knowledge about this receptor subtype in native environments is rather limited. The hypothalamic mHypoA-2/10 cell line was recently established and resembles appetite-regulating hypothalamic neurons of the paraventricular nucleus (PVN), where 5-HT2CR have been detected in vivo. Therefore, we tested mHypoA-2/10 cells for endogenous 5-HT2CR expression. Serotonin or the 5-HT2CR preferential agonist WAY-161,503 initiated cAMP response element (CRE)-dependent gene transcription with EC50 values of 15.5 ± 9.8 and 1.1 ± 0.9 nM, respectively. Both responses were blocked by two unrelated 5-HT2CR-selective antagonists (SB-242,084, RS-102,221) but not by a 5-HT2AR (EMD-281,014) or 5-HT2BR (RS-127,455) antagonists. By single-cell calcium imaging, we found that serotonin and WAY-161,503 induced robust calcium transients, which were also blunted by both 5-HT2CR antagonists. Additionally we revealed, first, that 5-HT2CR induced CRE activation via protein kinase C (PKC)-mediated engagement of extracellular-regulated kinases-1/2 and, second, that intrinsic activity of WAY-161,503 was in the range of 0.3-0.5 compared with serotonin, defining the frequently used 5-HT2CR agonist as a partial agonist of endogenous 5-HT2CR. In conclusion, we have shown that hypothalamic mHypoA-2/10 cells endogenously express 5-HT2CR and thus are the first cell line in which to analyze 5-HT2CR pharmacology, signaling, and regulation in its natural environment. Copyright © 2016 by The American Society for Pharmacology and Experimental Therapeutics.

  13. Mechanism of cAMP Partial Agonism in Protein Kinase G (PKG).

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    VanSchouwen, Bryan; Selvaratnam, Rajeevan; Giri, Rajanish; Lorenz, Robin; Herberg, Friedrich W; Kim, Choel; Melacini, Giuseppe

    2015-11-27

    Protein kinase G (PKG) is a major receptor of cGMP and controls signaling pathways often distinct from those regulated by cAMP. Hence, the selective activation of PKG by cGMP versus cAMP is critical. However, the mechanism of cGMP-versus-cAMP selectivity is only limitedly understood. Although the C-terminal cyclic nucleotide-binding domain B of PKG binds cGMP with higher affinity than cAMP, the intracellular concentrations of cAMP are typically higher than those of cGMP, suggesting that the cGMP-versus-cAMP selectivity of PKG is not controlled uniquely through affinities. Here, we show that cAMP is a partial agonist for PKG, and we elucidate the mechanism for cAMP partial agonism through the comparative NMR analysis of the apo, cGMP-, and cAMP-bound forms of the PKG cyclic nucleotide-binding domain B. We show that although cGMP activation is adequately explained by a two-state conformational selection model, the partial agonism of cAMP arises from the sampling of a third, partially autoinhibited state. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  14. Mechanism of cAMP Partial Agonism in Protein Kinase G (PKG)*♦

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    VanSchouwen, Bryan; Selvaratnam, Rajeevan; Giri, Rajanish; Lorenz, Robin; Herberg, Friedrich W.; Kim, Choel; Melacini, Giuseppe

    2015-01-01

    Protein kinase G (PKG) is a major receptor of cGMP and controls signaling pathways often distinct from those regulated by cAMP. Hence, the selective activation of PKG by cGMP versus cAMP is critical. However, the mechanism of cGMP-versus-cAMP selectivity is only limitedly understood. Although the C-terminal cyclic nucleotide-binding domain B of PKG binds cGMP with higher affinity than cAMP, the intracellular concentrations of cAMP are typically higher than those of cGMP, suggesting that the cGMP-versus-cAMP selectivity of PKG is not controlled uniquely through affinities. Here, we show that cAMP is a partial agonist for PKG, and we elucidate the mechanism for cAMP partial agonism through the comparative NMR analysis of the apo, cGMP-, and cAMP-bound forms of the PKG cyclic nucleotide-binding domain B. We show that although cGMP activation is adequately explained by a two-state conformational selection model, the partial agonism of cAMP arises from the sampling of a third, partially autoinhibited state. PMID:26370085

  15. Opioid receptor activation triggering downregulation of cAMP improves effectiveness of anti-cancer drugs in treatment of glioblastoma.

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    Friesen, Claudia; Hormann, Inis; Roscher, Mareike; Fichtner, Iduna; Alt, Andreas; Hilger, Ralf; Debatin, Klaus-Michael; Miltner, Erich

    2014-01-01

    Glioblastoma are the most frequent and malignant human brain tumors, having a very poor prognosis. The enhanced radio- and chemoresistance of glioblastoma and the glioblastoma stem cells might be the main reason why conventional therapies fail. The second messenger cyclic AMP (cAMP) controls cell proliferation, differentiation, and apoptosis. Downregulation of cAMP sensitizes tumor cells for anti-cancer treatment. Opioid receptor agonists triggering opioid receptors can activate inhibitory Gi proteins, which, in turn, block adenylyl cyclase activity reducing cAMP. In this study, we show that downregulation of cAMP by opioid receptor activation improves the effectiveness of anti-cancer drugs in treatment of glioblastoma. The µ-opioid receptor agonist D,L-methadone sensitizes glioblastoma as well as the untreatable glioblastoma stem cells for doxorubicin-induced apoptosis and activation of apoptosis pathways by reversing deficient caspase activation and deficient downregulation of XIAP and Bcl-xL, playing critical roles in glioblastomas' resistance. Blocking opioid receptors using the opioid receptor antagonist naloxone or increasing intracellular cAMP by 3-isobutyl-1-methylxanthine (IBMX) strongly reduced opioid receptor agonist-induced sensitization for doxorubicin. In addition, the opioid receptor agonist D,L-methadone increased doxorubicin uptake and decreased doxorubicin efflux, whereas doxorubicin increased opioid receptor expression in glioblastomas. Furthermore, opioid receptor activation using D,L-methadone inhibited tumor growth significantly in vivo. Our findings suggest that opioid receptor activation triggering downregulation of cAMP is a promising strategy to inhibit tumor growth and to improve the effectiveness of anti-cancer drugs in treatment of glioblastoma and in killing glioblastoma stem cells.

  16. Dissecting direct and indirect readout of cAMP receptor protein DNA binding using an inosine and 2,6-diaminopurine in vitro selection system

    DEFF Research Database (Denmark)

    Lindemose, Søren; Nielsen, Peter E.; Møllegaard, Niels Erik

    2008-01-01

    The DNA interaction of the Escherichia coli cyclic AMP receptor protein (CRP) represents a typical example of a dual recognition mechanism exhibiting both direct and indirect readout. We have dissected the direct and indirect components of DNA recognition by CRP employing in vitro selection...... is functionally intact. The majority of the selected sites contain the natural consensus sequence TGTGAN(6)TCACA (i.e. TITIDN(6)TCDCD). Thus, direct readout of the consensus sequence is independent of minor groove conformation. Consequently, the indirect readout known to occur in the TG/CA base pair step (primary...... kink site) in the consensus sequence is not affected by I-D substitutions. In contrast, the flanking regions are selected as I/C rich sequences (mostly I-tracts) instead of A/T rich sequences which are known to strongly increase CRP binding, thereby demonstrating almost exclusive indirect readout...

  17. Calcium and cAMP signaling induced by gamma-hydroxybutyrate receptor(s) stimulation in NCB-20 neurons.

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    Coune, P; Taleb, O; Mensah-Nyagan, A G; Maitre, M; Kemmel, V

    2010-04-28

    The NCB-20 neurohybridoma cells differentiated with dibutyryl-cyclic-AMP represent an interesting model to study several components of the gamma-hydroxybutyrate (GHB) system in brain. In particular, an active Na(+)-dependent uptake and a depolarization-evoked release of GHB is expressed by these cells, together with high affinity specific binding sites for this substance. However, only little is known about cellular mechanisms following GHB receptor(s) stimulation in these neurons. Electrophysiological data indicate that GHB can differently affect Ca(2+) currents. L-type calcium channels were typically inhibited by GHB when NCB-20 cells were depolarized. In contrast, when NCB-20 cells were at resting potential, GHB induced a specific Ca(2+) entry through T-type calcium channels. In this study, we investigated the effect induced on cytosolic free Ca(2+) level and cAMP production by GHB receptor(s) stimulated with micromolar concentrations of GHB or structural analogues of GHB. Ca(2+) movements studied by cellular imaging were dose-dependently increased but disappeared for GHB concentrations >25 microM. In addition, nanomolar doses of GHB inhibited forskolin-stimulated adenylate cyclase. This effect was also rapidly desensitized at higher GHB concentrations. Acting as an antagonist, NCS-382 decreased GHB receptor(s) mediated cAMP and calcium signals. The agonist NCS-356 mimicked GHB effects which were not affected by the GABA(B) receptor antagonist CGP-55-845. Our results reveal the occurrence of Ca(2+)-dependent adenylate cyclase inhibition in NCB-20 neurons after GHB receptor(s) stimulation by GHB concentrations NCB-20 neurons of GHB receptors belonging to GPCR family that may recruit various G protein subtypes. Copyright 2010 IBRO. Published by Elsevier Ltd. All rights reserved.

  18. LdrP, a cAMP receptor protein/FNR family transcriptional regulator, serves as a positive regulator for the light-inducible gene cluster in the megaplasmid of Thermus thermophilus.

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    Takano, Hideaki; Agari, Yoshihiro; Hagiwara, Kenta; Watanabe, Ren; Yamazaki, Ryuta; Beppu, Teruhiko; Shinkai, Akeo; Ueda, Kenji

    2014-12-01

    LdrP (TT_P0055) (LitR-dependent regulatory protein) is one of the four cAMP receptor protein (CRP)/FNR family transcriptional regulators retained by the extremely thermophilic bacterium Thermus thermophilus. Previously, we reported that LdrP served as a positive regulator for the light-induced transcription of crtB, a carotenoid biosynthesis gene encoded on the megaplasmid of this organism. Here, we showed that LdrP also functions as an activator of the expression of genes clustered around the crtB gene under the control of LitR, an adenosyl B12-bound light-sensitive regulator. Transcriptome analysis revealed the existence of 19 LitR-dependent genes on the megaplasmid. S1 nuclease protection assay confirmed that the promoters preceding TT_P0044 (P44), TT_P0049 (P49) and TT_P0070 (P70) were activated upon illumination in the WT strain. An ldrP mutant lost the ability to activate P44, P49 and P70, whilst disruption of litR resulted in constitutive transcription from these promoters irrespective of illumination, indicating that these genes were photo-dependently regulated by LdrP and LitR. An in vitro transcription experiment demonstrated that LdrP directly activated mRNA synthesis from P44 and P70 by the Thermus RNA polymerase holocomplex. The present evidence indicated that LdrP was the positive regulator essential for the transcription of the T. thermophilus light-inducible cluster encoded on the megaplasmid. © 2014 The Authors.

  19. Novel mechanisms and signaling pathways of esophageal ulcer healing: the role of prostaglandin EP2 receptors, cAMP, and pCREB.

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    Ahluwalia, Amrita; Baatar, Dolgor; Jones, Michael K; Tarnawski, Andrzej S

    2014-09-15

    Clinical studies indicate that prostaglandins of E class (PGEs) may promote healing of tissue injury e.g., gastroduodenal and dermal ulcers. However, the precise roles of PGEs, their E-prostanoid (EP) receptors, signaling pathways including cAMP and cAMP response element-binding protein (CREB), and their relation to VEGF and angiogenesis in the tissue injury healing process remain unknown, forming the rationale for this study. Using an esophageal ulcer model in rats, we demonstrated that esophageal mucosa expresses predominantly EP2 receptors and that esophageal ulceration triggers an increase in expression of the EP2 receptor, activation of CREB (the downstream target of the cAMP signaling), and enhanced VEGF gene expression. Treatment of rats with misoprostol, a PGE1 analog capable of activating EP receptors, enhanced phosphorylation of CREB, stimulated VEGF expression and angiogenesis, and accelerated esophageal ulcer healing. In cultured human esophageal epithelial (HET-1A) cells, misoprostol increased intracellular cAMP levels (by 163-fold), induced phosphorylation of CREB, and stimulated VEGF expression. A cAMP analog (Sp-cAMP) mimicked, whereas an inhibitor of cAMP-dependent protein kinase A (Rp-cAMP) blocked, these effects of misoprostol. These results indicate that the EP2/cAMP/protein kinase A pathway mediates the stimulatory effect of PGEs on angiogenesis essential for tissue injury healing via the induction of CREB activity and VEGF expression.

  20. The lactate receptor, G-protein-coupled receptor 81/hydroxycarboxylic acid receptor 1

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    Morland, Cecilie; Lauritzen, Knut Huso; Puchades, Maja

    2015-01-01

    , and schizophrenia and in the deposition of phosphorylated tau protein in Alzheimer's disease. HCAR1 could serve to ameliorate these conditions and might also act through downstream mechanisms other than cAMP. Lactate exits cells through monocarboxylate transporters in an equilibrating manner and through astrocyte......We have proposed that lactate is a “volume transmitter” in the brain and underpinned this by showing that the lactate receptor, G-protein-coupled receptor 81 (GPR81, also known as HCA1 or HCAR1), which promotes lipid storage in adipocytes, is also active in the mammalian brain. This includes...... the cerebral neocortex and the hippocampus, where it can be stimulated by physiological concentrations of lactate and by the HCAR1 agonist 3,5-dihydroxybenzoate to reduce cAMP levels. Cerebral HCAR1 is concentrated on the postsynaptic membranes of excitatory synapses and also is enriched at the blood...

  1. Lipoic acid attenuates inflammation via cAMP and protein kinase A signaling.

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    Sonemany Salinthone

    2010-09-01

    Full Text Available Abnormal regulation of the inflammatory response is an important component of diseases such as diabetes, Alzheimer's disease and multiple sclerosis (MS. Lipoic acid (LA has been shown to have antioxidant and anti-inflammatory properties and is being pursued as a therapy for these diseases. We first reported that LA stimulates cAMP production via activation of G-protein coupled receptors and adenylyl cyclases. LA also suppressed NK cell activation and cytotoxicity. In this study we present evidence supporting the hypothesis that the anti-inflammatory properties of LA are mediated by the cAMP/PKA signaling cascade. Additionally, we show that LA oral administration elevates cAMP levels in MS subjects.We determined the effects of LA on IL-6, IL-17 and IL-10 secretion using ELISAs. Treatment with 50 µg/ml and 100 µg/ml LA significantly reduced IL-6 levels by 19 and 34%, respectively, in T cell enriched PBMCs. IL-17 levels were also reduced by 35 and 50%, respectively. Though not significant, LA appeared to have a biphasic effect on IL-10 production. Thymidine incorporation studies showed LA inhibited T cell proliferation by 90%. T-cell activation was reduced by 50% as measured by IL-2 secretion. Western blot analysis showed that LA treatment increased phosphorylation of Lck, a downstream effector of protein kinase A. Pretreatment with a peptide inhibitor of PKA, PKI, blocked LA inhibition of IL-2 and IFN gamma production, indicating that PKA mediates these responses. Oral administration of 1200 mg LA to MS subjects resulted in increased cAMP levels in PBMCs four hours after ingestion. Average cAMP levels in 20 subjects were 43% higher than baseline.Oral administration of LA in vivo resulted in significant increases in cAMP concentration. The anti-inflammatory effects of LA are mediated in part by the cAMP/PKA signaling cascade. These novel findings enhance our understanding of the mechanisms of action of LA.

  2. A Universal Stress Protein (USP) in Mycobacteria Binds cAMP

    Science.gov (United States)

    Banerjee, Arka; Adolph, Ramona S.; Gopalakrishnapai, Jayashree; Kleinboelting, Silke; Emmerich, Christiane; Steegborn, Clemens; Visweswariah, Sandhya S.

    2015-01-01

    Mycobacteria are endowed with rich and diverse machinery for the synthesis, utilization, and degradation of cAMP. The actions of cyclic nucleotides are generally mediated by binding of cAMP to conserved and well characterized cyclic nucleotide binding domains or structurally distinct cGMP-specific and -regulated cyclic nucleotide phosphodiesterase, adenylyl cyclase, and E. coli transcription factor FhlA (GAF) domain-containing proteins. Proteins with cyclic nucleotide binding and GAF domains can be identified in the genome of mycobacterial species, and some of them have been characterized. Here, we show that a significant fraction of intracellular cAMP is bound to protein in mycobacterial species, and by using affinity chromatography techniques, we identify specific universal stress proteins (USP) as abundantly expressed cAMP-binding proteins in slow growing as well as fast growing mycobacteria. We have characterized the biochemical and thermodynamic parameters for binding of cAMP, and we show that these USPs bind cAMP with a higher affinity than ATP, an established ligand for other USPs. We determined the structure of the USP MSMEG_3811 bound to cAMP, and we confirmed through structure-guided mutagenesis, the residues important for cAMP binding. This family of USPs is conserved in all mycobacteria, and we suggest that they serve as “sinks” for cAMP, making this second messenger available for downstream effectors as and when ATP levels are altered in the cell. PMID:25802331

  3. Regulation of antidepressant activity by cAMP response element binding proteins.

    Science.gov (United States)

    Conti, Alana C; Blendy, Julie A

    2004-10-01

    Depression is a clinically and biologically heterogeneous disease that is one of the most prevalent and costly psychiatric disorders. It is the leading cause of disability regarding job performance and burden on family members in the United States and worldwide. Although the therapeutic efficacy of antidepressant drugs has been recognized for years, the exact molecular mechanisms of action remain elusive, making the systematic approach to the development of new drugs difficult. The acute increases in levels of monoamines brought about by various classes of antidepressants cannot account for the requirement of repeated, chronic administration for up to 2-6 wk before treatment benefits become evident. Furthermore, despite their efficacy, current antidepressant drugs improve symptoms in only 60% of patients treated. The development of new and better therapies depends on a thorough understanding of the neurobiology of depression and the molecular mechanisms underlying antidepressant drug action. Early studies focusing on alterations in the levels of receptors and second messengers helped define the important signaling pathways initiated by these drugs, whereas recent molecular studies suggested that long-term adaptations in cellular signaling mechanisms may be required for the onset and/or maintenance of antidepressant effects. Attention has now focused on downstream targets of Ca++ and cyclic adenosine monophosphate (cAMP) in the cell, such as the activation of transcription factors. This article discusses the transcription factor cAMP response element binding protein and a related protein, cyclic AMP response element modulator, and their roles as molecular mediators of antidepressant action.

  4. Propranolol Targets Hemangioma Stem Cells via cAMP and Mitogen-Activated Protein Kinase Regulation.

    Science.gov (United States)

    Munabi, Naikhoba C O; England, Ryan W; Edwards, Andrew K; Kitajewski, Alison A; Tan, Qian Kun; Weinstein, Andrew; Kung, Justin E; Wilcox, Maya; Kitajewski, Jan K; Shawber, Carrie J; Wu, June K

    2016-01-01

    Infantile hemangiomas (IHs) are the most common vascular tumor and arise from a hemangioma stem cell (HemSC). Propranolol has proved efficacious for problematic IHs. Propranolol is a nonselective β-adrenergic receptor (βAR) antagonist that can lower cAMP levels and activate the mitogen-activated protein kinase (MAPK) pathway downstream of βARs. We found that HemSCs express β1AR and β2AR in proliferating IHs and determined the role of these βARs and the downstream pathways in mediating propranolol's effects. In isolated HemSCs, propranolol suppressed cAMP levels and activated extracellular signal-regulated kinase (ERK)1/2 in a dose-dependent fashion. Propranolol, used at doses of Propranolol at ≥10(-5) M reduced cAMP levels and activated ERK1/2, and this correlated with HemSC apoptosis and cytotoxicity at ≥10(-4) M. Stimulation with a βAR agonist, isoprenaline, promoted HemSC proliferation and rescued the antiproliferative effects of propranolol, suggesting that propranolol inhibits βAR signaling in HemSCs. Treatment with a cAMP analog or a MAPK inhibitor partially rescued the HemSC cell viability suppressed by propranolol. A selective β2AR antagonist mirrored propranolol's effects on HemSCs in a dose-dependent fashion, and a selective β1AR antagonist had no effect, supporting a role for β2AR signaling in IH pathobiology. In a mouse model of IH, propranolol reduced the vessel caliber and blood flow assessed by ultrasound Doppler and increased activation of ERK1/2 in IH cells. We have thus demonstrated that propranolol acts on HemSCs in IH to suppress proliferation and promote apoptosis in a dose-dependent fashion via β2AR perturbation, resulting in reduced cAMP and MAPK activation. The present study investigated the action of propranolol in infantile hemangiomas (IHs). IHs are the most common vascular tumor in children and have been proposed to arise from a hemangioma stem cell (HemSC). Propranolol, a nonselective β-adrenergic receptor (

  5. Constellation of HCN channels and cAMP regulating proteins in dendritic spines of the primate prefrontal cortex: potential substrate for working memory deficits in schizophrenia.

    Science.gov (United States)

    Paspalas, Constantinos D; Wang, Min; Arnsten, Amy F T

    2013-07-01

    Schizophrenia associates with impaired prefrontal cortical (PFC) function and alterations in cyclic AMP (cAMP) signaling pathways. These include genetic insults to disrupted-in-schizophrenia (DISC1) and phosphodiesterases (PDE4s) regulating cAMP hydrolysis, and increased dopamine D1 receptor (D1R) expression that elevates cAMP. We used immunoelectron microscopy to localize DISC1, PDE4A, PDE4B, and D1R in monkey PFC and to view spatial interactions with hyperpolarization-activated cyclic nucleotide-gated (HCN) channels that gate network inputs when opened by cAMP. Physiological interactions between PDE4s and HCN channels were tested in recordings of PFC neurons in monkeys performing a spatial working memory task. The study reveals a constellation of cAMP-related proteins (DISC1, PDE4A, and D1R) and HCN channels next to excitatory synapses and the spine neck in thin spines of superficial PFC, where working memory microcircuits interconnect and spine loss is most evident in schizophrenia. In contrast, channels in dendrites were distant from synapses and cAMP-related proteins, and were associated with endosomal trafficking. The data suggest that a cAMP signalplex is selectively positioned in the spines to gate PFC pyramidal cell microcircuits. Single-unit recordings confirmed physiological interactions between cAMP and HCN channels, consistent with gating actions. These data may explain why PFC networks are especially vulnerable to genetic insults that dysregulate cAMP signaling.

  6. Prostaglandin E2 Inhibits Histamine-Evoked Ca2+ Release in Human Aortic Smooth Muscle Cells through Hyperactive cAMP Signaling Junctions and Protein Kinase A.

    Science.gov (United States)

    Taylor, Emily J A; Pantazaka, Evangelia; Shelley, Kathryn L; Taylor, Colin W

    2017-11-01

    In human aortic smooth muscle cells, prostaglandin E2 (PGE2) stimulates adenylyl cyclase (AC) and attenuates the increase in intracellular free Ca2+ concentration evoked by activation of histamine H1 receptors. The mechanisms are not resolved. We show that cAMP mediates inhibition of histamine-evoked Ca2+ signals by PGE2 Exchange proteins activated by cAMP were not required, but the effects were attenuated by inhibition of cAMP-dependent protein kinase (PKA). PGE2 had no effect on the Ca2+ signals evoked by protease-activated receptors, heterologously expressed muscarinic M3 receptors, or by direct activation of inositol 1,4,5-trisphosphate (IP3) receptors by photolysis of caged IP3 The rate of Ca2+ removal from the cytosol was unaffected by PGE2, but PGE2 attenuated histamine-evoked IP3 accumulation. Substantial inhibition of AC had no effect on the concentration-dependent inhibition of Ca2+ signals by PGE2 or butaprost (to activate EP2 receptors selectively), but it modestly attenuated responses to EP4 receptors, activation of which generated less cAMP than EP2 receptors. We conclude that inhibition of histamine-evoked Ca2+ signals by PGE2 occurs through "hyperactive signaling junctions," wherein cAMP is locally delivered to PKA at supersaturating concentrations to cause uncoupling of H1 receptors from phospholipase C. This sequence allows digital signaling from PGE2 receptors, through cAMP and PKA, to histamine-evoked Ca2+ signals. Copyright © 2017 by The Author(s).

  7. cAMP-Induced Desensitization of Surface cAMP Receptors in Dictyostelium : Different Second Messengers Mediate Receptor Phosphorylation, Loss of Ligand Binding, Degradation of Receptor, and Reduction of Receptor mRNA Levels

    NARCIS (Netherlands)

    Haastert, Peter J.M. van; Wang, Mei; Bominaar, Anthony A.; Devreotes, Peter N.; Schaap, Pauline

    Surface cAMP receptors on Dictyostelium cells are linked to several second messenger systems and mediate multiple physiological responses, including chemotaxis and differentiation. Activation of the receptor also triggers events which desensitize signal transduction. These events include the

  8. Chemotactic antagonists of cAMP inhibit Dictyostelium phospholipase C

    NARCIS (Netherlands)

    Bominaar, Anthony A.; Haastert, Peter J.M. van

    In Dictyostelium discoideum extracellular cAMP induces chemotaxis via a transmembrane signal transduction cascade consisting of surface cAMP receptors, G-proteins and effector enzymes including adenylyl cyclase, guanylyl cyclase and phospholipase C. Previously it was demonstrated that some cAMP

  9. Exchange Protein Activated by cAMP Enhances Long-Term Memory Formation Independent of Protein Kinase A

    Science.gov (United States)

    Ma, Nan; Abel, Ted; Hernandez, Pepe J.

    2009-01-01

    It is well established that cAMP signaling within neurons plays a major role in the formation of long-term memories--signaling thought to proceed through protein kinase A (PKA). However, here we show that exchange protein activated by cAMP (Epac) is able to enhance the formation of long-term memory in the hippocampus and appears to do so…

  10. The Steroid Hormone 20-Hydroxyecdysone Enhances Gene Transcription through the cAMP Response Element-binding Protein (CREB) Signaling Pathway*

    OpenAIRE

    Jing, Yu-Pu; Wang, Di; Han, Xiao-Lin; Dong, Du-Juan; Wang, Jin-Xing; Zhao, Xiao-Fan

    2016-01-01

    Animal steroid hormones regulate gene transcription through genomic pathways by binding to nuclear receptors. These steroid hormones also rapidly increase intracellular calcium and cyclic adenosine monophosphate (cAMP) levels and activate the protein kinase C (PKC) and protein kinase A (PKA) nongenomic pathways. However, the function and mechanism of the nongenomic pathways of the steroid hormones are unclear, and the relationship between the PKC and PKA pathways is also unclear. We propose t...

  11. Exchange Protein Directly Activated by cAMP (EPAC) Regulates Neuronal Polarization through Rap1B

    NARCIS (Netherlands)

    Munoz-Llancao, Pablo; Henriquez, Daniel R.; Wilson, Carlos; Bodaleo, Felipe; Boddeke, Erik W.; Lezoualc'h, Frank; Schmidt, Martina; Gonzalez-Billault, Christian

    2015-01-01

    Acquisition of neuronal polarity is a complex process involving cellular and molecular events. The second messenger cAMP is involved in axonal specification through activation of protein kinase A. However, an alternative cAMP-dependent mechanism involves the exchange protein directly activated by

  12. Down-regulation of protein kinase C by parathyroid hormone and mezerein differentially modulates cAMP production and phosphate transport in opossum kidney cells.

    Science.gov (United States)

    Cole, J A

    1997-08-01

    We examined the effects of prolonged exposure to parathyroid hormone (PTH) and the protein kinase C (PKC) activator mezerein (MEZ) on cyclic adenosine monophosphate (cAMP) production, PKC activity, and Na(+)-dependent phosphate (Na/Pi) transport in an opossum kidney cell line (OK/E). A 5 minute exposure to PTH stimulated, while a 6 h incubation reduced, cAMP production, Na/Pi transport was maximally inhibited under desensitizing conditions and was not affected by reintroduction of the hormone. MEZ pretreatment (6 h) enhanced PTH-, cholera toxin (CTX)-, and forskolin (FSK)-stimulated cAMP production, suggesting enhanced Gs alpha coupling and increased adenylyl cyclase activity. However, PKA- and PKC-dependent regulation of Na/Pi were blocked in MEZ-treated cells. The PTH-induced decrease in cAMP production was associated with a reduction in membrane-associated PKC activity while MEZ-induced increases in cAMP production were accompanied by decreases in membrane and cytosolic PKC activity. Enhanced cAMP production was not accompanied by significant changes in PTH/PTH related peptide (PTHrP) receptor affinity or number, nor was the loss of Na/Pi transport regulation associated with changes in PKA activity. The results indicate that down-regulation of PKC by PTH or MEZ differentially modulates cAMP production and regulation of Na/Pi transport. The distinct effects of PTH and MEZ on PKC activity suggest that agonist-specific activation and/or down-regulation of PKC isozyme(s) may be involved in the observed changes in cAMP production and Na/Pi transport.

  13. Identification of a novel human glucagon receptor promoter: regulation by cAMP and PGC-1alpha

    DEFF Research Database (Denmark)

    Mortensen, Ole Hartvig; Dichmann, Darwin Sorento; Abrahamsen, Niels

    2007-01-01

    inhibitory effects of cAMP on glucagon receptor mRNA expression is mediated at the level of gene transcription. The cAMP-mediated downregulation of the proximal promoter was examined by deletion analysis in the human hepatoma cell line HepG2 and the cAMP responsiveness was found to be located in a region...... between 1051 and 1016 base pairs upstream of the transcription start site, which contains several putative cAMP responsive elements. Expression of peroxisome proliferator-activated receptor gamma coactivator 1alpha (PGC-1alpha), known to be upregulated in the liver by fasting, was found to abolish the c......AMP-dependent downregulation of glucagon receptor mRNA expression in vitro, whereas overexpression of PGC-1beta had no effect....

  14. Glial-specific cAMP response of the glial fibrillary acidic protein gene cell lines.

    OpenAIRE

    Kaneko, R; Hagiwara, N; Leader, K; Sueoka, N

    1994-01-01

    Expression of the rat glial fibrillary acidic protein (GFAP) gene is responsive to the intracellular level of cAMP. We have examined the sequence 5'-upstream of the transcription start site of the rat GFAP-encoding gene to determine the elements responsible for regulating the cAMP response. The RT4 cell lines consist of a neural stem-cell type RT4-AC and its three derivative cell types, one glial-cell type, RT4-D, and two neuronal-cell types, RT4-B and RT4-E. GFAP is expressed in the stem-cel...

  15. Persistent cAMP-signals triggered by internalized G-protein-coupled receptors.

    Directory of Open Access Journals (Sweden)

    Davide Calebiro

    2009-08-01

    Full Text Available G-protein-coupled receptors (GPCRs are generally thought to signal to second messengers like cyclic AMP (cAMP from the cell surface and to become internalized upon repeated or prolonged stimulation. Once internalized, they are supposed to stop signaling to second messengers but may trigger nonclassical signals such as mitogen-activated protein kinase (MAPK activation. Here, we show that a GPCR continues to stimulate cAMP production in a sustained manner after internalization. We generated transgenic mice with ubiquitous expression of a fluorescent sensor for cAMP and studied cAMP responses to thyroid-stimulating hormone (TSH in native, 3-D thyroid follicles isolated from these mice. TSH stimulation caused internalization of the TSH receptors into a pre-Golgi compartment in close association with G-protein alpha(s-subunits and adenylyl cyclase III. Receptors internalized together with TSH and produced downstream cellular responses that were distinct from those triggered by cell surface receptors. These data suggest that classical paradigms of GPCR signaling may need revision, as they indicate that cAMP signaling by GPCRs may occur both at the cell surface and from intracellular sites, but with different consequences for the cell.

  16. Antagonists of chemoattractants reveal separate receptors for cAMP, folic acid and pterin in Dictyostelium

    NARCIS (Netherlands)

    Haastert, Peter J.M. van; Wit, René J.W. de; Konijn, Theo M.

    1982-01-01

    Adenosine 3’,5’-monophosphate (cAMP), folic acid and pterin are chemoattractants in the cellular slime molds. The cAMP analog, 3’-amino-cAMP, inhibits a chemotactic reaction to cAMP at a concentration at which the analog is chemotactically inactive. The antagonistic effect of 3’-amino-cAMP on the

  17. Association of dopamine D(3) receptors with actin-binding protein 280 (ABP-280).

    Science.gov (United States)

    Li, Ming; Li, Chuanyu; Weingarten, Paul; Bunzow, James R; Grandy, David K; Zhou, Qun Yong

    2002-03-01

    Proteins that bind to G protein-coupled receptors have been identified as regulators of receptor localization and signaling. In our previous studies, a cytoskeletal protein, actin-binding protein 280 (ABP-280), was found to associate with the third cytoplasmic loop of dopamine D(2) receptors. In this study, we demonstrate that ABP-280 also interacts with dopamine D(3) receptors, but not with D(4) receptors. Similar to the dopamine D(2) receptor, the D(3)/ABP-280 association is of signaling importance. In human melanoma M2 cells lacking ABP-280, D(3) receptors were unable to inhibit forskolin-stimulated cyclic AMP (cAMP) production significantly. D(4) receptors, however, exhibited a similar degree of inhibition of forskolin-stimulated cAMP production in ABP-280-deficient M2 cells and ABP-280-replent M2 subclones (A7 cells). Further experiments revealed that the D(3)/ABP-280 interaction was critically dependent upon a 36 amino acid carboxyl domain of the D(3) receptor third loop, which is conserved in the D(2) receptor but not in the D(4) receptor. Our results demonstrate a subtype-specific regulation of dopamine D(2)-family receptor signaling by the cytoskeletal protein ABP-280.

  18. Estradiol up-regulates L-type Ca2+channels via membrane-bound estrogen receptor / Phosphoinositide-3kinase / Akt / cAMP response element-binding protein signaling pathway.

    Science.gov (United States)

    Yang, Xiaoyan; Mao, Xiaofang; Xu, Gao; Xing, Shasha; Chattopadhyay, Ansuman; Jin, Si; Salama, Guy

    2018-01-09

    In long QT type-2 (LQT2), women are more prone to lethal arrhythmias called Torsade de Pointes (TdP) than men. We previously reported that 17-β-estradiol (E2) upregulates L-type Ca 2+ -channels and current (I Ca,L ) (∼30%) in rabbit ventricular myocytes by a classical genomic-mechanism mediated by estrogen-receptor-α (ER)α. In LQT2 ( I Kr -blockade or bradycardia), the higher Ca 2+ influx via I Ca,L , causes Ca 2+ -overload, spontaneous sarcoplasmic reticulum Ca 2+ -release, and re-activation of I Ca,L that trigger early afterdepolarizations (EADs) and TdP. The molecular mechanisms whereby E2 upregulates I Ca,L are poorly understood and are now investigated. H9C2 and rat myocytes were incubated with E2, ±ER antagonist, or inhibitors of downstream transcription factors 24 hours, followed by Western blots of Cav1.2α1C and voltage-clamp measurements of I Ca,L . Incubation of H9C2 cells with E2 (10∼100 nM) increased I Ca,L density and Cav1.2α1C expression which were suppressed by the ER-antagonist ICI-182,780 (1μM). Enhanced I Ca,L and Cav1.2α1C expression by E2 was suppressed by inhibitors of Pi3K (LY294002=30μM; pL via plasma-membrane ER, and activating a Pi3K, Akt and CREB signaling. The promoter regions of CACNA1C gene (human-rabbit-rat) contain adjacent/overlapping binding-sites for p-CREB and ERα which suggest a synergistic regulation by these pathways. Copyright © 2018. Published by Elsevier Inc.

  19. Identification of small-molecule agonists of human relaxin family receptor 1 (RXFP1) by using a homogenous cell-based cAMP assay.

    Science.gov (United States)

    Chen, Catherine Z; Southall, Noel; Xiao, Jingbo; Marugan, Juan J; Ferrer, Marc; Hu, Xin; Jones, Raisa E; Feng, Shu; Agoulnik, Irina U; Zheng, Wei; Agoulnik, Alexander I

    2013-07-01

    The relaxin hormone is involved in a variety of biological functions, including female reproduction and parturition, as well as regulation of cardiovascular, renal, pulmonary, and hepatic functions. It regulates extracellular matrix remodeling, cell invasiveness, proliferation, differentiation, and overall tissue homeostasis. The G protein-coupled receptor (GPCR) relaxin family receptor 1 (RXFP1) is a cognate relaxin receptor that mainly signals through cyclic AMP second messenger. Although agonists of the receptor could have a wide range of pharmacologic utility, until now there have been no reported small-molecule agonists for relaxin receptors. Here, we report the development of a quantitative high-throughput platform for an RXFP1 agonist screen based on homogenous cell-based HTRF cyclic AMP (cAMP) assay technology. Two small molecules of similar structure were independently identified from a screen of more than 365 677 compounds. Neither compound showed activity in a counterscreen with HEK293T cells transfected with an unrelated GPCR vasopressin 1b receptor. These small-molecule agonists also demonstrated selectivity against the RXFP2 receptor, providing a basis for future medicinal chemistry optimization of selective relaxin receptor agonists.

  20. D1-like dopamine receptors downregulate Na+-K+-ATPase activity and increase cAMP production in the posterior gills of the blue crab Callinectes sapidus.

    Science.gov (United States)

    Arnaldo, Francis B; Villar, Van Anthony M; Konkalmatt, Prasad R; Owens, Shaun A; Asico, Laureano D; Jones, John E; Yang, Jian; Lovett, Donald L; Armando, Ines; Jose, Pedro A; Concepcion, Gisela P

    2014-09-15

    Dopamine-mediated regulation of Na(+)-K(+)-ATPase activity in the posterior gills of some crustaceans has been reported to be involved in osmoregulation. The dopamine receptors of invertebrates are classified into three groups based on their structure and pharmacology: D1- and D2-like receptors and a distinct invertebrate receptor subtype (INDR). We tested the hypothesis that a D1-like receptor is expressed in the blue crab Callinectes sapidus and regulates Na(+)-K(+)-ATPase activity. RT-PCR, using degenerate primers, showed the presence of D1βR mRNA in the posterior gill. The blue crab posterior gills showed positive immunostaining for a dopamine D5 receptor (D5R or D1βR) antibody in the basolateral membrane and cytoplasm. Confocal microscopy showed colocalization of Na(+)-K(+)-ATPase and D1βR in the basolateral membrane. To determine the effect of D1-like receptor stimulation on Na(+)-K(+)-ATPase activity, intact crabs acclimated to low salinity for 6 days were given an intracardiac infusion of the D1-like receptor agonist fenoldopam, with or without the D1-like receptor antagonist SCH23390. Fenoldopam increased cAMP production twofold and decreased Na(+)-K(+)-ATPase activity by 50% in the posterior gills. This effect was blocked by coinfusion with SCH23390, which had no effect on Na(+)-K(+)-ATPase activity by itself. Fenoldopam minimally decreased D1βR protein expression (10%) but did not affect Na(+)-K(+)-ATPase α-subunit protein expression. This study shows the presence of functional D1βR in the posterior gills of euryhaline crabs chronically exposed to low salinity and highlights the evolutionarily conserved function of the dopamine receptors on sodium homeostasis. Copyright © 2014 the American Physiological Society.

  1. Dissociations in the effects of β2-adrenergic receptor agonists on cAMP formation and superoxide production in human neutrophils: support for the concept of functional selectivity.

    Directory of Open Access Journals (Sweden)

    Irena Brunskole Hummel

    Full Text Available In neutrophils, activation of the β2-adrenergic receptor (β2AR, a Gs-coupled receptor, inhibits inflammatory responses, which could be therapeutically exploited. The aim of this study was to evaluate the effects of various β2AR ligands on adenosine-3',5'-cyclic monophosphate (cAMP accumulation and N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP-induced superoxide anion (O2(•- production in human neutrophils and to probe the concept of ligand-specific receptor conformations (also referred to as functional selectivity or biased signaling in a native cell system. This is an important question because so far, evidence for functional selectivity has been predominantly obtained with recombinant systems, due to the inherent difficulties to genetically manipulate human native cells. cAMP concentration was determined by HPLC/tandem mass spectrometry, and O2(•- formation was assessed by superoxide dismutase-inhibitable reduction of ferricytochrome c. β2AR agonists were generally more potent in inhibiting fMLP-induced O2(•- production than in stimulating cAMP accumulation. (--Ephedrine and dichloroisoproterenol were devoid of any agonistic activity in the cAMP assay, but partially inhibited fMLP-induced O2(•- production. Moreover, (--adrenaline was equi-efficacious in both assays whereas the efficacy of salbutamol was more than two-fold higher in the O2(•- assay. Functional selectivity was visualized by deviations of ligand potencies and efficacies from linear correlations for various parameters. We obtained no evidence for involvement of protein kinase A in the inhibition of fMLP-induced O2(•- production after β2AR-stimulation although cAMP-increasing substances inhibited O2(•- production. Taken together, our data corroborate the concept of ligand-specific receptor conformations with unique signaling capabilities in native human cells and suggest that the β2AR inhibits O2(•- production in a cAMP-independent manner.

  2. Broncho Vaxom (OM-85) modulates rhinovirus docking proteins on human airway epithelial cells via Erk1/2 mitogen activated protein kinase and cAMP.

    Science.gov (United States)

    Roth, Michael; Pasquali, Christian; Stolz, Daiana; Tamm, Michael

    2017-01-01

    Bronchial epithelial cells (BEC) are primary target for Rhinovirus infection through attaching to cell membrane proteins. OM-85, a bacterial extract, improves recovery of asthma and COPD patients after viral infections, but only part of the mechanism was addressed, by focusing on defined immune cells. We therefore determined the effect of OM-85 on isolated primary human BEC of controls (n = 8), asthma patients (n = 10) and COPD patients (n = 9). BEC were treated with OM-85 alone (24 hours) or infected with Rhinovirus. BEC survival was monitored by manual cell counting and Rhinovirus replication by lytic activity. Immuno-blotting and ELISA were used to determine the expression of Rhinovirus interacting proteins: intracellular adhesion molecule (ICAM), major histocompatibility complex class II (MHC-2), complement component C1q receptor (C1q-R), inducible T-Cell co-stimulator (ICOS), its ligand ICOSL, and myeloid differentiation primary response gene 88 (Myd88); as well as for signal transducers Erk1/2, p38, JNK mitogen activated protein kinases MAPK), and cAMP. OM-85 significantly reduced Rhinovirus-induced BEC death and virus replication. OM-85 significantly increased the expression of virus interacting proteins C1q-R and β-defensin in all 3 probes and groups, which was prevented by either Erk1/2 MAPK or cAMP inhibition. In addition, OM-85 significantly reduced Rhinovirus induced expression of ICAM1 involving p38 MAPK. In BEC OM-85 had no significant effect on the expression of ICOS, ICOSL and MHC-2 membrane proteins nor on the adaptor protein MyD88. The OM-85-induced increased of C1q-R and β-defensin, both important for antigen presentation and phagocytosis, supports its activity in host cell's defence against Rhinovirus infection.

  3. New kids on the block: The Popeye domain containing (POPDC) protein family acting as a novel class of cAMP effector proteins in striated muscle.

    Science.gov (United States)

    Brand, Thomas; Schindler, Roland

    2017-12-01

    The cyclic 3',5'-adenosine monophosphate (cAMP) signalling pathway constitutes an ancient signal transduction pathway present in prokaryotes and eukaryotes. Previously, it was thought that in eukaryotes three effector proteins mediate cAMP signalling, namely protein kinase A (PKA), exchange factor directly activated by cAMP (EPAC) and the cyclic-nucleotide gated channels. However, recently a novel family of cAMP effector proteins emerged and was termed the Popeye domain containing (POPDC) family, which consists of three members POPDC1, POPDC2 and POPDC3. POPDC proteins are transmembrane proteins, which are abundantly present in striated and smooth muscle cells. POPDC proteins bind cAMP with high affinity comparable to PKA. Presently, their biochemical activity is poorly understood. However, mutational analysis in animal models as well as the disease phenotype observed in patients carrying missense mutations suggests that POPDC proteins are acting by modulating membrane trafficking of interacting proteins. In this review, we will describe the current knowledge about this gene family and also outline the apparent gaps in our understanding of their role in cAMP signalling and beyond. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

  4. Function of the cytoplasmic tail of human calcitonin receptor-like receptor in complex with receptor activity-modifying protein 2

    Energy Technology Data Exchange (ETDEWEB)

    Kuwasako, Kenji, E-mail: kuwasako@fc.miyazaki-u.ac.jp [Frontier Science Research Center, University of Miyazaki, 5200 Kihara, Kiyotake, Miyazaki 889-1692 (Japan); Kitamura, Kazuo; Nagata, Sayaka; Hikosaka, Tomomi [Division of Circulation and Body Fluid Regulation, Faculty of Medicine, University of Miyazaki, 5200 Kihara, Kiyotake, Miyazaki 889-1692 (Japan); Kato, Johji [Frontier Science Research Center, University of Miyazaki, 5200 Kihara, Kiyotake, Miyazaki 889-1692 (Japan)

    2010-02-12

    Receptor activity-modifying protein 2 (RAMP2) enables calcitonin receptor-like receptor (CRLR) to form an adrenomedullin (AM)-specific receptor. Here we investigated the function of the cytoplasmic C-terminal tail (C-tail) of human (h)CRLR by co-transfecting its C-terminal mutants into HEK-293 cells stably expressing hRAMP2. Deleting the C-tail from CRLR disrupted AM-evoked cAMP production or receptor internalization, but did not affect [{sup 125}I]AM binding. We found that CRLR residues 428-439 are required for AM-evoked cAMP production, though deleting this region had little effect on receptor internalization. Moreover, pretreatment with pertussis toxin (100 ng/mL) led to significant increases in AM-induced cAMP production via wild-type CRLR/RAMP2 complexes. This effect was canceled by deleting CRLR residues 454-457, suggesting Gi couples to this region. Flow cytometric analysis revealed that CRLR truncation mutants lacking residues in the Ser/Thr-rich region extending from Ser{sup 449} to Ser{sup 467} were unable to undergo AM-induced receptor internalization and, in contrast to the effect on wild-type CRLR, overexpression of GPCR kinases-2, -3 and -4 failed to promote internalization of CRLR mutants lacking residues 449-467. Thus, the hCRLR C-tail is crucial for AM-evoked cAMP production and internalization of the CRLR/RAMP2, while the receptor internalization is dependent on the aforementioned GPCR kinases, but not Gs coupling.

  5. Camps 2.0: exploring the sequence and structure space of prokaryotic, eukaryotic, and viral membrane proteins.

    Science.gov (United States)

    Neumann, Sindy; Hartmann, Holger; Martin-Galiano, Antonio J; Fuchs, Angelika; Frishman, Dmitrij

    2012-03-01

    Structural bioinformatics of membrane proteins is still in its infancy, and the picture of their fold space is only beginning to emerge. Because only a handful of three-dimensional structures are available, sequence comparison and structure prediction remain the main tools for investigating sequence-structure relationships in membrane protein families. Here we present a comprehensive analysis of the structural families corresponding to α-helical membrane proteins with at least three transmembrane helices. The new version of our CAMPS database (CAMPS 2.0) covers nearly 1300 eukaryotic, prokaryotic, and viral genomes. Using an advanced classification procedure, which is based on high-order hidden Markov models and considers both sequence similarity as well as the number of transmembrane helices and loop lengths, we identified 1353 structurally homogeneous clusters roughly corresponding to membrane protein folds. Only 53 clusters are associated with experimentally determined three-dimensional structures, and for these clusters CAMPS is in reasonable agreement with structure-based classification approaches such as SCOP and CATH. We therefore estimate that ∼1300 structures would need to be determined to provide a sufficient structural coverage of polytopic membrane proteins. CAMPS 2.0 is available at http://webclu.bio.wzw.tum.de/CAMPS2.0/. Copyright © 2011 Wiley Periodicals, Inc.

  6. The Vasopressin Type-2 Receptor and Prostaglandin Receptors EP2 and EP4 can Increase Aquaporin-2 Plasma Membrane Targeting Through a cAMP Independent Pathway

    DEFF Research Database (Denmark)

    Olesen, Emma Tina Bisgaard; Moeller, Hanne Bjerregaard; Assentoft, Mette

    2016-01-01

    AMP-dependent. However, on the basis of recent reports, it was hypothesized in the current study that increased cAMP levels are not necessary for AQP2 membrane targeting. The role and dynamics of cAMP signaling on AQP2 membrane targeting in Madin-Darby Canine Kidney and mouse cortical collecting duct (mpkCCD14) cells......Apical membrane targeting of the collecting duct water channel aquaporin-2 (AQP2) is essential for body water balance. As this event is regulated by Gs coupled 7-transmembrane receptors such as the vasopressin type 2 receptor (V2R) and the prostanoid receptors EP2 and EP4, it is believed to be c...... was examined using selective agonists against the V2R (dDAVP), EP2 (butaprost) and EP4 (CAY10580). During EP2 stimulation, AQP2 membrane targeting continually increased during 80 min of stimulation; whereas cAMP levels reached a plateau after 10 min. EP4 stimulation caused a rapid and transient increase in AQP...

  7. Receptors, G proteins, and their interactions

    NARCIS (Netherlands)

    Hollmann, Markus W.; Strumper, Danja; Herroeder, Susanne; Durieux, Marcel E.

    2005-01-01

    Membrane receptors coupling to intracellular G proteins (G protein-coupled receptors) form one of the major classes of membrane signaling proteins. They are of great importance to the practice of anesthesiology because they are involved in many systems of relevance to the specialty (cardiovascular

  8. Broncho Vaxom (OM-85 modulates rhinovirus docking proteins on human airway epithelial cells via Erk1/2 mitogen activated protein kinase and cAMP.

    Directory of Open Access Journals (Sweden)

    Michael Roth

    Full Text Available Bronchial epithelial cells (BEC are primary target for Rhinovirus infection through attaching to cell membrane proteins. OM-85, a bacterial extract, improves recovery of asthma and COPD patients after viral infections, but only part of the mechanism was addressed, by focusing on defined immune cells.We therefore determined the effect of OM-85 on isolated primary human BEC of controls (n = 8, asthma patients (n = 10 and COPD patients (n = 9.BEC were treated with OM-85 alone (24 hours or infected with Rhinovirus. BEC survival was monitored by manual cell counting and Rhinovirus replication by lytic activity. Immuno-blotting and ELISA were used to determine the expression of Rhinovirus interacting proteins: intracellular adhesion molecule (ICAM, major histocompatibility complex class II (MHC-2, complement component C1q receptor (C1q-R, inducible T-Cell co-stimulator (ICOS, its ligand ICOSL, and myeloid differentiation primary response gene 88 (Myd88; as well as for signal transducers Erk1/2, p38, JNK mitogen activated protein kinases MAPK, and cAMP.OM-85 significantly reduced Rhinovirus-induced BEC death and virus replication. OM-85 significantly increased the expression of virus interacting proteins C1q-R and β-defensin in all 3 probes and groups, which was prevented by either Erk1/2 MAPK or cAMP inhibition. In addition, OM-85 significantly reduced Rhinovirus induced expression of ICAM1 involving p38 MAPK. In BEC OM-85 had no significant effect on the expression of ICOS, ICOSL and MHC-2 membrane proteins nor on the adaptor protein MyD88.The OM-85-induced increased of C1q-R and β-defensin, both important for antigen presentation and phagocytosis, supports its activity in host cell's defence against Rhinovirus infection.

  9. Gene Expression Patterns Define Key Transcriptional Events InCell-Cycle Regulation By cAMP And Protein Kinase A

    Energy Technology Data Exchange (ETDEWEB)

    Zambon, Alexander C.; Zhang, Lingzhi; Minovitsky, Simon; Kanter, Joan R.; Prabhakar, Shyam; Salomonis, Nathan; Vranizan, Karen; Dubchak Inna,; Conklin, Bruce R.; Insel, Paul A.

    2005-06-01

    Although a substantial number of hormones and drugs increase cellular cAMP levels, the global impact of cAMP and its major effector mechanism, protein kinase A (PKA), on gene expression is not known. Here we show that treatment of murine wild-type S49 lymphoma cells for 24 h with 8-(4-chlorophenylthio)-cAMP (8-CPTcAMP), a PKA-selective cAMP analog, alters the expression of approx equal to 4,500 of approx. equal to 13,600 unique genes. By contrast, gene expression was unaltered in Kin- S49 cells (that lack PKA) incubated with 8-CPTcAMP. Changes in mRNA and protein expression of several cell cycle regulators accompanied cAMP-induced G1-phase cell-cycle arrest of wild-type S49 cells. Within 2h, 8-CPT-cAMP altered expression of 152 genes that contain evolutionarily conserved cAMP-response elements within 5 kb of transcriptional start sites, including the circadian clock gene Per1. Thus, cAMP through its activation of PKA produces extensive transcriptional regulation in eukaryotic cells. These transcriptional networks include a primary group of cAMP-response element-containing genes and secondary networks that include the circadian clock.

  10. Transcriptional control of adrenal steroidogenesis: novel connection between Janus kinase (JAK) 2 protein and protein kinase A (PKA) through stabilization of cAMP response element-binding protein (CREB) transcription factor.

    Science.gov (United States)

    Lefrancois-Martinez, Anne-Marie; Blondet-Trichard, Antonine; Binart, Nadine; Val, Pierre; Chambon, Céline; Sahut-Barnola, Isabelle; Pointud, Jean-Christophe; Martinez, Antoine

    2011-09-23

    In the adrenal gland, adrenocorticotropin (ACTH) acting through the cAMP protein kinase (PKA) transduction pathway is the main regulator of genes involved in glucocorticoid synthesis. The prolactin (PRL) receptor is expressed in the adrenal cortex of most mammals, but experimental proof that PRL ensures direct control on glucocorticoid synthesis in rodents remains elusive. To unravel the physiological importance of PRL in adrenocortical functions, we measured steroidogenic capacity of Prlr-deficient mice (Prlr(-/-)) and explored the influence of JAK/STAT signaling, the major PRL transduction pathway, on the steroidogenic activity of adrenocortical cell cultures. We demonstrate that lack of Prlr does not affect basal (nor stress-induced) corticosterone levels in mice. PRL triggers JAK2/STAT5-dependent transcription in adrenal cells, but this does not influence corticosterone release. In contrast, pharmacological or siRNA-mediated inhibition of JAK2 reveals its essential role in both basal and ACTH/cAMP-induced steroidogenesis. We demonstrate that nuclear JAK2 regulates the amount of active transcription factor CREB (cAMP response element-binding protein) through tyrosine phosphorylation and prevention of proteasomal degradation, which in turn leads to transcriptional activation of the rate-limiting steroidogenic Star gene. Hence, we describe a novel link between PKA and JAK2 by which nuclear JAK2 signaling controls adrenal steroidogenesis by increasing the stability of CREB.

  11. Protein intake during training sessions has no effect on performance and recovery during a strenuous training camp for elite cyclists

    DEFF Research Database (Denmark)

    Hansen, Mette; Bangsbo, Jens; Jensen, Jørgen

    2016-01-01

    during cycling at a training camp for top cyclists did not result in marked performance benefits compared to intake of carbohydrates when a recovery drink containing adequate protein and carbohydrate was ingested immediately after each training session in both groups. These findings suggest......BACKGROUND: Training camps for top-class endurance athletes place high physiological demands on the body. Focus on optimizing recovery between training sessions is necessary to minimize the risk of injuries and improve adaptations to the training stimuli. Carbohydrate supplementation during...... sessions is generally accepted as being beneficial to aid performance and recovery, whereas the effect of protein supplementation and timing is less well understood. We studied the effects of protein ingestion during training sessions on performance and recovery of elite cyclists during a strenuous...

  12. Activation of protein kinase A and exchange protein directly activated by cAMP promotes adipocyte differentiation of human mesenchymal stem cells

    DEFF Research Database (Denmark)

    Jia, Bingbing; Madsen, Lise; Petersen, Rasmus Koefoed

    2012-01-01

    Human mesenchymal stem cells are primary multipotent cells capable of differentiating into several cell types including adipocytes when cultured under defined in vitro conditions. In the present study we investigated the role of cAMP signaling and its downstream effectors, protein kinase A (PKA......) and exchange protein directly activated by cAMP (Epac) in adipocyte conversion of human mesenchymal stem cells derived from adipose tissue (hMADS). We show that cAMP signaling involving the simultaneous activation of both PKA- and Epac-dependent signaling is critical for this process even in the presence...... of the strong adipogenic inducers insulin, dexamethasone, and rosiglitazone, thereby clearly distinguishing the hMADS cells from murine preadipocytes cell lines, where rosiglitazone together with dexamethasone and insulin strongly promotes adipocyte differentiation. We further show that prostaglandin I(2) (PGI...

  13. Protein Connectivity in Chemotaxis Receptor Complexes.

    Directory of Open Access Journals (Sweden)

    Stephan Eismann

    2015-12-01

    Full Text Available The chemotaxis sensory system allows bacteria such as Escherichia coli to swim towards nutrients and away from repellents. The underlying pathway is remarkably sensitive in detecting chemical gradients over a wide range of ambient concentrations. Interactions among receptors, which are predominantly clustered at the cell poles, are crucial to this sensitivity. Although it has been suggested that the kinase CheA and the adapter protein CheW are integral for receptor connectivity, the exact coupling mechanism remains unclear. Here, we present a statistical-mechanics approach to model the receptor linkage mechanism itself, building on nanodisc and electron cryotomography experiments. Specifically, we investigate how the sensing behavior of mixed receptor clusters is affected by variations in the expression levels of CheA and CheW at a constant receptor density in the membrane. Our model compares favorably with dose-response curves from in vivo Förster resonance energy transfer (FRET measurements, demonstrating that the receptor-methylation level has only minor effects on receptor cooperativity. Importantly, our model provides an explanation for the non-intuitive conclusion that the receptor cooperativity decreases with increasing levels of CheA, a core signaling protein associated with the receptors, whereas the receptor cooperativity increases with increasing levels of CheW, a key adapter protein. Finally, we propose an evolutionary advantage as explanation for the recently suggested CheW-only linker structures.

  14. Exchange Protein Directly Activated by cAMP (epac) : A Multidomain cAMP Mediator in the Regulation of Diverse Biological Functions

    NARCIS (Netherlands)

    Schmidt, Martina; Dekker, Frank J.; Maarsingh, Harm

    Since the discovery nearly 60 years ago, cAMP is envisioned as one of the most universal and versatile second messengers. The tremendous feature of cAMP to tightly control highly diverse physiologic processes, including calcium homeostasis, metabolism, secretion, muscle contraction, cell fate, and

  15. cAMP signalling in the normal and tumorigenic pituitary gland.

    Science.gov (United States)

    Formosa, R; Vassallo, J

    2014-07-05

    cAMP signalling plays a key role in the normal physiology of the pituitary gland, regulating cellular growth and proliferation, hormone production and release. Deregulation of the cAMP signalling pathway has been reported to be a common occurrence in pituitary tumorigenesis. Several mechanisms have been implicated including somatic mutations, gene-gene interactions and gene-environmental interactions. Somatic mutations in G-proteins and protein kinases directly alter cAMP signalling, while malfunctioning of other signalling pathways such as the Raf/MAPK/ERK, PI3K/Akt/mTOR and Wnt pathways which normally interact with the cAMP pathway may mediate indirect effects on cAMP and varying downstream effectors. The aryl hydrocarbon receptor signalling pathway has been implicated in pituitary tumorigenesis and we review its role in general and specifically in relation to cAMP de-regulation. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

  16. Modulation of dopamine D(2) receptor signaling by actin-binding protein (ABP-280).

    Science.gov (United States)

    Li, M; Bermak, J C; Wang, Z W; Zhou, Q Y

    2000-03-01

    Proteins that bind to G protein-coupled receptors have recently been identified as regulators of receptor anchoring and signaling. In this study, actin-binding protein 280 (ABP-280), a widely expressed cytoskeleton-associated protein that plays an important role in regulating cell morphology and motility, was found to associate with the third cytoplasmic loop of dopamine D(2) receptors. The specificity of this interaction was originally identified in a yeast two-hybrid screen and confirmed by protein binding. The functional significance of the D(2) receptor-ABP-280 association was evaluated in human melanoma cells lacking ABP-280. D(2) receptor agonists were less potent in inhibiting forskolin-stimulated cAMP production in these cells. Maximal inhibitory responses of D(2) receptor activation were also reduced. Further yeast two-hybrid experiments showed that ABP-280 association is critically dependent on the carboxyl domain of the D(2) receptor third cytoplasmic loop, where there is a potential serine phosphorylation site (S358). Serine 358 was replaced with aspartic acid to mimic the effects of receptor phosphorylation. This mutant (D(2)S358D) displayed compromised binding to ABP-280 and coupling to adenylate cyclase. PKC activation also generated D(2) receptor signaling attenuation, but only in ABP-containing cells, suggesting a PKC regulatory role in D(2)-ABP association. A mechanism for these results may be derived from a role of ABP-280 in the clustering of D(2) receptors, as determined by immunocytochemical analysis in ABP-deficient and replete cells. Our results suggest a new molecular mechanism of modulating D(2) receptor signaling by cytoskeletal protein interaction.

  17. Expression of Tas1 taste receptors in mammalian spermatozoa: functional role of Tas1r1 in regulating basal Ca²⁺ and cAMP concentrations in spermatozoa.

    Directory of Open Access Journals (Sweden)

    Dorke Meyer

    Full Text Available BACKGROUND: During their transit through the female genital tract, sperm have to recognize and discriminate numerous chemical compounds. However, our current knowledge of the molecular identity of appropriate chemosensory receptor proteins in sperm is still rudimentary. Considering that members of the Tas1r family of taste receptors are able to discriminate between a broad diversity of hydrophilic chemosensory substances, the expression of taste receptors in mammalian spermatozoa was examined. METHODOLOGY/PRINCIPAL FINDINGS: The present manuscript documents that Tas1r1 and Tas1r3, which form the functional receptor for monosodium glutamate (umami in taste buds on the tongue, are expressed in murine and human spermatozoa, where their localization is restricted to distinct segments of the flagellum and the acrosomal cap of the sperm head. Employing a Tas1r1-deficient mCherry reporter mouse strain, we found that Tas1r1 gene deletion resulted in spermatogenic abnormalities. In addition, a significant increase in spontaneous acrosomal reaction was observed in Tas1r1 null mutant sperm whereas acrosomal secretion triggered by isolated zona pellucida or the Ca²⁺ ionophore A23187 was not different from wild-type spermatozoa. Remarkably, cytosolic Ca²⁺ levels in freshly isolated Tas1r1-deficient sperm were significantly higher compared to wild-type cells. Moreover, a significantly higher basal cAMP concentration was detected in freshly isolated Tas1r1-deficient epididymal spermatozoa, whereas upon inhibition of phosphodiesterase or sperm capacitation, the amount of cAMP was not different between both genotypes. CONCLUSIONS/SIGNIFICANCE: Since Ca²⁺ and cAMP control fundamental processes during the sequential process of fertilization, we propose that the identified taste receptors and coupled signaling cascades keep sperm in a chronically quiescent state until they arrive in the vicinity of the egg - either by constitutive receptor activity and

  18. Novel receptors for bacterial protein toxins.

    Science.gov (United States)

    Schmidt, Gudula; Papatheodorou, Panagiotis; Aktories, Klaus

    2015-02-01

    While bacterial effectors are often directly introduced into eukaryotic target cells by various types of injection machines, toxins enter the cytosol of host cells from endosomal compartments or after retrograde transport via Golgi from the ER. A first crucial step of toxin-host interaction is receptor binding. Using optimized protocols and new methods novel toxin receptors have been identified, including metalloprotease ADAM 10 for Staphylococcus aureus α-toxin, laminin receptor Lu/BCAM for Escherichia coli cytotoxic necrotizing factor CNF1, lipolysis stimulated lipoprotein receptor (LSR) for Clostridium difficile transferase CDT and low-density lipoprotein receptor-related protein (LRP) 1 for Clostridium perfringens TpeL toxin. Copyright © 2014 Elsevier Ltd. All rights reserved.

  19. Effect of Increased Cyclic AMP Concentration on Muscle Protein Synthesis and Beta-Adrenergic Receptor Expression in Chicken Skeletal Muscle Cells in Culture

    Science.gov (United States)

    Young, R. B.; Vaughn, J. R.; Bridge, K. Y.; Smith, C. K.

    1998-01-01

    Analogies of epinephrine are known to cause hypertrophy of skeletal muscle when fed to animals. These compounds presumably exert their physiological action through interaction with the P-adrenergic receptor. Since the intracellular signal generated by the Beta-adrenergic receptor is cyclic AMP (cAMP), experiments were initiated in cell culture to determine if artificial elevation of cAMP by treatment with forskolin would alter muscle protein metabolism and P-adrenergic receptor expression. Chicken skeletal muscle cells after 7 days in culture were treated with 0.2-30 micrometers forskolin for a total of three days. At the end of the treatment period, both the concentration of cAMP and the quantity of myosin heavy chain (MHC) were measured. Concentration of cAMP in forskolin-treated cells increased up to 10-fold in a dose dependent manner. In contrast, the quantity of MHC was increased approximately 50% above control cells at 0.2 micrometers forskolin, but exhibited a gradual decline at higher levels of forskolin so that the quantity of MHC in cells treated with 30 micrometers forskolin was not significantly different from controls. Curiously, the intracellular concentration of cAMP which elicited the maximum increase in the quantity of MHC was only 40% higher than cAMP concentration in control cells.

  20. The Popeye Domain Containing Genes and cAMP Signaling

    Directory of Open Access Journals (Sweden)

    Thomas Brand

    2014-05-01

    Full Text Available 3'-5'-cyclic adenosine monophosphate (cAMP is a second messenger, which plays an important role in the heart. It is generated in response to activation of G-protein-coupled receptors (GPCRs. Initially, it was thought that protein kinase A (PKA exclusively mediates cAMP-induced cellular responses such as an increase in cardiac contractility, relaxation, and heart rate. With the identification of the exchange factor directly activated by cAMP (EPAC and hyperpolarizing cyclic nucleotide-gated (HCN channels as cAMP effector proteins it became clear that a protein network is involved in cAMP signaling. The Popeye domain containing (Popdc genes encode yet another family of cAMP-binding proteins, which are prominently expressed in the heart. Loss-of-function mutations in mice are associated with cardiac arrhythmia and impaired skeletal muscle regeneration. Interestingly, the cardiac phenotype, which is present in both, Popdc1 and Popdc2 null mutants, is characterized by a stress-induced sinus bradycardia, suggesting that Popdc proteins participate in cAMP signaling in the sinuatrial node. The identification of the two-pore channel TREK-1 and Caveolin 3 as Popdc-interacting proteins represents a first step into understanding the mechanisms of heart rate modulation triggered by Popdc proteins.

  1. Involvement of Cyclic AMP Receptor Protein in Regulation of the rmf Gene Encoding the Ribosome Modulation Factor in Escherichia coli

    OpenAIRE

    Shimada, Tomohiro; Yoshida, Hideji; Ishihama, Akira

    2013-01-01

    The decrease in overall translation in stationary-phase Escherichia coli is accompanied with the formation of functionally inactive 100S ribosomes mediated by the ribosome modulation factor (RMF). At present, however, little is known regarding the regulation of stationary-phase-coupled RMF expression. In the course of a systematic screening of regulation targets of DNA-binding transcription factors from E. coli, we realized that CRP (cyclic AMP [cAMP] receptor protein), the global regulator f...

  2. Dynamic fluctuations provide the basis of a conformational switch mechanism in apo cyclic AMP receptor protein.

    Directory of Open Access Journals (Sweden)

    Burcu Aykaç Fas

    Full Text Available Escherichia coli cyclic AMP Receptor Protein (CRP undergoes conformational changes with cAMP binding and allosterically promotes CRP to bind specifically to the DNA. In that, the structural and dynamic properties of apo CRP prior to cAMP binding are of interest for the comprehension of the activation mechanism. Here, the dynamics of apo CRP monomer/dimer and holo CRP dimer were studied by Molecular Dynamics (MD simulations and Gaussian Network Model (GNM. The interplay of the inter-domain hinge with the cAMP and DNA binding domains are pre-disposed in the apo state as a conformational switch in the CRP's allosteric communication mechanism. The hinge at L134-D138 displaying intra- and inter-subunit coupled fluctuations with the cAMP and DNA binding domains leads to the emergence of stronger coupled fluctuations between the two domains and describes an on state. The flexible regions at K52-E58, P154/D155 and I175 maintain the dynamic coupling of the two domains. With a shift in the inter-domain hinge position towards the N terminus, nevertheless, the latter correlations between the domains loosen and become disordered; L134-D138 dynamically interacts only with the cAMP and DNA binding domains of its own subunit, and an off state is assumed. We present a mechanistic view on how the structural dynamic units are hierarchically built for the allosteric functional mechanism; from apo CRP monomer to apo-to-holo CRP dimers.

  3. Dynamic Fluctuations Provide the Basis of a Conformational Switch Mechanism in Apo Cyclic AMP Receptor Protein

    Science.gov (United States)

    Aykaç Fas, Burcu; Tutar, Yusuf; Haliloğlu, Türkan

    2013-01-01

    Escherichia coli cyclic AMP Receptor Protein (CRP) undergoes conformational changes with cAMP binding and allosterically promotes CRP to bind specifically to the DNA. In that, the structural and dynamic properties of apo CRP prior to cAMP binding are of interest for the comprehension of the activation mechanism. Here, the dynamics of apo CRP monomer/dimer and holo CRP dimer were studied by Molecular Dynamics (MD) simulations and Gaussian Network Model (GNM). The interplay of the inter-domain hinge with the cAMP and DNA binding domains are pre-disposed in the apo state as a conformational switch in the CRP's allosteric communication mechanism. The hinge at L134-D138 displaying intra- and inter-subunit coupled fluctuations with the cAMP and DNA binding domains leads to the emergence of stronger coupled fluctuations between the two domains and describes an on state. The flexible regions at K52-E58, P154/D155 and I175 maintain the dynamic coupling of the two domains. With a shift in the inter-domain hinge position towards the N terminus, nevertheless, the latter correlations between the domains loosen and become disordered; L134-D138 dynamically interacts only with the cAMP and DNA binding domains of its own subunit, and an off state is assumed. We present a mechanistic view on how the structural dynamic units are hierarchically built for the allosteric functional mechanism; from apo CRP monomer to apo-to-holo CRP dimers. PMID:23874183

  4. The CytR repressor antagonizes cyclic AMP-cyclic AMP receptor protein activation of the deoCp2 promoter of Escherichia coli K-12

    DEFF Research Database (Denmark)

    Søgaard-Andersen, Lotte; Martinussen, J; Møllegaard, N E

    1990-01-01

    We have investigated the regulation of the Escherichia coli deoCp2 promoter by the CytR repressor and the cyclic AMP (cAMP) receptor protein (CRP) complexed to cAMP. Promoter regions controlled by these two proteins characteristically contain tandem cAMP-CRP binding sites. Here we show that (i) Cyt......AMP-CRP complex; (iii) introduction of point mutations in either CRP target resulted in loss of CytR regulation; and (iv) regulation by CytR of deletion mutants lacking CRP-2 could be specifically reestablished by increasing the intracellular concentration of CytR. These findings indicate that both CRP targets...

  5. A multi-angular mass spectrometric view at cyclic nucleotide signaling proteins : Structure/function and protein interactions of cAMP- and cGMP-dependent protein kinase

    NARCIS (Netherlands)

    Scholten, A.

    2006-01-01

    The primary focus of this thesis is the two kinases PKA and PKG, cAMP and cGMP dependent protein kinase respectively. PKA and PKG are studied both at structure/function level as well as at the level of interaction with other proteins in tissue. Our primary methods are all based on mass spectrometry.

  6. MAPK signaling drives inflammation in LPS-stimulated cardiomyocytes: the route of crosstalk to G-protein-coupled receptors.

    Directory of Open Access Journals (Sweden)

    W Joshua Frazier

    Full Text Available Profound cardiovascular dysfunction is an important cause of mortality from septic shock. The molecular underpinnings of cardiac dysfunction during the inflammatory surge of early sepsis are not fully understood. MAPKs are important signal transducers mediating inflammation whereas G-protein signaling pathways modulate the cardiac response to stress. Using H9c2 cardiomyocytes, we investigated the interaction of MAPK and G-protein signaling in a sepsis model to test the hypothesis that the cardiomyocyte inflammatory response is controlled by MAPKs via G-protein-mediated events. We found that LPS stimulated proinflammatory cytokine production was markedly exacerbated by siRNA knockdown of the MAPK negative regulator Mkp-1. Cytokine production was blunted when cells were treated with p38 inhibitor. Two important cellular signaling molecules typically regulated by G-protein-coupled receptors, cAMP and PKC activity, were also stimulated by LPS and inflammatory cytokines TNF-α and IL-6, through a process regulated by Mkp-1 and p38. Interestingly, neutralizing antibodies against Gα(s and Gα(q blocked the increase in cellular cAMP and PKC activation, respectively, in response to inflammatory stimuli, indicating a critical role of G-protein coupled receptors in this process. LPS stimulation increased COX-2 in H9c2 cells, which also express prostaglandin receptors. Blockade of G-protein-coupled EP4 prostaglandin receptor by AH 23848 prevented LPS-induced cAMP increase. These data implicate MAPKs and G-proteins in the cardiomyocyte inflammatory response to LPS as well as crosstalk via COX-2-generated PGE(2. These data add to our understanding of the pathogenesis of septic shock and have the potential to guide the selection of future therapeutics.

  7. Dynamic feature of mitotic arrest deficient 2-like protein 2 (MAD2L2) and structural basis for its interaction with chromosome alignment-maintaining phosphoprotein (CAMP).

    Science.gov (United States)

    Hara, Kodai; Taharazako, Shota; Ikeda, Masanori; Fujita, Hiroki; Mikami, Yoshiko; Kikuchi, Sotaro; Hishiki, Asami; Yokoyama, Hideshi; Ishikawa, Yoshinobu; Kanno, Shin-Ichiro; Tanaka, Kozo; Hashimoto, Hiroshi

    2017-10-27

    Mitotic arrest deficient 2-like protein 2 (MAD2L2), also termed MAD2B or REV7, is involved in multiple cellular functions including translesion DNA synthesis (TLS), signal transduction, transcription, and mitotic events. MAD2L2 interacts with chromosome alignment-maintaining phosphoprotein (CAMP), a kinetochore-microtubule attachment protein in mitotic cells, presumably through a novel "WK" motif in CAMP. Structures of MAD2L2 in complex with binding regions of the TLS proteins REV3 and REV1 have revealed that MAD2L2 has two faces for protein-protein interactions that are regulated by its C-terminal region; however, the mechanisms underlying the MAD2L2-CAMP interaction and the mitotic role of MAD2L2 remain unknown. Here we have determined the structures of human MAD2L2 in complex with a CAMP fragment in two crystal forms. The overall structure of the MAD2L2-CAMP complex in both crystal forms was essentially similar to that of the MAD2L2-REV3 complex. However, the residue interactions between MAD2L2 and CAMP were strikingly different from those in the MAD2L2-REV3 complex. Furthermore, structure-based interaction analyses revealed an unprecedented mechanism involving CAMP's WK motif. Surprisingly, in one of the crystal forms, the MAD2L2-CAMP complex formed a dimeric structure in which the C-terminal region of MAD2L2 was swapped and adopted an immature structure. The structure provides direct evidence for the dynamic nature of MAD2L2 structure, which in turn may have implications for the protein-protein interaction mechanism and the multiple functions of this protein. This work is the first structural study of MAD2L2 aside from its role in TLS and might pave the way to clarify MAD2L2's function in mitosis. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  8. Hydrogen sulfide inhibits A2A adenosine receptor agonist induced β-amyloid production in SH-SY5Y neuroblastoma cells via a cAMP dependent pathway.

    Directory of Open Access Journals (Sweden)

    Bhushan Vijay Nagpure

    Full Text Available Alzheimer's disease (AD is the leading cause of senile dementia in today's society. Its debilitating symptoms are manifested by disturbances in many important brain functions, which are influenced by adenosine. Hence, adenosinergic system is considered as a potential therapeutic target in AD treatment. In the present study, we found that sodium hydrosulfide (NaHS, an H2S donor, 100 µM attenuated HENECA (a selective A2A receptor agonist, 10-200 nM induced β-amyloid (1-42 (Aβ42 production in SH-SY5Y cells. NaHS also interfered with HENECA-stimulated production and post-translational modification of amyloid precursor protein (APP by inhibiting its maturation. Measurement of the C-terminal APP fragments generated from its enzymatic cleavage by β-site amyloid precursor protein cleaving enzyme 1 (BACE1 showed that NaHS did not have any significant effect on β-secretase activity. However, the direct measurements of HENECA-elevated γ-secretase activity and mRNA expressions of presenilins suggested that the suppression of Aβ42 production in NaHS pretreated cells was mediated by inhibiting γ-secretase. NaHS induced reductions were accompanied by similar decreases in intracellular cAMP levels and phosphorylation of cAMP responsive element binding protein (CREB. NaHS significantly reduced the elevated cAMP and Aβ42 production caused by forskolin (an adenylyl cyclase, AC agonist alone or forskolin in combination with IBMX (a phosphodiesterase inhibitor, but had no effect on those caused by IBMX alone. Moreover, pretreatment with NaHS significantly attenuated HENECA-elevated AC activity and mRNA expressions of various AC isoforms. These data suggest that NaHS may preferentially suppress AC activity when it was stimulated. In conclusion, H2S attenuated HENECA induced Aβ42 production in SH-SY5Y neuroblastoma cells through inhibiting γ-secretase via a cAMP dependent pathway.

  9. Insights into cellular signalling by G protein coupled receptor transactivation of cell surface protein kinase receptors.

    Science.gov (United States)

    Chaplin, Rebecca; Thach, Lyna; Hollenberg, Morley D; Cao, Yingnan; Little, Peter J; Kamato, Danielle

    2017-06-01

    G protein coupled receptor (GPCR) signalling is mediated by transactivation independent and transactivation dependent pathways. GPCRs transactivate protein tyrosine kinase receptors (PTKRs) and protein serine/threonine kinase receptors (PS/TKR). Since the initial observations of transactivation dependent signalling, there has been an effort to understand the mechanisms behind this phenomena. GPCR signalling has evolved to include biased signalling. Biased signalling, whereby selected ligands can activate the same GPCR that can generate multiple signals, but drive only a unique response. To date, there has been no focus on the ability of biased agonists to activate the PTKR and PS/TKR transactivation pathways differentially. As such, this represents a novel direction for future research. This review will discuss the main mechanisms of GPCR mediated receptor transactivation and the pathways involved in intracellular responses.

  10. G Protein-coupled Receptor Biased Agonism.

    Science.gov (United States)

    Hodavance, Sima Y; Gareri, Clarice; Torok, Rachel D; Rockman, Howard A

    2016-03-01

    G protein-coupled receptors are the largest family of targets for current therapeutics. The classic model of their activation was binary, where agonist binding induced an active conformation and subsequent downstream signaling. Subsequently, the revised concept of biased agonism emerged, where different ligands at the same G protein-coupled receptor selectively activate one downstream pathway versus another. Advances in understanding the mechanism of biased agonism have led to the development of novel ligands, which have the potential for improved therapeutic and safety profiles. In this review, we summarize the theory and most recent breakthroughs in understanding biased signaling, examine recent laboratory investigations concerning biased ligands across different organ systems, and discuss the promising clinical applications of biased agonism.

  11. Receptor-like protein-tyrosine phosphatase alpha specifically inhibits insulin-increased prolactin gene expression

    DEFF Research Database (Denmark)

    Jacob, K K; Sap, J; Stanley, F M

    1998-01-01

    A physiologically relevant response to insulin, stimulation of prolactin promoter activity in GH4 pituitary cells, was used as an assay to study the specificity of protein-tyrosine phosphatase function. Receptor-like protein-tyrosine phosphatase alpha (RPTPalpha) blocks the effect of insulin...... to increase prolactin gene expression but potentiates the effects of epidermal growth factor and cAMP on prolactin promoter activity. RPTPalpha was the only protein-tyrosine phosphatase tested that did this. Thus, the effect of RPTPalpha on prolactin-chloramphenicol acetyltransferase (CAT) promoter activity...... is specific by two criteria. A number of potential RPTPalpha targets were ruled out by finding (a) that they are not affected or (b) that they are not on the pathway to insulin-increased prolactin-CAT activity. The negative effect of RPTPalpha on insulin activation of the prolactin promoter is not due...

  12. Involvement of Src family of kinases and cAMP phosphodiesterase in the luteinizing hormone/chorionic gonadotropin receptor-mediated signaling in the corpus luteum of monkey

    Directory of Open Access Journals (Sweden)

    Kunal Shah B

    2012-03-01

    Full Text Available Abstract Background In higher primates, during non-pregnant cycles, it is indisputable that circulating LH is essential for maintenance of corpus luteum (CL function. On the other hand, during pregnancy, CL function gets rescued by the LH analogue, chorionic gonadotropin (CG. The molecular mechanisms involved in the control of luteal function during spontaneous luteolysis and rescue processes are not completely understood. Emerging evidence suggests that LH/CGR activation triggers proliferation and transformation of target cells by various signaling molecules as evident from studies demonstrating participation of Src family of tyrosine kinases (SFKs and MAP kinases in hCG-mediated actions in Leydig cells. Since circulating LH concentration does not vary during luteal regression, it was hypothesized that decreased responsiveness of luteal cells to LH might occur due to changes in LH/CGR expression dynamics, modulation of SFKs or interference with steroid biosynthesis. Methods Since, maintenance of structure and function of CL is dependent on the presence of functional LH/CGR its expression dynamics as well as mRNA and protein expressions of SFKs were determined throughout the luteal phase. Employing well characterized luteolysis and CL rescue animal models, activities of SFKs, cAMP phosphodiesterase (cAMP-PDE and expression of SR-B1 (a membrane receptor associated with trafficking of cholesterol ester were examined. Also, studies were carried out to investigate the mechanisms responsible for decline in progesterone biosynthesis in CL during the latter part of the non-pregnant cycle. Results and discussion The decreased responsiveness of CL to LH during late luteal phase could not be accounted for by changes in LH/CGR mRNA levels, its transcript variants or protein. Results obtained employing model systems depicting different functional states of CL revealed increased activity of SFKs [pSrc (Y-416] and PDE as well as decreased expression of SR-B1

  13. The Regulator of Calcineurin 1 (RCAN1/DSCR1) Activates the cAMP Response Element-binding Protein (CREB) Pathway*

    Science.gov (United States)

    Kim, Seon Sook; Seo, Su Ryeon

    2011-01-01

    cAMP response element-binding protein (CREB) is one of the best known transcription factors in the development and function of the nervous system. In this report, we found that the regulator of calcineurin 1 (RCAN1), which is overexpressed in the brain of patients with Down syndrome, increased the phosphorylation of CREB and cAMP response element-mediated gene transcription in response to the activation of the intracellular cAMP pathway. Furthermore, we found that the increased activation of CREB signaling by RCAN1 depended on the ability of RCAN1 to inhibit calcineurin activity. Our data provide the first evidence that RCAN1 acts as an important regulatory component in the control of CREB signaling. PMID:21890628

  14. The regulator of calcineurin 1 (RCAN1/DSCR1) activates the cAMP response element-binding protein (CREB) pathway.

    Science.gov (United States)

    Kim, Seon Sook; Seo, Su Ryeon

    2011-10-28

    cAMP response element-binding protein (CREB) is one of the best known transcription factors in the development and function of the nervous system. In this report, we found that the regulator of calcineurin 1 (RCAN1), which is overexpressed in the brain of patients with Down syndrome, increased the phosphorylation of CREB and cAMP response element-mediated gene transcription in response to the activation of the intracellular cAMP pathway. Furthermore, we found that the increased activation of CREB signaling by RCAN1 depended on the ability of RCAN1 to inhibit calcineurin activity. Our data provide the first evidence that RCAN1 acts as an important regulatory component in the control of CREB signaling.

  15. cAMP response element binding protein is required for differentiation of respiratory epithelium during murine development.

    Directory of Open Access Journals (Sweden)

    A Daniel Bird

    Full Text Available The cAMP response element binding protein 1 (Creb1 transcription factor regulates cellular gene expression in response to elevated levels of intracellular cAMP. Creb1(-/- fetal mice are phenotypically smaller than wildtype littermates, predominantly die in utero and do not survive after birth due to respiratory failure. We have further investigated the respiratory defect of Creb1(-/- fetal mice during development. Lungs of Creb1(-/- fetal mice were pale in colour and smaller than wildtype controls in proportion to their reduced body size. Creb1(-/- lungs also did not mature morphologically beyond E16.5 with little or no expansion of airway luminal spaces, a phenotype also observed with the Creb1(-/- lung on a Crem(-/- genetic background. Creb1 was highly expressed throughout the lung at all stages examined, however activation of Creb1 was detected primarily in distal lung epithelium. Cell differentiation of E17.5 Creb1(-/- lung distal epithelium was analysed by electron microscopy and showed markedly reduced numbers of type-I and type-II alveolar epithelial cells. Furthermore, immunomarkers for specific lineages of proximal epithelium including ciliated, non-ciliated (Clara, and neuroendocrine cells showed delayed onset of expression in the Creb1(-/- lung. Finally, gene expression analyses of the E17.5 Creb1(-/- lung using whole genome microarray and qPCR collectively identified respiratory marker gene profiles and provide potential novel Creb1-regulated genes. Together, these results demonstrate a crucial role for Creb1 activity for the development and differentiation of the conducting and distal lung epithelium.

  16. G-protein coupled receptor signaling architecture of mammalian immune cells.

    Directory of Open Access Journals (Sweden)

    Natalia Polouliakh

    Full Text Available A series of recent studies on large-scale networks of signaling and metabolic systems revealed that a certain network structure often called "bow-tie network" are observed. In signaling systems, bow-tie network takes a form with diverse and redundant inputs and outputs connected via a small numbers of core molecules. While arguments have been made that such network architecture enhances robustness and evolvability of biological systems, its functional role at a cellular level remains obscure. A hypothesis was proposed that such a network function as a stimuli-reaction classifier where dynamics of core molecules dictate downstream transcriptional activities, hence physiological responses against stimuli. In this study, we examined whether such hypothesis can be verified using experimental data from Alliance for Cellular Signaling (AfCS that comprehensively measured GPCR related ligands response for B-cell and macrophage. In a GPCR signaling system, cAMP and Ca2+ act as core molecules. Stimuli-response for 32 ligands to B-Cells and 23 ligands to macrophages has been measured. We found that ligands with correlated changes of cAMP and Ca2+ tend to cluster closely together within the hyperspaces of both cell types and they induced genes involved in the same cellular processes. It was found that ligands inducing cAMP synthesis activate genes involved in cell growth and proliferation; cAMP and Ca2+ molecules that increased together form a feedback loop and induce immune cells to migrate and adhere together. In contrast, ligands without a core molecules response are scattered throughout the hyperspace and do not share clusters. G-protein coupling receptors together with immune response specific receptors were found in cAMP and Ca2+ activated clusters. Analyses have been done on the original software applicable for discovering 'bow-tie' network architectures within the complex network of intracellular signaling where ab initio clustering has been

  17. PDE4-Mediated cAMP Signalling

    Directory of Open Access Journals (Sweden)

    Bracy A. Fertig

    2018-01-01

    Full Text Available cAMP is the archetypal and ubiquitous second messenger utilised for the fine control of many cardiovascular cell signalling systems. The ability of cAMP to elicit cell surface receptor-specific responses relies on its compartmentalisation by cAMP hydrolysing enzymes known as phosphodiesterases. One family of these enzymes, PDE4, is particularly important in the cardiovascular system, where it has been extensively studied and shown to orchestrate complex, localised signalling that underpins many crucial functions of the heart. In the cardiac myocyte, cAMP activates PKA, which phosphorylates a small subset of mostly sarcoplasmic substrate proteins that drive β-adrenergic enhancement of cardiac function. The phosphorylation of these substrates, many of which are involved in cardiac excitation-contraction coupling, has been shown to be tightly regulated by highly localised pools of individual PDE4 isoforms. The spatial and temporal regulation of cardiac signalling is made possible by the formation of macromolecular “signalosomes”, which often include a cAMP effector, such as PKA, its substrate, PDE4 and an anchoring protein such as an AKAP. Studies described in the present review highlight the importance of this relationship for individual cardiac PKA substrates and we provide an overview of how this signalling paradigm is coordinated to promote efficient adrenergic enhancement of cardiac function. The role of PDE4 also extends to the vascular endothelium, where it regulates vascular permeability and barrier function. In this distinct location, PDE4 interacts with adherens junctions to regulate their stability. These highly specific, non-redundant roles for PDE4 isoforms have far reaching therapeutic potential. PDE inhibitors in the clinic have been plagued with problems due to the active site-directed nature of the compounds which concomitantly attenuate PDE activity in all highly localised “signalosomes”.

  18. The G protein-coupled receptor, class C, group 6, subtype A (GPRC6A) receptor

    DEFF Research Database (Denmark)

    Clemmensen, C; Smajilovic, S; Wellendorph, P

    2014-01-01

    GPRC6A (G protein-coupled receptor, class C, group 6, subtype A) is a class C G protein-coupled receptor, that has been cloned from human, mouse and rat. Several groups have shown that the receptor is activated by a range of basic and small aliphatic L-α-amino acids of which L-arginine, L-lysine...

  19. CB1 Cannabinoid Receptors and their Associated Proteins

    Science.gov (United States)

    Howlett, Allyn C.; Blume, Lawrence C.; Dalton, George D.

    2011-01-01

    CB1 receptors are G-protein coupled receptors (GPCRs) abundant in neurons, in which they modulate neurotransmission. The CB1 receptor influence on memory and learning is well recognized, and disease states associated with CB1 receptors are observed in addiction disorders, motor dysfunction, schizophrenia, and in bipolar, depression, and anxiety disorders. Beyond the brain, CB1 receptors also function in liver and adipose tissues, vascular as well as cardiac tissue, reproductive tissues and bone. Signal transduction by CB1 receptors occurs through interaction with Gi/o proteins to inhibit adenylyl cyclase, activate mitogen-activated protein kinases (MAPK), inhibit voltage-gated Ca2+ channels, activate K+ currents (Kir), and influence Nitric Oxide (NO) signaling. CB1 receptors are observed in internal organelles as well as plasma membrane. β-Arrestins, adaptor protein AP-3, and G-protein receptor-associated sorting protein 1 (GASP1) modulate cellular trafficking. Cannabinoid Receptor Interacting Protein 1a (CRIP1a) is an accessory protein whose function has not been delineated. Factor Associated with Neutral sphingomyelinase (FAN) regulates ceramide signaling. Such diversity in cellular signaling and modulation by interacting proteins suggests that agonists and allosteric modulators could be developed to specifically regulate unique, cell type-specific responses. PMID:20166926

  20. Parathyroid hormone stimulates juxtaglomerular cell cAMP accumulation without stimulating renin release

    Science.gov (United States)

    Atchison, Douglas K.; Harding, Pamela; Cecilia Ortiz-Capisano, M.; Peterson, Edward L.

    2012-01-01

    Parathyroid hormone (PTH) is positively coupled to the generation of cAMP via its actions on the PTH1R and PTH2R receptors. Renin secretion from juxtaglomerular (JG) cells is stimulated by elevated intracellular cAMP, and every stimulus that increases renin secretion is thought to do so via increasing cAMP. Thus we hypothesized that PTH increases renin release from primary cultures of mouse JG cells by elevating intracellular cAMP via the PTH1R receptor. We found PTH1R, but not PTH2R, mRNA expressed in JG cells. While PTH increased JG cell cAMP content from (log10 means ± SE) 3.27 ± 0.06 to 3.92 ± 0.12 fmol/mg protein (P renin release. The PTH1R-specific agonist, parathyroid hormone-related protein (PTHrP), also increased JG cell cAMP from 3.13 ± 0.09 to 3.93 ± 0.09 fmol/mg protein (P renin release. PTH2R receptor agonists had no effect on cAMP or renin release. PTHrP increased cAMP in the presence of both low and high extracellular calcium from 3.31 ± 0.17 to 3.83 ± 0.20 fmol/mg protein (P renin release. PTHrP increased JG cell cAMP in the presence of adenylyl cyclase-V inhibition from 2.85 ± 0.17 to 3.44 ± 0.14 fmol/mg protein (P renin release. As a positive control, forskolin increased JG cell cAMP from 3.39 ± 0.13 to 4.48 ± 0.07 fmol/mg protein (P renin release from 2.96 ± 0.10 to 3.29 ± 0.08 ng ANG I·mg prot−1·h−1 (P renin release. These data suggest compartmentalization of cAMP signaling in JG cells. PMID:22896038

  1. Semiotic Selection of Mutated or Misfolded Receptor Proteins

    DEFF Research Database (Denmark)

    Giorgi, Franco; Bruni, Luis Emilio; Maggio, Roberto

    2013-01-01

    Receptor oligomerization plays a key role in maintaining genome stability and restricting protein mutagenesis. When properly folded, protein monomers assemble as oligomeric receptors and interact with environmental ligands. In a gene-centered view, the ligand specificity expressed by these recept......Receptor oligomerization plays a key role in maintaining genome stability and restricting protein mutagenesis. When properly folded, protein monomers assemble as oligomeric receptors and interact with environmental ligands. In a gene-centered view, the ligand specificity expressed...... for receptor monomers to assemble along the membrane and to sustain meaningful relationships with environmental ligands. How could a cell lineage deal with these loss-of-function mutations during evolution and restrain gene redundancy accordingly? In this paper, we will be arguing that the easiest way...... focused on the significance and semiotic nature of the interplay between membrane receptors and the epigenetic control of gene expression, as mediated by the control of mismatched repairing and protein folding mechanisms....

  2. Coordinated induction of GST and MRP2 by cAMP in Caco-2 cells: Role of protein kinase A signaling pathway and toxicological relevance

    Energy Technology Data Exchange (ETDEWEB)

    Arana, Maite Rocío, E-mail: arana@ifise-conicet.gov.ar [Instituto de Fisiología Experimental (CONICET), Facultad de Ciencias Bioquímicas y Farmacéuticas (UNR), Suipacha 570, 2000 Rosario (Argentina); Tocchetti, Guillermo Nicolás, E-mail: gtocchetti@live.com.ar [Instituto de Fisiología Experimental (CONICET), Facultad de Ciencias Bioquímicas y Farmacéuticas (UNR), Suipacha 570, 2000 Rosario (Argentina); Domizi, Pablo, E-mail: domizi@ibr-conicet.gov.ar [Instituto de Biología Molecular y Celular de Rosario (CONICET), Facultad de Ciencias Bioquímicas y Farmacéuticas (UNR), Suipacha 570, 2000 Rosario (Argentina); Arias, Agostina, E-mail: agoarias@yahoo.com.ar [Instituto de Fisiología Experimental (CONICET), Facultad de Ciencias Bioquímicas y Farmacéuticas (UNR), Suipacha 570, 2000 Rosario (Argentina); Rigalli, Juan Pablo, E-mail: jprigalli@gmail.com [Instituto de Fisiología Experimental (CONICET), Facultad de Ciencias Bioquímicas y Farmacéuticas (UNR), Suipacha 570, 2000 Rosario (Argentina); Ruiz, María Laura, E-mail: ruiz@ifise-conicet.gov.ar [Instituto de Fisiología Experimental (CONICET), Facultad de Ciencias Bioquímicas y Farmacéuticas (UNR), Suipacha 570, 2000 Rosario (Argentina); and others

    2015-09-01

    The cAMP pathway is a universal signaling pathway regulating many cellular processes including metabolic routes, growth and differentiation. However, its effects on xenobiotic biotransformation and transport systems are poorly characterized. The effect of cAMP on expression and activity of GST and MRP2 was evaluated in Caco-2 cells, a model of intestinal epithelium. Cells incubated with the cAMP permeable analog dibutyryl cyclic AMP (db-cAMP: 1,10,100 μM) for 48 h exhibited a dose–response increase in GST class α and MRP2 protein expression. Incubation with forskolin, an activator of adenylyl cyclase, confirmed the association between intracellular cAMP and upregulation of MRP2. Consistent with increased expression of GSTα and MRP2, db-cAMP enhanced their activities, as well as cytoprotection against the common substrate 1-chloro-2,4-dinitrobenzene. Pretreatment with protein kinase A (PKA) inhibitors totally abolished upregulation of MRP2 and GSTα induced by db-cAMP. In silico analysis together with experiments consisting of treatment with db-cAMP of Caco-2 cells transfected with a reporter construct containing CRE and AP-1 sites evidenced participation of these sites in MRP2 upregulation. Further studies involving the transcription factors CREB and AP-1 (c-JUN, c-FOS and ATF2) demonstrated increased levels of total c-JUN and phosphorylation of c-JUN and ATF2 by db-cAMP, which were suppressed by a PKA inhibitor. Co-immunoprecipitation and ChIP assay studies demonstrated that db-cAMP increased c-JUN/ATF2 interaction, with further recruitment to the region of the MRP2 promoter containing CRE and AP-1 sites. We conclude that cAMP induces GSTα and MRP2 expression and activity in Caco-2 cells via the PKA pathway, thus regulating detoxification of specific xenobiotics. - Highlights: • cAMP positively modulates the expression and activity of GST and MRP2 in Caco-2 cells. • Such induction resulted in increased cytoprotection against chemical injury. • PKA

  3. Genetically-encoded tools for cAMP probing and modulation in living systems.

    Directory of Open Access Journals (Sweden)

    Valeriy M Paramonov

    2015-09-01

    Full Text Available Intracellular 3'-5'-cyclic adenosine monophosphate (cAMP is one of the principal second messengers downstream of a manifold of signal transduction pathways, including the ones triggered by G protein-coupled receptors. Not surprisingly, biochemical assays for cAMP have been instrumental for basic research and drug discovery for decades, providing insights into cellular physiology and guiding pharmaceutical industry. However, despite impressive track record, the majority of conventional biochemical tools for cAMP probing share the same fundamental shortcoming - all the measurements require sample disruption for cAMP liberation. This common bottleneck, together with inherently low spatial resolution of measurements (as cAMP is typically analyzed in lysates of thousands of cells, underpin the ensuing limitations of the conventional cAMP assays: 1 genuine kinetic measurements of cAMP levels over time in a single given sample are unfeasible; 2 inability to obtain precise information on cAMP spatial distribution and transfer at subcellular levels, let alone the attempts to pinpoint dynamic interactions of cAMP and its effectors. At the same time, tremendous progress in synthetic biology over the recent years culminated in drastic refinement of our toolbox, allowing us not only to bypass the limitations of conventional assays, but to put intracellular cAMP life-span under tight control – something, that seemed scarcely attainable before. In this review article we discuss the main classes of modern genetically-encoded tools tailored for cAMP probing and modulation in living systems. We examine the capabilities and weaknesses of these different tools in the context of their operational characteristics and applicability to various experimental set-ups involving living cells, providing the guidance for rational selection of the best tools for particular needs.

  4. Impaired bidirectional synaptic plasticity and procedural memory formation in striatum-specific cAMP response element-binding protein-deficient mice

    OpenAIRE

    Pittenger, Christopher; Fasano, Stefania; Mazzocchi-Jones, David Martin; Dunnett, Stephen Bruce; Kandel, Eric R.; Brambilla, Riccardo

    2006-01-01

    The striatum has a well documented role in procedural learning and memory. However, the synaptic and molecular mechanisms of acquisition and storage of this form of memory remain poorly understood. We examined procedural memory and plasticity in transgenic mice reversibly expressing a dominant-negative cAMP response element-binding protein (CREB) mutant in the dorsal striatum. In these transgenic mice, corticostriatal long-term potentiation and depression are abolished, indicating that CREB f...

  5. GPCRDB: an information system for G protein-coupled receptors

    NARCIS (Netherlands)

    Isberg, V.; Vroling, B.; Kant, R.; Li, K.; Vriend, G.; Gloriam, D.

    2014-01-01

    For the past 20 years, the GPCRDB (G protein-coupled receptors database; http://www.gpcr.org/7tm/) has been a 'one-stop shop' for G protein-coupled receptor (GPCR)-related data. The GPCRDB contains experimental data on sequences, ligand-binding constants, mutations and oligomers, as well as many

  6. Activation of exchange protein activated by cAMP in the rat basolateral amygdala impairs reconsolidation of a memory associated with self-administered cocaine.

    Directory of Open Access Journals (Sweden)

    Xun Wan

    Full Text Available The intracellular mechanisms underlying memory reconsolidation critically involve cAMP signaling. These events were originally attributed to PKA activation by cAMP, but the identification of Exchange Protein Activated by cAMP (Epac, as a distinct mediator of cAMP signaling, suggests that cAMP-regulated processes that subserve memory reconsolidation are more complex. Here we investigated how activation of Epac with 8-pCPT-cAMP (8-CPT impacts reconsolidation of a memory that had been associated with cocaine self-administration. Rats were trained to lever press for cocaine on an FR-1 schedule, in which each cocaine delivery was paired with a tone+light cue. Lever pressing was then extinguished in the absence of cue presentations and cocaine delivery. Following the last day of extinction, rats were put in a novel context, in which the conditioned cue was presented to reactivate the cocaine-associated memory. Immediate bilateral infusions of 8-CPT into the basolateral amygdala (BLA following reactivation disrupted subsequent cue-induced reinstatement in a dose-dependent manner, and modestly reduced responding for conditioned reinforcement. When 8-CPT infusions were delayed for 3 hours after the cue reactivation session or were given after a cue extinction session, no effect on cue-induced reinstatement was observed. Co-administration of 8-CPT and the PKA activator 6-Bnz-cAMP (10 nmol/side rescued memory reconsolidation while 6-Bnz alone had no effect, suggesting an antagonizing interaction between the two cAMP signaling substrates. Taken together, these studies suggest that activation of Epac represents a parallel cAMP-dependent pathway that can inhibit reconsolidation of cocaine-cue memories and reduce the ability of the cue to produce reinstatement of cocaine-seeking behavior.

  7. A G-protein-coupled receptor regulation pathway in cytochrome P450-mediated permethrin-resistance in mosquitoes, Culex quinquefasciatus.

    Science.gov (United States)

    Li, Ting; Cao, Chuanwang; Yang, Ting; Zhang, Lee; He, Lin; Xi, Zhiyong; Bian, Guowu; Liu, Nannan

    2015-12-10

    Rhodopsin-like G protein-coupled receptors (GPCRs) are known to be involved in the GPCR signal transduction system and regulate many essential physiological processes in organisms. This study, for the first time, revealed that knockdown of the rhodopsin-like GPCR gene in resistant mosquitoes resulted in a reduction of mosquitoes' resistance to permethrin, simultaneously reducing the expression of two cAMP-dependent protein kinase A genes (PKAs) and four resistance related cytochrome P450 genes. The function of rhodopsin-like GPCR was further confirmed using transgenic lines of Drosophila melanogaster, in which the tolerance to permethrin and the expression of Drosophila resistance P450 genes were both increased. The roles of GPCR signaling pathway second messenger cyclic adenosine monophosphate (cAMP) and downstream effectors PKAs in resistance were investigated using cAMP production inhibitor Bupivacaine HCl and the RNAi technique. Inhibition of cAMP production led to significant decreases in both the expression of four resistance P450 genes and two PKA genes and mosquito resistance to permethrin. Knockdown of the PKA genes had shown the similar effects on permethrin resistance and P450 gene expression. Taken together, our studies revealed, for the first time, the role of the GPCR/cAMP/PKA-mediated regulatory pathway governing P450 gene expression and P450-mediated resistance in Culex mosquitoes.

  8. Proteomic and Metabolic Analyses of S49 Lymphoma Cells Reveal Novel Regulation of Mitochondria by cAMP and Protein Kinase A.

    Science.gov (United States)

    Wilderman, Andrea; Guo, Yurong; Divakaruni, Ajit S; Perkins, Guy; Zhang, Lingzhi; Murphy, Anne N; Taylor, Susan S; Insel, Paul A

    2015-09-04

    Cyclic AMP (cAMP), acting via protein kinase A (PKA), regulates many cellular responses, but the role of mitochondria in such responses is poorly understood. To define such roles, we used quantitative proteomic analysis of mitochondria-enriched fractions and performed functional and morphologic studies of wild-type (WT) and kin(-) (PKA-null) murine S49 lymphoma cells. Basally, 75 proteins significantly differed in abundance between WT and kin(-) S49 cells. WT, but not kin(-), S49 cells incubated with the cAMP analog 8-(4-chlorophenylthio)adenosine cAMP (CPT-cAMP) for 16 h have (a) increased expression of mitochondria-related genes and proteins, including ones in pathways of branched-chain amino acid and fatty acid metabolism and (b) increased maximal capacity of respiration on branched-chain keto acids and fatty acids. CPT-cAMP also regulates the cellular rate of ATP-utilization, as the rates of both ATP-linked respiration and proton efflux are decreased in WT but not kin(-) cells. CPT-cAMP protected WT S49 cells from glucose or glutamine deprivation, In contrast, CPT-cAMP did not protect kin(-) cells or WT cells treated with the PKA inhibitor H89 from glutamine deprivation. Under basal conditions, the mitochondrial structure of WT and kin(-) S49 cells is similar. Treatment with CPT-cAMP produced apoptotic changes (i.e. decreased mitochondrial density and size and loss of cristae) in WT, but not kin(-) cells. Together, these findings show that cAMP acts via PKA to regulate multiple aspects of mitochondrial function and structure. Mitochondrial perturbation thus likely contributes to cAMP/PKA-mediated cellular responses. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  9. A cAMP Biosensor-Based High-Throughput Screening Assay for Identification of Gs-Coupled GPCR Ligands and Phosphodiesterase Inhibitors

    DEFF Research Database (Denmark)

    Vedel, Line; Bräuner-Osborne, Hans; Mathiesen, Jesper Mosolff

    2015-01-01

    Cyclic adenosine 3',5'-monophosphate (cAMP) is an important second messenger, and quantification of intracellular cAMP levels is essential in studies of G protein-coupled receptors (GPCRs). The intracellular cAMP levels are regulated by the adenylate cyclase (AC) upon activation of either Gs- or Gi......-coupled GPCRs, which leads to increased or decreased cAMP levels, respectively. Here we describe a real-time Förster resonance energy transfer (FRET)-based cAMP high-throughput screening (HTS) assay for identification and characterization of Gs-coupled GPCR ligands and phosphodiesterase (PDE) inhibitors...... also observed for the other representative Gs-coupled GPCRs tested, GLP-1R and GlucagonR. The FRET-based cAMP biosensor assay is robust, reproducible, and inexpensive with good Z factors and is highly applicable for HTS....

  10. 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.

  11. Human GIP(3-30)NH inhibits G protein-dependent as well as G protein-independent signaling and is selective for the GIP receptor with high-affinity binding to primate but not rodent GIP receptors

    DEFF Research Database (Denmark)

    Gabe, Maria Buur Nordskov; Sparre-Ulrich, Alexander Hovard; Pedersen, Mie Fabricius

    2018-01-01

    GIP(3-30)NH2is a high affinity antagonist of the GIP receptor (GIPR) in humans inhibiting insulin secretion via G protein-dependent pathways. However, its ability to inhibit G protein-independent signaling is unknown. Here we determine its action on arrestin-recruitment and receptor internalization...... using human125I-GIP(3-30)NH2. The selectivity of human GIP(3-30)NH2was examined by testing for agonistic and antagonistic properties on 62 human GPCRs. Human GIP(3-30)NH2inhibited GIP(1-42)-induced cAMP and β-arrestin 1 and 2 recruitment on the human GIPR and Schild plot analysis showed competitive...

  12. Antiproliferative effect of brief exposure to cholera toxin in vascular smooth muscle cells: role of cAMP and protein kinase A.

    Science.gov (United States)

    Thorin-Trescases, N; Orlov, S N; Taurin, S; Dulin, N O; Allen, B G; deBlois, D; Tremblay, J; Pshezhetsky, A V; Hamet, P

    2001-06-01

    The effect of cholera toxin (CTX), an activator of the adenylate cyclase-coupled G protein alpha(s) subunit, was studied on cultured vascular smooth muscle cell (VSMC) proliferation. Continuous exposure (48 h) to CTX as well as 2-min pretreatment of VSMC with CTX led to the same level of cAMP production, inhibition of DNA synthesis, and arrest in the G1 phase without induction of necrosis or apoptosis in VSMC. Protein kinase A (PKA) activity in CTX-pretreated cells was transiently elevated by 3-fold after 3 h of incubation, whereas after 48 h it was reduced by 2-fold compared with baseline values without modulation of the expression of its catalytic alpha subunit. The PKA inhibitors H89 and KT 5720 did not protect VSMC from the antiproliferative effect of CTX. Two-dimensional electrophoresis was used to analyze the influence of CTX on protein phosphorylation. After 3 h of incubation of CTX-pretreated cells, we observed both newly-phosphorylated and dephosphorylated proteins (77 and 50 protein species, respectively). After 24 h of incubation, the number of phosphorylated proteins in CTX-treated cells was decreased to 39, whereas the number of dephosphorylated proteins was increased to 106. In conclusion, brief exposure to CTX leads to full-scale activation of cAMP signaling and evokes VSMC arrest in the G1 phase.

  13. cAMP response element binding protein (CREB activates transcription via two distinct genetic elements of the human glucose-6-phosphatase gene

    Directory of Open Access Journals (Sweden)

    Stefano Luisa

    2005-01-01

    Full Text Available Abstract Background The enzyme glucose-6-phosphatase catalyzes the dephosphorylation of glucose-6-phosphatase to glucose, the final step in the gluconeogenic and glycogenolytic pathways. Expression of the glucose-6-phosphatase gene is induced by glucocorticoids and elevated levels of intracellular cAMP. The effect of cAMP in regulating glucose-6-phosphatase gene transcription was corroborated by the identification of two genetic motifs CRE1 and CRE2 in the human and murine glucose-6-phosphatase gene promoter that resemble cAMP response elements (CRE. Results The cAMP response element is a point of convergence for many extracellular and intracellular signals, including cAMP, calcium, and neurotrophins. The major CRE binding protein CREB, a member of the basic region leucine zipper (bZIP family of transcription factors, requires phosphorylation to become a biologically active transcriptional activator. Since unphosphorylated CREB is transcriptionally silent simple overexpression studies cannot be performed to test the biological role of CRE-like sequences of the glucose-6-phosphatase gene. The use of a constitutively active CREB2/CREB fusion protein allowed us to uncouple the investigation of target genes of CREB from the variety of signaling pathways that lead to an activation of CREB. Here, we show that this constitutively active CREB2/CREB fusion protein strikingly enhanced reporter gene transcription mediated by either CRE1 or CRE2 derived from the glucose-6-phosphatase gene. Likewise, reporter gene transcription was enhanced following expression of the catalytic subunit of cAMP-dependent protein kinase (PKA in the nucleus of transfected cells. In contrast, activating transcription factor 2 (ATF2, known to compete with CREB for binding to the canonical CRE sequence 5'-TGACGTCA-3', did not transactivate reporter genes containing CRE1, CRE2, or both CREs derived from the glucose-6-phosphatase gene. Conclusions Using a constitutively active CREB2

  14. Receptor activity-modifying protein dependent and independent activation mechanisms in the coupling of calcitonin gene-related peptide and adrenomedullin receptors to Gs.

    Science.gov (United States)

    Woolley, Michael J; Reynolds, Christopher A; Simms, John; Walker, Christopher S; Mobarec, Juan Carlos; Garelja, Michael L; Conner, Alex C; Poyner, David R; Hay, Debbie L

    2017-10-15

    Calcitonin gene-related peptide (CGRP) or adrenomedullin (AM) receptors are heteromers of the calcitonin receptor-like receptor (CLR), a class B G protein-coupled receptor, and one of three receptor activity-modifying proteins (RAMPs). How CGRP and AM activate CLR and how this process is modulated by RAMPs is unclear. We have defined how CGRP and AM induce Gs-coupling in CLR-RAMP heteromers by measuring the effect of targeted mutagenesis in the CLR transmembrane domain on cAMP production, modeling the active state conformations of CGRP and AM receptors in complex with the Gs C-terminus and conducting molecular dynamics simulations in an explicitly hydrated lipidic bilayer. The largest effects on receptor signaling were seen with H295A5.40b, I298A5.43b, L302A5.47b, N305A5.50b, L345A6.49b and E348A6.52b, F349A6.53b and H374A7.47b (class B numbering in superscript). Many of these residues are likely to form part of a group in close proximity to the peptide binding site and link to a network of hydrophilic and hydrophobic residues, which undergo rearrangements to facilitate Gs binding. Residues closer to the extracellular loops displayed more pronounced RAMP or ligand-dependent effects. Mutation of H3747.47b to alanine increased AM potency 100-fold in the CGRP receptor. The molecular dynamics simulation showed that TM5 and TM6 pivoted around TM3. The data suggest that hydrophobic interactions are more important for CLR activation than other class B GPCRs, providing new insights into the mechanisms of activation of this class of receptor. Furthermore the data may aid in the understanding of how RAMPs modulate the signaling of other class B GPCRs. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

  15. Losartan decreases vasopressin-mediated cAMP accumulation in the thick ascending limb of the loop of Henle in rats with congestive heart failure

    DEFF Research Database (Denmark)

    Torp, M; Brønd, L; Hadrup, N

    2007-01-01

    receptor type-1 (AT(1)) blockade with losartan. AIM: In this study, we investigated whether CHF rats displayed changes in AVP stimulated cAMP formation in the TAL and examined the role of AT(1) receptor blockade on this system. METHOD: CHF was induced by ligation of the left anterior descending coronary...... artery (LAD). SHAM-operated rats were used as controls. Half of the rats were treated with losartan (10 mg kg day(-1) i.p.). RESULTS: CHF rats were characterized by increased left ventricular end diastolic pressure. Measurement of cAMP in isolated outer medullary TAL showed that both basal and AVP (10......(-6) m) stimulated cAMP levels were significantly increased in CHF rats (25.52 +/- 4.49 pmol cAMP microg(-1) protein, P Losartan significantly reduced the basal level of cAMP in CHF rats (CHF: 12.56 +/- 1.93 fmol...

  16. The identification of novel proteins that interact with the GLP-1 receptor and restrain its activity.

    Science.gov (United States)

    Huang, X; Dai, F F; Gaisano, G; Giglou, K; Han, J; Zhang, M; Kittanakom, S; Wong, V; Wei, L; Showalter, A D; Sloop, K W; Stagljar, I; Wheeler, M B

    2013-09-01

    Glucagon-like peptide 1 receptor (GLP-1R) controls diverse physiological functions in tissues including the pancreatic islets, brain, and heart. To understand the mechanisms that control glucagon-like peptide 1 (GLP-1) signaling better, we sought to identify proteins that interact with the GLP-1R using a membrane-based split ubiquitin yeast two-hybrid (MYTH) assay. A screen of a human fetal brain cDNA prey library with an unliganded human GLP-1R as bait in yeast revealed 38 novel interactor protein candidates. These interactions were confirmed in mammalian Chinese hamster ovarian cells by coimmunoprecipitation. Immunofluorescence was used to show subcellular colocalization of the interactors with GLP-1R. Cluster analysis revealed that the interactors were primarily associated with signal transduction, metabolism, and cell development. When coexpressed with the GLP-1R in Chinese hamster ovarian cells, 15 interactors significantly altered GLP-1-induced cAMP accumulation. Surprisingly, all 15 proteins inhibited GLP-1-activated cAMP. Given GLP-1's prominent role as an incretin, we then focused on 3 novel interactors, SLC15A4, APLP1, and AP2M1, because they are highly expressed and localized to the membrane in mouse insulinoma β-cells. Small interfering RNA-mediated knockdown of each candidate gene significantly enhanced GLP-1-induced insulin secretion. In conclusion, we have generated a novel GLP-1R-protein interactome, identifying several interactors that suppress GLP-1R signaling. We suggest that the inhibition of these interactors may serve as a novel strategy to enhance GLP-1R activity.

  17. cAMP response element-binding protein and Yes-associated protein form a feedback loop that promotes neurite outgrowth.

    Science.gov (United States)

    Chen, Lei; Feng, Peimin; Peng, Anjiao; Qiu, Xiangmiao; Zhu, Xi; He, Shixu; Zhou, Dong

    2017-08-31

    The cAMP response element-binding (CREB) protein is a member of the CREB/activating transcription factor family that is activated by various extracellular stimuli. It has been shown that CREB-dependent transcription stimulation plays a key role in neuronal differentiation and plasticity, but the underlying mechanisms remain largely elusive. Here, we show that Yes-associated protein (YAP) is a direct target induced by CREB upon retinoic acid (RA)-induced neurite outgrowth stimuli in N2a cells. Interestingly, YAP knockout using the CRISPR/Cas9 system inhibits neuronal differentiation and reduced neurite length. We further show that YAP could directly bind to CREB via its N-terminal region, and loss of YAP results in instability of phosphorylated CREB upon neurite outgrowth stimuli. Transient expression of YAP could largely restore CREB expression and neurite outgrowth in YAP knockout cells. Together, our results suggest that CREB and YAP form a positive feedback loop that is critical to maintain the stability of phosphorylated CREB and promote neurite outgrowth. © 2017 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.

  18. The melanocortin receptors and their accessory proteins

    Directory of Open Access Journals (Sweden)

    Shwetha eRamachandrappa

    2013-02-01

    Full Text Available The five melanocortin receptors named MC1R-MC5R have diverse physiological roles encompassing pigmentation, steroidogenesis, energy homeostasis and feeding behaviour as well as exocrine function. Since their identification almost 20 years ago much has been learnt about these receptors. As well as interacting with their endogenous ligands the melanocortin peptides, there is now a growing list of important peptides that can modulate the way these receptors signal, acting as agonists, antagonists and inverse agonists. The discovery of MRAPs as a novel accessory factor to the melanocortin receptors provides further insight into the regulation of these important GPCRs.

  19. The urokinase receptor associated protein (uPARAP/endo180)

    DEFF Research Database (Denmark)

    Engelholm, L H; Nielsen, B S; Danø, K

    2001-01-01

    of this proteolytic system. uPARAP is a high molecular weight type-1 membrane protein, belonging to the macrophage mannose receptor protein family. On the surface of certain cells, uPARAP forms a ternary complex with the pro-form of the urokinase-type plasminogen activator (uPA) and its primary receptor (uPAR). While......The urokinase-mediated plasminogen activation system plays a central role in the extracellular proteolytic degradation reactions in cancer invasion. In this review article we discuss a number of recent findings identifying a new cellular receptor protein, uPARAP, that interacts with components...

  20. Protein intake during training sessions has no effect on performance and recovery during a strenuous training camp for elite cyclists

    DEFF Research Database (Denmark)

    Hansen, Mette; Bangsbo, Jens; Jensen, Jørgen

    2016-01-01

    BACKGROUND: Training camps for top-class endurance athletes place high physiological demands on the body. Focus on optimizing recovery between training sessions is necessary to minimize the risk of injuries and improve adaptations to the training stimuli. Carbohydrate supplementation during sessi...

  1. Alteration of Receptor/G-protein Interaction by Putative Endogenous Protein Kinase Activity in Dictyostelium discoideum Membranes

    NARCIS (Netherlands)

    Haastert, Peter J.M. van

    1987-01-01

    Membranes of Dictyostelium discoideum cells were incubated under phosphorylation conditions and washed, and the effects on cAMP binding to chemotactic receptors in the absence and presence of guanosine 5’-O-(3-thiotriphosphate) (GTPγS) were investigated. Most experiments were done with adenosine

  2. New factors influencing G protein coupled receptors' system functions

    African Journals Online (AJOL)

    New factors such as the G protein coupled receptor (GPCR) surrounding's chemical environment, cell membrane constituents, the existent gap junction, endogenous receptor affinity status and animal species have been shown to influence the GPCR physiology and variations of those factors can modify the functions of the ...

  3. Farnesoid X receptor induces Takeda G-protein receptor 5 cross-talk to regulate bile acid synthesis and hepatic metabolism.

    Science.gov (United States)

    Pathak, Preeti; Liu, Hailiang; Boehme, Shannon; Xie, Cen; Krausz, Kristopher W; Gonzalez, Frank; Chiang, John Y L

    2017-06-30

    The bile acid-activated receptors, nuclear farnesoid X receptor (FXR) and the membrane Takeda G-protein receptor 5 (TGR5), are known to improve glucose and insulin sensitivity in obese and diabetic mice. However, the metabolic roles of these two receptors and the underlying mechanisms are incompletely understood. Here, we studied the effects of the dual FXR and TGR5 agonist INT-767 on hepatic bile acid synthesis and intestinal secretion of glucagon-like peptide-1 (GLP-1) in wild-type, Fxr-/-, and Tgr5-/- mice. INT-767 efficaciously stimulated intracellular Ca2+ levels, cAMP activity, and GLP-1 secretion and improved glucose and lipid metabolism more than did the FXR-selective obeticholic acid and TGR5-selective INT-777 agonists. Interestingly, INT-767 reduced expression of the genes in the classic bile acid synthesis pathway but induced those in the alternative pathway, which is consistent with decreased taurocholic acid and increased tauromuricholic acids in bile. Furthermore, FXR activation induced expression of FXR target genes, including fibroblast growth factor 15, and unexpectedly Tgr5 and prohormone convertase 1/3 gene expression in the ileum. We identified an FXR-responsive element on the Tgr5 gene promoter. Fxr-/- and Tgr5-/- mice exhibited reduced GLP-1 secretion, which was stimulated by INT-767 in the Tgr5-/- mice but not in the Fxr-/- mice. Our findings uncovered a novel mechanism in which INT-767 activation of FXR induces Tgr5 gene expression and increases Ca2+ levels and cAMP activity to stimulate GLP-1 secretion and improve hepatic glucose and lipid metabolism in high-fat diet-induced obese mice. Activation of both FXR and TGR5 may therefore represent an effective therapy for managing hepatic steatosis, obesity, and diabetes. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  4. Novel RNAi-mediated approach to G protein-coupled receptor deorphanization: proof of principle and characterization of a planarian 5-HT receptor.

    Directory of Open Access Journals (Sweden)

    Mostafa Zamanian

    Full Text Available G protein-coupled receptors (GPCRs represent the largest known superfamily of membrane proteins extending throughout the Metazoa. There exists ample motivation to elucidate the functional properties of GPCRs given their role in signal transduction and their prominence as drug targets. In many target organisms, these efforts are hampered by the unreliable nature of heterologous receptor expression platforms. We validate and describe an alternative loss-of-function approach for ascertaining the ligand and G protein coupling properties of GPCRs in their native cell membrane environment. Our efforts are focused on the phylum Platyhelminthes, given the heavy health burden exacted by pathogenic flatworms, as well as the role of free-living flatworms as model organisms for the study of developmental biology. RNA interference (RNAi was used in conjunction with a biochemical endpoint assay to monitor cAMP modulation in response to the translational suppression of individual receptors. As proof of principle, this approach was used to confirm the neuropeptide GYIRFamide as the cognate ligand for the planarian neuropeptide receptor GtNPR-1, while revealing its endogenous coupling to Gα(i/o. The method was then extended to deorphanize a novel Gα(s-coupled planarian serotonin receptor, DtSER-1. A bioinformatics protocol guided the selection of receptor candidates mediating 5-HT-evoked responses. These results provide functional data on a neurotransmitter central to flatworm biology, while establishing the great potential of an RNAi-based deorphanization protocol. Future work can help optimize and adapt this protocol for higher-throughput platforms as well as other phyla.

  5. Real-Time G-Protein-Coupled Receptor Imaging to Understand and Quantify Receptor Dynamics

    Directory of Open Access Journals (Sweden)

    María S. Aymerich

    2011-01-01

    Full Text Available Understanding the trafficking of G-protein-coupled receptors (GPCRs and their regulation by agonists and antagonists is fundamental to develop more effective drugs. Optical methods using fluorescent-tagged receptors and spinning disk confocal microscopy are useful tools to investigate membrane receptor dynamics in living cells. The aim of this study was to develop a method to characterize receptor dynamics using this system which offers the advantage of very fast image acquisition with minimal cell perturbation. However, in short-term assays photobleaching was still a problem. Thus, we developed a procedure to perform a photobleaching-corrected image analysis. A study of short-term dynamics of the long isoform of the dopamine type 2 receptor revealed an agonist-induced increase in the mobile fraction of receptors with a rate of movement of 0.08 μm/s For long-term assays, the ratio between the relative fluorescence intensity at the cell surface versus that in the intracellular compartment indicated that receptor internalization only occurred in cells co-expressing G protein-coupled receptor kinase 2. These results indicate that the lateral movement of receptors and receptor internalization are not directly coupled. Thus, we believe that live imaging of GPCRs using spinning disk confocal image analysis constitutes a powerful tool to study of receptor dynamics.

  6. Cellular prion protein and NMDA receptor modulation: protecting against excitotoxicity

    Directory of Open Access Journals (Sweden)

    Stefanie A.G. Black

    2014-08-01

    Full Text Available Although it is well established that misfolding of the cellular prion protein (PrPC into the beta-sheet-rich, aggregated scrapie conformation (PrPSc causes a variety of transmissible spongiform encephalopathies (TSEs, the physiological roles of PrPC are still incompletely understood. There is accumulating evidence describing the roles of PrPC in neurodegeneration and neuroinflammation. Recently, we identified a functional regulation of NMDA receptors by PrPC that involves formation of a physical protein complex between these proteins. Excessive NMDA receptor activity during conditions such as ischemia mediates enhanced Ca2+ entry into cells and contributes to excitotoxic neuronal death. In addition, NMDA receptors and/or PrPC play critical roles in neuroinflammation and glial cell toxicity. Inhibition of NMDA receptor activity protects against PrPSc-induced neuronal death. Moreover, in mice lacking PrPC, infarct size is increased after focal cerebral ischemia, and absence of PrPC increases susceptibility of neurons to NMDA receptor-dependent death. Recently, PrPC was found to be a receptor for oligomeric beta-amyloid (Abeta peptides, suggesting a role for PrPC in Alzheimer’s disease. Our recent findings suggest that Abeta peptides enhance NMDA receptor current by perturbing the normal copper- and PrPC-dependent regulation of these receptors. Here, we review evidence highlighting a role for PrPC in preventing NMDA receptor-mediated excitotoxicity and inflammation. There is a need for more detailed molecular characterization of PrPC-mediated regulation of NMDA receptors, such as determining which NMDA receptor subunits mediate pathogenic effects upon loss of PrPC-mediated regulation and identifying PrPC binding site(s on the receptor. This knowledge will allow development of novel therapeutic interventions for not only TSEs, but also for Alzheimer’s disease and other neurodegenerative disorders involving dysfunction of PrPC.

  7. Molecular Simulations Reveal an Unresolved Conformation of the Type IA Protein Kinase A Regulatory Subunit and Suggest Its Role in the cAMP Regulatory Mechanism.

    Science.gov (United States)

    Hirakis, Sophia P; Malmstrom, Robert D; Amaro, Rommie E

    2017-08-01

    We identify a previously unresolved, unrecognized, and highly stable conformation of the protein kinase A (PKA) regulatory subunit RIα. This conformation, which we term the "Flipback" structure, bridges conflicting characteristics in crystallographic structures and solution experiments of the PKA RIα heterotetramer. Our simulations reveal a hinge residue, G235, in the B/C helix that is conserved through all isoforms of RI. Brownian dynamics simulations suggest that the Flipback conformation plays a role in cAMP association to the A domain of the R subunit.

  8. The involvement of cAMP in the growth inhibition of filamentous fungus Rhizopus nigricans by steroids.

    Science.gov (United States)

    Jeraj, Natasa; Lenasi, Helena; Breskvar, Katja

    2005-01-01

    Several steroids, in particular progesterone, are toxic for the filamentous fungus Rhizopus nigricans and, at high concentrations, inhibit its growth. Previous studies on this microorganism revealed progesterone specific receptors coupled to G proteins at the plasma membrane. In this study, the next step of steroid signalling in R. nigricans following G protein activation is investigated, together with the possible impact of this pathway on fungal growth inhibition. The intracellular level of cAMP decreased in the presence of steroids, demonstrating the probable involvement of cAMP signalling in the response of R. nigricans to steroids. Results of the growth analysis in the presence of cAMP increasing agents suggest that the role of cAMP in fungal growth inhibition by steroids cannot be ruled out, but it would appear to be minor and not make a major contribution to growth inhibition.

  9. Probing Biased/Partial Agonism at the G Protein-Coupled A2B Adenosine Receptorˇ

    Science.gov (United States)

    Gao, Zhan-Guo; Balasubramanian, Ramachandran; Kiselev, Evgeny; Wei, Qiang; Jacobson, Kenneth A.

    2014-01-01

    G protein-coupled A2B adenosine receptor (AR) regulates numerous important physiological functions, but its activation by diverse A2BAR agonists is poorly profiled. We probed potential partial and/or biased agonism in cell lines expressing variable levels of endogenous or recombinant A2BAR. In cAMP accumulation assays, both 5′-substituted NECA and C2-substituted MRS3997 are full agonists. However, only 5′-substituted adenosine analogs are full agonists in calcium mobilization, ERK1/2 phosphorylation and β-arrestin translocation. A2BAR overexpression in HEK293 cells markedly increased the agonist potency and maximum effect in cAMP accumulation, but less in calcium and ERK1/2. A2BAR siRNA silencing was more effective in reducing the maximum cAMP effect of non-nucleoside agonist BAY60-6583 than NECA's. A quantitative ‘operational model’ characterized C2-substituted MRS3997 as either balanced (cAMP accumulation, ERK1/2) or strongly biased agonist (against calcium, β-arrestin). N6-Substitution biased against ERK1/2 (weakly) and calcium and β-arrestin (strongly) pathways. BAY60-6583 is ERK1/2-biased, suggesting a mechanism distinct from adenosine derivatives. BAY60-6583, as A2BAR antagonist in MIN-6 mouse pancreatic β cells expressing low A2BAR levels, induced insulin release. This is the first relatively systematic study of structure-efficacy relationships of this emerging drug target. PMID:24853985

  10. 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

  11. Receptor Quaternary Organization Explains G Protein-Coupled Receptor Family Structure

    Directory of Open Access Journals (Sweden)

    James H. Felce

    2017-09-01

    Full Text Available The organization of Rhodopsin-family G protein-coupled receptors (GPCRs at the cell surface is controversial. Support both for and against the existence of dimers has been obtained in studies of mostly individual receptors. Here, we use a large-scale comparative study to examine the stoichiometric signatures of 60 receptors expressed by a single human cell line. Using bioluminescence resonance energy transfer- and single-molecule microscopy-based assays, we found that a relatively small fraction of Rhodopsin-family GPCRs behaved as dimers and that these receptors otherwise appear to be monomeric. Overall, the analysis predicted that fewer than 20% of ∼700 Rhodopsin-family receptors form dimers. The clustered distribution of the dimers in our sample and a striking correlation between receptor organization and GPCR family size that we also uncover each suggest that receptor stoichiometry might have profoundly influenced GPCR expansion and diversification.

  12. 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

    Chemokines (chemotactic cytokines) and their associated G protein-coupled receptors (GPCRs) work in a concerted manner to govern immune cell positioning in time and space. Promiscuity of both ligands and receptors, but also biased signaling within the chemokine system, adds to the complexity of how...

  13. Sigma 1 receptor modulation of G-protein-coupled receptor signaling: potentiation of opioid transduction independent from receptor binding.

    Science.gov (United States)

    Kim, Felix J; Kovalyshyn, Ivanka; Burgman, Maxim; Neilan, Claire; Chien, Chih-Cheng; Pasternak, Gavril W

    2010-04-01

    sigma Ligands modulate opioid actions in vivo, with agonists diminishing morphine analgesia and antagonists enhancing the response. Using human BE(2)-C neuroblastoma cells that natively express opioid receptors and human embryonic kidney (HEK) cells transfected with a cloned mu opioid receptor, we now demonstrate a similar modulation of opioid function, as assessed by guanosine 5'-O-(3-[(35)S]thio)triphosphate ([(35)S]GTP gamma S) binding, by sigma(1) receptors. sigma Ligands do not compete opioid receptor binding. Administered alone, neither sigma agonists nor antagonists significantly stimulated [(35)S]GTP gamma S binding. Yet sigma receptor selective antagonists, but not agonists, shifted the EC(50) of opioid-induced stimulation of [(35)S]GTP gamma S binding by 3- to 10-fold to the left. This enhanced potency was seen without a change in the efficacy of the opioid, as assessed by the maximal stimulation of [(35)S]GTP gamma S binding. sigma(1) Receptors physically associate with mu opioid receptors, as shown by coimmunoprecipitation studies in transfected HEK cells, implying a direct interaction between the proteins. Thus, sigma receptors modulate opioid transduction without influencing opioid receptor binding. RNA interference knockdown of sigma(1) in BE(2)-C cells also potentiated mu opioid-induced stimulation of [(35)S]GTP gamma S binding. These modulatory actions are not limited to mu and delta opioid receptors. In mouse brain membrane preparations, sigma(1)-selective antagonists also potentiated both opioid receptor and muscarinic acetylcholine receptor-mediated stimulation of [(35)S]GTP gamma S binding, suggesting a broader role for sigma receptors in modulating G-protein-coupled receptor signaling.

  14. The urokinase receptor associated protein (uPARAP/endo180)

    DEFF Research Database (Denmark)

    Engelholm, L H; Nielsen, B S; Danø, K

    2001-01-01

    The urokinase-mediated plasminogen activation system plays a central role in the extracellular proteolytic degradation reactions in cancer invasion. In this review article we discuss a number of recent findings identifying a new cellular receptor protein, uPARAP, that interacts with components...... of this proteolytic system. uPARAP is a high molecular weight type-1 membrane protein, belonging to the macrophage mannose receptor protein family. On the surface of certain cells, uPARAP forms a ternary complex with the pro-form of the urokinase-type plasminogen activator (uPA) and its primary receptor (uPAR). While...... the biological consequences of this reaction have not yet been verified experimentally, a likely event is ligand internalization because uPARAP is a constitutively recycling internalization receptor. uPARAP also binds at least one component, collagen type V, in the extracellular matrix meshwork, pointing...

  15. SOCS proteins in regulation of receptor tyrosine kinase signaling

    DEFF Research Database (Denmark)

    Kazi, Julhash U.; Kabir, Nuzhat N.; Flores Morales, Amilcar

    2014-01-01

    proteins, SOCS1-7, and cytokine-inducible SH2-containing protein (CIS). A key feature of this family of proteins is the presence of an SH2 domain and a SOCS box. Recent studies suggest that SOCS proteins also play a role in RTK signaling. Activation of RTK results in transcriptional activation of SOCS......-encoding genes. These proteins associate with RTKs through their SH2 domains and subsequently recruit the E3 ubiquitin machinery through the SOCS box, and thereby limit receptor stability by inducing ubiquitination. In a similar fashion, SOCS proteins negatively regulate mitogenic signaling by RTKs. It is also...

  16. Consulting to summer camps.

    Science.gov (United States)

    Ditter, Bob

    2007-10-01

    There has been an increased need for consultation to summer camps from the allied health/mental health fields because camps are available to children with medical and psychological illnesses. Factors in camp programs that are necessary for effective consultation and the various roles a consultant may serve within the camp community are discussed in this article.

  17. Victory Junction Gang Camp

    Science.gov (United States)

    Shell, Ryan

    2007-01-01

    This article describes the Victory Junction Gang Camp, a not-for-profit, NASCAR-themed camp for children with chronic medical conditions that serves 24 different disease groups. The mission of the camp is to give children life-changing camping experiences that are exciting, fun, and empowering in a safe and medically sound environment. While doing…

  18. 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...

  19. 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...

  20. Endothelial protein C receptor in renal tubular epithelial cells and ...

    African Journals Online (AJOL)

    The endothelial protein C receptor (EPCR) plays an important role within the protein C pathway in regulating coagulation and inflammation. It was reported that EPCR was expressed in large vessels, placenta, heart, liver and lung endothelial cell. However, there are a few studies concerned about renal epithelial cells.

  1. Melanocortin receptor binding determinants in the agouti protein.

    Science.gov (United States)

    Kiefer, L L; Veal, J M; Mountjoy, K G; Wilkison, W O

    1998-01-27

    The agouti protein plays an important role in the development of diabetes and obesity in rodents and has been shown to be a potent antagonist of melanocortin receptors. For this reason alanine-scanning mutagenesis was performed on the agouti protein carboxyl terminus to locate residues important for melanocortin receptor binding inhibition. When agouti residues Arg116 and Phe118 are changed to alanine, very large decreases in agouti affinity for melanocortin receptor 1, 3, and 4 result. Mutation of Phe117 to alanine causes a similar increase in agouti KI app at melanocortin receptor 4. Substitution of agouti residue Asp108 with alanine results in large increases in KI app for all three melanocortin receptors examined. All of these residues are conserved in the agouti-related transcript, ART, whose expression is up-regulated in animal models of obesity. The three-dimensional structure of the agouti carboxyl terminus was modeled, and residues which decrease receptor binding by a factor of > or = 15 when mutated to alanine localize to one side of the structure. These agouti variants with altered receptor selectivity may be useful in determining the role of melanocortin receptors in diabetes and obesity.

  2. 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...... a quantitative characterization of the kinetics and affinity of interactions between GPCRs and one of the best characterized PDZ scaffold proteins, postsynaptic density protein 95 (PSD-95), using fluorescence polarization (FP) and surface plasmon resonance (SPR). By comparing these in vitro findings....... The approach can easily be transferred to other receptors and scaffold proteins and this could help accelerate the discovery and quantitative characterization of GPCR-PDZ interactions....

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

    Science.gov (United States)

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

    2008-05-13

    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 processes and GPCR up- and downregulation. GPCR function can also be regulated by several proteins that directly interact with the receptor and thereby modulate receptor activity. An additional mechanism by which receptor signalling is regulated involves an emerging class of proteins, the so-called regulators of G protein signalling (RGS). In this review we will describe some of these control mechanisms in more detail with some specific examples in the cardiovascular system. In addition, we will provide an overview on RGS proteins and the involvement of RGS proteins in cardiovascular function.

  4. 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

  5. G-protein coupled estrogen receptor (GPER) inhibits final oocyte maturation in common carp, Cyprinus carpio.

    Science.gov (United States)

    Majumder, Suravi; Das, Sumana; Moulik, Sujata Roy; Mallick, Buddhadev; Pal, Puja; Mukherjee, Dilip

    2015-01-15

    GPR-30, now named as GPER (G protein-coupled estrogen receptor) was first identified as an orphan receptor and subsequently shown to be required for estrogen-mediated signaling in certain cancer cells. Later studies demonstrated that GPER has the characteristics of a high affinity estrogen membrane receptor on Atlantic croaker and zebra fish oocytes and mediates estrogen inhibition of oocyte maturation in these two distantly related teleost. To determine the broad application of these findings to other teleost, expression of GPER mRNA and its involvement in 17β-estradiol mediated inhibition of oocyte maturation in other cyprinid, Cyprinus carpio was investigated. Carp oocytes at pre-vitellogenic, late-vitellogenic and post-vitellogenic stages of development contained GPER mRNA and its transcribed protein with a maximum at late-vitellogenic oocytes. Ovarian follicular cells did not express GPER mRNA. Carp oocytes GPER mRNA was essentially identical to that found in other perciformes and cyprinid fish oocytes. Both spontaneous and 17,20β-dihydroxy-4-pregnen-3-one (17,20β-P)-induced oocyte maturation in carp was significantly decreased when they were incubated with either E2, or GPER agonist G-1. On the other hand spontaneous oocyte maturation was significantly increased when carp ovarian follicles were incubated with an aromatase inhibitor, fadrozole, GPER antagonist, G-15 and enzymatic removal of the ovarian follicle cell layers. This increase in oocyte maturation was partially reversed by co-treatment with E2. Consistent with previous findings with human and fish GPR30, E2 treatment in carp oocytes caused increase in cAMP production and simultaneously decrease in oocyte maturation, which was inhibited by the addition of 17,20β-P. The results suggest that E2 and GPER play a critical role in regulating re-entry in to meiotic cell cycle in carp oocytes. Copyright © 2014 Elsevier Inc. All rights reserved.

  6. The E92K Melanocortin 1 Receptor Mutant Induces cAMP Production and Arrestin Recruitment but Not ERK Activity Indicating Biased Constitutive Signaling

    Science.gov (United States)

    Benned-Jensen, Tau; Mokrosinski, Jacek; Rosenkilde, Mette M.

    2011-01-01

    Background The melanocortin 1 receptor (MC1R) constitutes a key regulator of melanism. Consequently, many naturally-occurring MC1R mutations are associated with a change in color. An example is the Glu-to-Lys substitution found at position II:20/2.60 in the top of transmembrane helix II which has been identified in melanic mice and several other species. This mutation induces a pronounced increase in MC1R constitutive activity suggesting a link between constitutive activity and melanism which is corroborated by the attenuation of α-melanocyte stimulating hormone (αMSH) induced activation. However, the mechanism by which the mutation induces constitutive activity is currently not known. Methodology/Principal Findings Here we characterize the constitutive activity, cell surface expression and internalization of the mouse mutant, Mc1r E92K. As previously reported, only positively charged residues at position II:20/2.60 induced an increase in constitutive activity as measured by cAMP accumulation and CREB activation. Furthermore, the mutation induced a constitutive recruitment of β-arrestin. This phenomenon is only observed in MC1R, however, as the equivalent mutations in MC2-5R had no effect on receptor signaling. Interestingly, the mutation did not induce constitutive ERK1/2 phosphorylation or increase the internalization rate indicating the constitutive activity to be biased. Finally, to identify regions of importance for the increased constitutive activity of Mc1r E92K, we employed a chimeric approach and identified G102 and L110 in the extracellular loop 1 to be selectively important for the constitutive activity as this, but not αMSH-mediated activation, was abolished upon Ala substitution. Conclusions/Significance It is concluded that the E92K mutation induces an active conformation distinct from that induced by αMSH and that the extracellular loop 1 is involved in maintaining this conformational state. In turn, the results suggest that in MC1R, which lacks

  7. Insulin-like growth factor-1 (IGF-1 induces the activation/phosphorylation of Akt kinase and cAMP response element-binding protein (CREB by activating different signaling pathways in PC12 cells

    Directory of Open Access Journals (Sweden)

    Zheng Wen-Hua

    2006-06-01

    Full Text Available Abstract Background Insulin-like growth factor-1 (IGF-1 is a polypeptide growth factor with a variety of functions in both neuronal and non-neuronal cells. IGF-1 plays anti-apoptotic and other functions by activating multiple signaling pathways including Akt kinase, a serine/threonine kinase essential for cell survival. The nuclear transcription factor cAMP response element-binding protein (CREB may also be involved although relationships between these two proteins in IGF-1 receptor signaling and protection is not clear, especially in neuronal cells. Results IGF-1, in a concentration- and time-dependent manner, induces the activation/phosphorylation of Akt and CREB in PC12 cells by activating different signaling pathways. IGF-1 induced a sustained phosphorylation of Akt while only a transient one was seen for CREB. The phosphorylation of Akt is mediated by the PI3 kinase pathway while that of CREB is dependent on the activation of both MAPK kinase and p38 MAPK. Moreover, the stimulation of PKC attenuated the phosphorylation of Akt induced by IGF-1 while enhancing that of CREB. Survival assays with various kinase inhibitors suggested that the activation/phosphorylation of both Akt and CREB contributes to IGF-1 mediated cell survival in PC12 cells. Conclusion These data suggest that IGF-1 induced the activation of Akt and CREB using distinct pathways in PC12 cells.

  8. Plant nuclear hormone receptors: a role for small molecules in protein-protein interactions.

    Science.gov (United States)

    Lumba, Shelley; Cutler, Sean; McCourt, Peter

    2010-01-01

    Plant hormones are a group of chemically diverse small molecules that direct processes ranging from growth and development to biotic and abiotic stress responses. Surprisingly, genome analyses suggest that classic animal nuclear hormone receptor homologs do not exist in plants. It now appears that plants have co-opted several protein families to perceive hormones within the nucleus. In one solution to the problem, the hormones auxin and jasmonate (JA) act as “molecular glue” that promotes protein-protein interactions between receptor F-boxes and downstream corepressor targets. In another solution, gibberellins (GAs) bind and elicit a conformational change in a novel soluble receptor family related to hormone-sensitive lipases. Abscisic acid (ABA), like GA, also acts through an allosteric mechanism involving a START-domain protein. The molecular identification of plant nuclear hormone receptors will allow comparisons with animal nuclear receptors and testing of fundamental questions about hormone function in plant development and evolution.

  9. Activation of Exchange Protein Activated by Cyclic-AMP Enhances Long-Lasting Synaptic Potentiation in the Hippocampus

    Science.gov (United States)

    Gelinas, Jennifer N.; Banko, Jessica L.; Peters, Melinda M.; Klann, Eric; Weeber, Edwin J.; Nguyen, Peter V.

    2008-01-01

    cAMP is a critical second messenger implicated in synaptic plasticity and memory in the mammalian brain. Substantial evidence links increases in intracellular cAMP to activation of cAMP-dependent protein kinase (PKA) and subsequent phosphorylation of downstream effectors (transcription factors, receptors, protein kinases) necessary for long-term…

  10. Reciprocal Roles of Angiotensin II and Angiotensin II Receptors Blockade (ARB in Regulating Cbfa1/RANKL via cAMP Signaling Pathway: Possible Mechanism for Hypertension-Related Osteoporosis and Antagonistic Effect of ARB on Hypertension-Related Osteoporosis

    Directory of Open Access Journals (Sweden)

    Ji-Yao Li

    2011-06-01

    Full Text Available Hypertension is a risk factor for osteoporosis. Animal and epidemiological studies demonstrate that high blood pressure is associated with increased calcium loss, elevated parathyroid hormone, and increased calcium movement from bone. However, the mechanism responsible for hypertension-related osteoporosis remains elusive. Recent epidemiological studies indicate the benefits of Angiotensin II Receptors Blockade (ARB on decreasing fracture risks. Since receptors for angiotensin II, the targets of ARB, are expressed in both osteoblasts and osteoclasts, we postulated that angiotensin II plays an important role in hypertension-related osteoporosis. Cbfa1 and RANKL, the important factors for maintaining bone homeostasis and key mediators in controlling osteoblast and osteoclast differentiation, are both regulated by cAMP-dependent signaling. Angiotensin II along with factors such as LDL, HDL, NO and homocysteine that are commonly altered both in hypertension and osteoporosis, can down-regulate the expression of Cbfa1 but up-regulate RANKL expression via the cAMP signaling pathway. We thus hypothesized that, by altering the ratio of Cbfa1/RANKL expression via the cAMP-dependent pathway, angiotensin II differently regulates osteoblast and osteoclast differentiation leading to enhanced bone resorption and reduced bone formation. Since ARB can antagonize the adverse effect of angiotensin II on bone by lowering cAMP levels and modifying other downstream targets, including LDL, HDL, NO and Cbfa1/RANKL, we propose the hypothesis that the antagonistic effects of ARB may also be exerted via cAMP signaling pathway.

  11. The effect of mitragynine on cAMP formation and mRNA expression of mu-opioid receptors mediated by chronic morphine treatment in SK-N-SH neuroblastoma cell.

    Science.gov (United States)

    Jamil, Mohd Fadzly Amar; Subki, Mohd Ferdaues Mohd; Lan, Tan Mei; Majid, Mohamed Isa Abdul; Adenan, Mohd Ilham

    2013-06-21

    [corrected] Mitragynine is an indole alkaloid compound of Mitragyna speciosa (M. speciosa) Korth. (Rubiaceae). This plant is native to the southern regions of Thailand and northern regions of Malaysia and is frequently used to manage the withdrawal symptoms in both countries. To investigate the effect of mitragynine after chronic morphine treatment on cyclic AMP (cAMP) level and mRNA expression of mu-opioid receptor (MOR) in human neuroblastoma SK-N-SH cell. Mitragynine was isolated from the Mitragyna speciosa plant using the acid-base extraction method. The cAMP level upon forskolin stimulation in the cells was determined using the Calbiochem(®) Direct Immunoassay Kit. The mRNA expression of the MOR was carried out using quantitative RT-PCR. Cotreatment and pretreatment of morphine and mitragynine significantly reduced the production of cAMP level at a lower concentration of mitragynine while the higher concentration of this compound could lead to the development of tolerance and dependence as shown by the increase of the cAMP level production in foskolin stimulation. In MOR mRNA expression study, cotreatment of morphine with mitragynine significantly reduced the down-regulation of MOR mRNA expression as compared to morphine treatment only. These finding suggest that mitragynine could possibly avoid the tolerance and dependence on chronic morphine treatment by reducing the up-regulation of cAMP level as well as reducing the down-regulation of MOR at a lower concentration of mitragynine. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

  12. The repertoire of trace amine G-protein-coupled receptors

    DEFF Research Database (Denmark)

    Gloriam, David E.; Bjarnadóttir, Thóra K; Yan, Yi-Lin

    2005-01-01

    Trace amines, such as tyramine, beta-phenylethylamine, tryptamine, and octopamine, are present in trace levels in nervous systems and bind a specific family of G-protein-coupled receptors (GPCR), but the function or origin of this system is not well understood. We searched the genomes of several ...... ancestor of vertebrate TA-receptors arose before the split of the ray-finned and lobe-finned fishes. The evolutionary history of the TA-receptors is more complex than for most other GPCR families and here we suggest a mechanism by which they may have arisen....

  13. Regulation of Insulin Receptor Trafficking by Bardet Biedl Syndrome Proteins.

    Directory of Open Access Journals (Sweden)

    Rachel D Starks

    2015-06-01

    Full Text Available Insulin and its receptor are critical for the regulation of metabolic functions, but the mechanisms underlying insulin receptor (IR trafficking to the plasma membrane are not well understood. Here, we show that Bardet Biedl Syndrome (BBS proteins are necessary for IR localization to the cell surface. We demonstrate that the IR interacts physically with BBS proteins, and reducing the expression of BBS proteins perturbs IR expression in the cell surface. We show the consequence of disrupting BBS proteins for whole body insulin action and glucose metabolism using mice lacking different BBS genes. These findings demonstrate the importance of BBS proteins in underlying IR cell surface expression. Our data identify defects in trafficking and localization of the IR as a novel mechanism accounting for the insulin resistance commonly associated with human BBS. This is supported by the reduced surface expression of the IR in fibroblasts derived from patients bearing the M390R mutation in the BBS1 gene.

  14. Multifactorial Regulation of G Protein-Coupled Receptor Endocytosis

    Science.gov (United States)

    Zhang, Xiaohan; Kim, Kyeong-Man

    2017-01-01

    Endocytosis is a process by which cells absorb extracellular materials via the inward budding of vesicles formed from the plasma membrane. Receptor-mediated endocytosis is a highly selective process where receptors with specific binding sites for extracellular molecules internalize via vesicles. G protein-coupled receptors (GPCRs) are the largest single family of plasma-membrane receptors with more than 1000 family members. But the molecular mechanisms involved in the regulation of GPCRs are believed to be highly conserved. For example, receptor phosphorylation in collaboration with β-arrestins plays major roles in desensitization and endocytosis of most GPCRs. Nevertheless, a number of subsequent studies showed that GPCR regulation, such as that by endocytosis, occurs through various pathways with a multitude of cellular components and processes. This review focused on i) functional interactions between homologous and heterologous pathways, ii) methodologies applied for determining receptor endocytosis, iii) experimental tools to determine specific endocytic routes, iv) roles of small guanosine triphosphate-binding proteins in GPCR endocytosis, and v) role of post-translational modification of the receptors in endocytosis. PMID:28035080

  15. Toward fluorescent probes for G-protein-coupled receptors (GPCRs).

    Science.gov (United States)

    Ma, Zhao; Du, Lupei; Li, Minyong

    2014-10-23

    G-protein-coupled receptors (GPCRs), a superfamily of cell-surface receptors that are the targets of about 40% of prescription drugs on the market, can sense numerous critical extracellular signals. Recent breakthroughs in structural biology, especially in holo-form X-ray crystal structures, have contributed to our understanding of GPCR signaling. However, actions of GPCRs at the cellular and molecular level, interactions between GPCRs, and the role of protein dynamics in receptor activities still remain controversial. To overcome these dilemmas, fluorescent probes of GPCRs have been employed, which have advantages of in vivo safety and real-time monitoring. Various probes that depend on specific mechanisms and/or technologies have been used to study GPCRs. The present review focuses on surveying the design and applications of fluorescent probes for GPCRs that are derived from small molecules or using protein-labeling techniques, as well as discussing some design strategies for new probes.

  16. G protein-coupled receptor accessory proteins and signaling: pharmacogenomic insights.

    Science.gov (United States)

    Thompson, Miles D; Cole, David E C; Jose, Pedro A; Chidiac, Peter

    2014-01-01

    The identification and characterization of the genes encoding G protein-coupled receptors (GPCRs) and the proteins necessary for the processes of ligand binding, GPCR activation, inactivation, and receptor trafficking to the membrane are discussed in the context of human genetic disease. In addition to functional GPCR variants, the identification of genetic disruptions affecting proteins necessary to GPCR functions have provided insights into the function of these pathways. Gsα and Gβ subunit polymorphisms have been found to result in complex phenotypes. Disruptions in accessory proteins that normally modify or organize heterotrimeric G-protein coupling may also result in disease states. These include the contribution of variants of the regulator of G protein signaling (RGS) protein to hypertension; the role variants of the activator of G protein signaling (AGS) proteins to phenotypes (such as the type III AGS8 variant to hypoxia); the contribution of G protein-coupled receptor kinase (GRK) proteins, such as GRK4, in disorders such as hypertension. The role of accessory proteins in GPCR structure and function is discussed in the context of genetic disorders associated with disruption of the genes that encode them. An understanding of the pharmacogenomics of GPCR and accessory protein signaling provides the basis for examining both GPCR pharmacogenetics and the genetics of monogenic disorders that result from disruption of given receptor systems.

  17. All three LDL receptor homology regions of the LDL receptor-related protein bind multiple ligands.

    Science.gov (United States)

    Croy, Johnny E; Shin, William D; Knauer, Mary F; Knauer, Daniel J; Komives, Elizabeth A

    2003-11-11

    The three complete human LDL receptor homology regions of the LDL receptor-related protein (sLRP2, sLRP3, and sLRP4) have been expressed in Pichia pastoris SMD1168 with constitutive coexpression of the receptor-associated protein (RAP). Each sLRP was purified to homogeneity after deglycosylation using a combination of anion-exchange and size exclusion chromatography. Mass spectrometry and N-terminal sequencing confirmed the identity of each fragment at purified yields of several milligrams per liter. Despite the large number of disulfide linkages and glycosylation sites in each LDL receptor homology region (sLRP), all were shown to be competent for binding to several LRP1 ligands. Each sLRP also bound human RAP, which is thought to be a generalized receptor antagonist, in solution-binding experiments. As expected, sLRP2 bound the receptor-binding domain of alpha(2)-macroglobulin (residues 1304-1451). All three sLRPs bound human apolipoprotein-enriched beta very low density lipoprotein, the canonical ligand for this receptor. All three sLRPs also bound lactoferrin and thrombin-protease nexin 1 complexes. Only sLRP4 bound thrombin-antithrombin III complexes. The results show that binding-competent LDL receptor homology regions (sLRPs) can be produced in high yield in P. pastoris and readily purified. Each sLRP has binding sites for multiple ligands, but not all ligand binding could be competed by RAP.

  18. Afamin stimulates osteoclastogenesis and bone resorption via Gi-coupled receptor and Ca2+/calmodulin-dependent protein kinase (CaMK) pathways.

    Science.gov (United States)

    Kim, B J; Lee, Y S; Lee, S Y; Park, S Y; Dieplinger, H; Yea, K; Lee, S H; Koh, J M; Kim, G S

    2013-11-01

    Afamin was recently identified as a novel osteoclast-derived coupling factor that can stimulate the in vitro and in vivo migration of preosteoblasts. In order to understand in more detail the biological roles of afamin in bone metabolism, we investigated its effects on osteoclastic differentiation and bone resorption. Osteoclasts were differentiated from mouse bone marrow cells. Tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells were considered as osteoclasts, and the resorption area was determined by incubating the cells on dentine discs. The intracellular cAMP level was determined using a direct enzyme immunoassay. Signaling pathways were investigated using western blot and RT-PCR. Recombinant afamin was administered exogenously to bone cell cultures. Afamin stimulated both osteoclastogenesis and in vitro bone resorption. Consistently, the expressions of osteoclast differentiation markers were significantly increased by afamin. Although afamin mainly affected the late-differentiation stages of osteoclastogenesis, the expression levels of receptor activator of nuclear factor-κB ligand (RANKL)-dependent signals were not changed. Afamin markedly decreased the levels of intracellular cAMP with reversal by pretreatment with pertussis toxin (PTX), a specific inhibitor of Gi-coupled receptor signaling. In addition, PTX almost completely blocked afamin-stimulated osteoclastogenesis. Furthermore, pretreatment with KN93 and STO609 - Ca2+/cal - mo dulin-dependent protein kinase (CaMK) and CaMK kinase inhibitors, respectively - significantly prevented decreases in the intracellular cAMP level by afamin while attenuating afamin-stimulated osteoclastogenesis. Afamin enhances osteoclastogenesis by decreasing intracellular cAMP levels via Gi-coupled receptor and CaMK pathways.

  19. Pancreatic Beta Cell G-Protein Coupled Receptors and Second Messenger Interactions: A Systems Biology Computational Analysis.

    Directory of Open Access Journals (Sweden)

    Leonid E Fridlyand

    Full Text Available Insulin secretory in pancreatic beta-cells responses to nutrient stimuli and hormonal modulators include multiple messengers and signaling pathways with complex interdependencies. Here we present a computational model that incorporates recent data on glucose metabolism, plasma membrane potential, G-protein-coupled-receptors (GPCR, cytoplasmic and endoplasmic reticulum calcium dynamics, cAMP and phospholipase C pathways that regulate interactions between second messengers in pancreatic beta-cells. The values of key model parameters were inferred from published experimental data. The model gives a reasonable fit to important aspects of experimentally measured metabolic and second messenger concentrations and provides a framework for analyzing the role of metabolic, hormones and neurotransmitters changes on insulin secretion. Our analysis of the dynamic data provides support for the hypothesis that activation of Ca2+-dependent adenylyl cyclases play a critical role in modulating the effects of glucagon-like peptide 1 (GLP-1, glucose-dependent insulinotropic polypeptide (GIP and catecholamines. The regulatory properties of adenylyl cyclase isoforms determine fluctuations in cytoplasmic cAMP concentration and reveal a synergistic action of glucose, GLP-1 and GIP on insulin secretion. On the other hand, the regulatory properties of phospholipase C isoforms determine the interaction of glucose, acetylcholine and free fatty acids (FFA (that act through the FFA receptors on insulin secretion. We found that a combination of GPCR agonists activating different messenger pathways can stimulate insulin secretion more effectively than a combination of GPCR agonists for a single pathway. This analysis also suggests that the activators of GLP-1, GIP and FFA receptors may have a relatively low risk of hypoglycemia in fasting conditions whereas an activator of muscarinic receptors can increase this risk. This computational analysis demonstrates that study of

  20. Moonlighting Proteins and Protein–Protein Interactions as Neurotherapeutic Targets in the G Protein-Coupled Receptor Field

    Science.gov (United States)

    Fuxe, Kjell; Borroto-Escuela, Dasiel O; Romero-Fernandez, Wilber; Palkovits, Miklós; Tarakanov, Alexander O; Ciruela, Francisco; Agnati, Luigi F

    2014-01-01

    There is serious interest in understanding the dynamics of the receptor–receptor and receptor–protein interactions in space and time and their integration in GPCR heteroreceptor complexes of the CNS. Moonlighting proteins are special multifunctional proteins because they perform multiple autonomous, often unrelated, functions without partitioning into different protein domains. Moonlighting through receptor oligomerization can be operationally defined as an allosteric receptor–receptor interaction, which leads to novel functions of at least one receptor protomer. GPCR-mediated signaling is a more complicated process than previously described as every GPCR and GPCR heteroreceptor complex requires a set of G protein interacting proteins, which interacts with the receptor in an orchestrated spatio-temporal fashion. GPCR heteroreceptor complexes with allosteric receptor–receptor interactions operating through the receptor interface have become major integrative centers at the molecular level and their receptor protomers act as moonlighting proteins. The GPCR heteroreceptor complexes in the CNS have become exciting new targets for neurotherapeutics in Parkinson's disease, schizophrenia, drug addiction, and anxiety and depression opening a new field in neuropsychopharmacology. PMID:24105074

  1. Bursicon, the insect cuticle-hardening hormone, is a heterodimeric cystine knot protein that activates G protein-coupled receptor LGR2.

    Science.gov (United States)

    Luo, Ching-Wei; Dewey, Elizabeth M; Sudo, Satoko; Ewer, John; Hsu, Sheau Yu; Honegger, Hans-Willi; Hsueh, Aaron J W

    2005-02-22

    All arthropods periodically molt to replace their exoskeleton (cuticle). Immediately after shedding the old cuticle, the neurohormone bursicon causes the hardening and darkening of the new cuticle. Here we show that bursicon, to our knowledge the first heterodimeric cystine knot hormone found in insects, consists of two proteins encoded by the genes burs and pburs (partner of burs). The pburs/burs heterodimer from Drosophila melanogaster binds with high affinity and specificity to activate the G protein-coupled receptor DLGR2, leading to the stimulation of cAMP signaling in vitro and tanning in neck-ligated blowflies. Native bursicon from Periplaneta americana is also a heterodimer. In D. melanogaster the levels of pburs, burs, and DLGR2 transcripts are increased before ecdysis, consistent with their role in postecdysial cuticle changes. Immunohistochemical analyses in diverse insect species revealed the colocalization of pburs- and burs-immunoreactivity in some of the neurosecretory neurons that also express crustacean cardioactive peptide. Forty-three years after its initial description, the elucidation of the molecular identity of bursicon and the verification of its receptor allow for studies of bursicon actions in regulating cuticle tanning, wing expansion, and as yet unknown functions. Because bursicon subunit genes are homologous to the vertebrate bone morphogenetic protein antagonists, our findings also facilitate investigation on the function of these proteins during vertebrate development.

  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. Applications of molecular replacement to G protein-coupled receptors

    Energy Technology Data Exchange (ETDEWEB)

    Kruse, Andrew C.; Manglik, Aashish; Kobilka, Brian K., E-mail: kobilka@stanford.edu [Stanford University, 279 Campus Drive, Stanford, CA 94305 (United States); Weis, William I., E-mail: kobilka@stanford.edu [Stanford University, 279 Campus Drive, Stanford, CA 94305 (United States); Stanford University, Fairchild Building, Stanford, CA 94305 (United States)

    2013-11-01

    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.

  4. Structural organization of G-protein-coupled receptors

    Science.gov (United States)

    Lomize, Andrei L.; Pogozheva, Irina D.; Mosberg, Henry I.

    1999-07-01

    Atomic-resolution structures of the transmembrane 7-α-helical domains of 26 G-protein-coupled receptors (GPCRs) (including opsins, cationic amine, melatonin, purine, chemokine, opioid, and glycoprotein hormone receptors and two related proteins, retinochrome and Duffy erythrocyte antigen) were calculated by distance geometry using interhelical hydrogen bonds formed by various proteins from the family and collectively applied as distance constraints, as described previously [Pogozheva et al., Biophys. J., 70 (1997) 1963]. The main structural features of the calculated GPCR models are described and illustrated by examples. Some of the features reflect physical interactions that are responsible for the structural stability of the transmembrane α-bundle: the formation of extensive networks of interhelical H-bonds and sulfur-aromatic clusters that are spatially organized as 'polarity gradients' the close packing of side-chains throughout the transmembrane domain; and the formation of interhelical disulfide bonds in some receptors and a plausible Zn2+ binding center in retinochrome. Other features of the models are related to biological function and evolution of GPCRs: the formation of a common 'minicore' of 43 evolutionarily conserved residues; a multitude of correlated replacements throughout the transmembrane domain; an Na+-binding site in some receptors, and excellent complementarity of receptor binding pockets to many structurally dissimilar, conformationally constrained ligands, such as retinal, cyclic opioid peptides, and cationic amine ligands. The calculated models are in good agreement with numerous experimental data.

  5. Structural Studies of G Protein-Coupled Receptors.

    Science.gov (United States)

    Zhang, Dandan; Zhao, Qiang; Wu, Beili

    2015-10-01

    G protein-coupled receptors (GPCRs) constitute the largest and the most physiologically important membrane protein family that recognizes a variety of environmental stimuli, and are drug targets in the treatment of numerous diseases. Recent progress on GPCR structural studies shed light on molecular mechanisms of GPCR ligand recognition, activation and allosteric modulation, as well as structural basis of GPCR dimerization. In this review, we will discuss the structural features of GPCRs and structural insights of different aspects of GPCR biological functions.

  6. VPAC receptors: structure, molecular pharmacology and interaction with accessory proteins.

    Science.gov (United States)

    Couvineau, Alain; Laburthe, Marc

    2012-05-01

    The vasoactive intestinal peptide (VIP) is a neuropeptide with wide distribution in both central and peripheral nervous systems, where it plays important regulatory role in many physiological processes. VIP displays a large biological functions including regulation of exocrine secretions, hormone release, fetal development, immune responses, etc. VIP appears to exert beneficial effect in neuro-degenerative and inflammatory diseases. The mechanism of action of VIP implicates two subtypes of receptors (VPAC1 and VPAC2), which are members of class B receptors belonging to the super-family of GPCR. This article reviews the current knowledge regarding the structure and molecular pharmacology of VPAC receptors. The structure-function relationship of VPAC1 receptor has been extensively studied, allowing to understand the molecular basis for receptor affinity, specificity, desensitization and coupling to adenylyl cyclase. Those studies have clearly demonstrated the crucial role of the N-terminal ectodomain (N-ted) of VPAC1 receptor in VIP recognition. By using different approaches including directed mutagenesis, photoaffinity labelling, NMR, molecular modelling and molecular dynamic simulation, it has been shown that the VIP molecule interacts with the N-ted of VPAC1 receptor, which is itself structured as a 'Sushi' domain. VPAC1 receptor also interacts with a few accessory proteins that play a role in cell signalling of receptors. Recent advances in the structural characterization of VPAC receptor and more generally of class B GPCRs will lead to the design of new molecules, which could have considerable interest for the treatment of inflammatory and neuro-degenerative diseases. © 2011 The Authors. British Journal of Pharmacology © 2011 The British Pharmacological Society.

  7. Membrane cholesterol access into a G-protein-coupled receptor

    Science.gov (United States)

    Guixà-González, Ramon; Albasanz, José L.; Rodriguez-Espigares, Ismael; Pastor, Manuel; Sanz, Ferran; Martí-Solano, Maria; Manna, Moutusi; Martinez-Seara, Hector; Hildebrand, Peter W.; Martín, Mairena; Selent, Jana

    2017-02-01

    Cholesterol is a key component of cell membranes with a proven modulatory role on the function and ligand-binding properties of G-protein-coupled receptors (GPCRs). Crystal structures of prototypical GPCRs such as the adenosine A2A receptor (A2AR) have confirmed that cholesterol finds stable binding sites at the receptor surface suggesting an allosteric role of this lipid. Here we combine experimental and computational approaches to show that cholesterol can spontaneously enter the A2AR-binding pocket from the membrane milieu using the same portal gate previously suggested for opsin ligands. We confirm the presence of cholesterol inside the receptor by chemical modification of the A2AR interior in a biotinylation assay. Overall, we show that cholesterol's impact on A2AR-binding affinity goes beyond pure allosteric modulation and unveils a new interaction mode between cholesterol and the A2AR that could potentially apply to other GPCRs.

  8. Genetic polymorphism of bone morphogenetic protein receptor 1B ...

    African Journals Online (AJOL)

    The Indonesian fat-tailed sheep (IFTS) is a local sheep that has been long time raised and well adapted to the extreme environments of Lombok Island. The present study was conducted to determine the polymorphism of bone morphogenetic protein receptor 1B (BMPR-1B) gene and its association with litter size in the IFTS ...

  9. Endothelial protein C receptor in renal tubular epithelial cells and ...

    African Journals Online (AJOL)

    Jane

    2011-07-20

    Jul 20, 2011 ... However, troglitazone had protective effects of EPCR on injured cells. Key words: Endothelial protein C receptor, renal tubular epithelial cell, troglitazone, tumor necrosis factor-α, interleukin-1β; high glucose. ..... induce apoptosis of vascular smooth muscle cells through an extracellular signal- regulated ...

  10. Divergent cAMP signaling differentially regulates serotonin-induced spinal motor plasticity.

    Science.gov (United States)

    Fields, D P; Mitchell, G S

    2017-02-01

    Spinal metabotropic serotonin receptors encode transient experiences into long-lasting changes in motor behavior (i.e. motor plasticity). While interactions between serotonin receptor subtypes are known to regulate plasticity, the significance of molecular divergence in downstream G protein coupled receptor signaling is not well understood. Here we tested the hypothesis that distinct cAMP dependent signaling pathways differentially regulate serotonin-induced phrenic motor facilitation (pMF); a well-studied model of spinal motor plasticity. Specifically, we studied the capacity of cAMP-dependent protein kinase A (PKA) and exchange protein activated by cAMP (EPAC) to regulate 5-HT2A receptor-induced pMF within adult male rats. Although spinal PKA, EPAC and 5-HT2A each elicit pMF when activated alone, concurrent PKA and 5-HT2A activation interact via mutual inhibition thereby blocking pMF expression. Conversely, concurrent EPAC and 5-HT2A activation enhance pMF expression reflecting additive contributions from both mechanisms. Thus, we demonstrate that distinct downstream cAMP signaling pathways enable differential regulation of 5-HT2A-induced pMF. Conditional activation of independent signaling mechanisms may explain experience amendable changes in plasticity expression (i.e. metaplasticity), an emerging concept thought to enable flexible motor control within the adult central nervous system. Published by Elsevier Ltd.

  11. Direct interactions between calcitonin-like receptor (CLR) and CGRP-receptor component protein (RCP) regulate CGRP receptor signaling.

    Science.gov (United States)

    Egea, Sophie C; Dickerson, Ian M

    2012-04-01

    Calcitonin gene-related peptide (CGRP) is a neuropeptide with multiple neuroendocrine roles, including vasodilation, migraine, and pain. The receptor for CGRP is a G protein-coupled receptor (GPCR) that requires three proteins for function. CGRP binds to a heterodimer composed of the GPCR calcitonin-like receptor (CLR) and receptor activity-modifying protein (RAMP1), a single transmembrane protein required for pharmacological specificity and trafficking of the CLR/RAMP1 complex to the cell surface. In addition, the CLR/RAMP1 complex requires a third protein named CGRP-receptor component protein (RCP) for signaling. Previous studies have demonstrated that depletion of RCP from cells inhibits CLR signaling, and in vivo studies have demonstrated that expression of RCP correlates with CLR signaling and CGRP efficacy. It is not known whether RCP interacts directly with CLR to exert its effect. The current studies identified a direct interaction between RCP and an intracellular domain of CLR using yeast two-hybrid analysis and coimmunoprecipitation. When this interacting domain of CLR was expressed as a soluble fusion protein, it coimmunoprecipitated with RCP and inhibited signaling from endogenous CLR. Expression of this dominant-negative domain of CLR did not significantly inhibit trafficking of CLR to the cell surface, and thus RCP may not have a chaperone function for CLR. Instead, RCP may regulate CLR signaling in the cell membrane, and direct interaction between RCP and CLR is required for CLR activation. To date, RCP has been found to interact only with CLR and represents a novel neuroendocrine regulatory step in GPCR signaling.

  12. G-protein-coupled receptors and tyrosine kinases: crossroads in cell signaling and regulation.

    Science.gov (United States)

    Gavi, Shai; Shumay, Elena; Wang, Hsien-yu; Malbon, Craig C

    2006-03-01

    G-protein-coupled receptors and protein tyrosine kinases represent two prominent pathways for cellular signaling. As our knowledge of cell signaling pathways mediated by the superfamily of G-protein-coupled receptors and the smaller family of receptor tyrosine kinases expands, so does our appreciation of how these two major signaling platforms share information and modulate each other, otherwise termed "cross-talk". Cross-talk between G-protein-coupled receptors and tyrosine kinases can occur at several levels, including the receptor-to-receptor level, and at crucial downstream points (e.g. phosphatidylinositol-3-kinase, Akt/protein kinase B and the mitogen-activated protein kinase cascade). Regulation of G-protein-coupled receptors by non-receptor tyrosine kinases, such as Src family members, also operates in signaling. A broader understanding of how G-protein-coupled receptors and tyrosine kinases cross-talk reveals new insights into signaling modalities in both health and disease.

  13. Camp and Human Behavior.

    Science.gov (United States)

    Henderson, Karla A.

    1995-01-01

    Addresses the potential of camp to promote self-esteem and nurture a sense of community. Summarizes articles in this journal issue that focus on individual and group behavior including homesickness, how camps can promote positive attitudes toward disabled campers, and a camp program that provides respite care for families of children with AIDS.…

  14. The localization and concentration of the PDE2-encoded high-affinity cAMP phosphodiesterase is regulated by cAMP-dependent protein kinase A in the yeast Saccharomyces cerevisiae.

    Science.gov (United States)

    Hu, Yun; Liu, Enkai; Bai, Xiaojia; Zhang, Aili

    2010-03-01

    The genome of the yeast Saccharomyces cerevisiae encodes two cyclic AMP (cAMP) phosphodiesterases, a low-affinity one, Pde1, and a high-affinity one, Pde2. Pde1 has been ascribed a function for downregulating agonist-induced cAMP accumulation in a protein kinase A (PKA)-governed negative feedback loop, whereas Pde2 controls the basal cAMP level in the cell. Here we show that PKA regulates the localization and protein concentration of Pde2. Pde2 is accumulated in the nucleus in wild-type cells growing on glucose, or in strains with hyperactive PKA. In contrast, in derepressed wild-type cells or cells with attenuated PKA activity, Pde2 is distributed over the nucleus and cytoplasm. We also show evidence indicating that the Pde2 protein level is positively correlated with PKA activity. The increase in the Pde2 protein level in high-PKA strains and in cells growing on glucose was due to its increased half-life. These results suggest that, like its low-affinity counterpart, the high-affinity phosphodiesterase may also play an important role in the PKA-controlled feedback inhibition of intracellular cAMP.

  15. Cyclic AMP receptor protein-aequorin molecular switch for cyclic AMP.

    Science.gov (United States)

    Scott, Daniel; Hamorsky, Krystal Teasley; Ensor, C Mark; Anderson, Kimberly W; Daunert, Sylvia

    2011-03-16

    Molecular switches are designer molecules that combine the functionality of two individual proteins into one, capable of manifesting an "on/off" signal in response to a stimulus. These switches have unique properties and functionalities and thus, can be employed as nanosensors in a variety of applications. To that end, we have developed a bioluminescent molecular switch for cyclic AMP. Bioluminescence offers many advantages over fluorescence and other detection methods including the fact that there is essentially zero background signal in physiological fluids, allowing for more sensitive detection and monitoring. The switch was created by combining the properties of the cyclic AMP receptor protein (CRP), a transcriptional regulatory protein from E. Coli that binds selectively to cAMP with those of aequorin, a bioluminescent photoprotein native of the jellyfish Aequorea victoria . Genetic manipulation to split the genetic coding sequence of aequorin in two and genetically attach the fragments to the N and C termini of CRP resulted in a hybrid protein molecular switch. The conformational change experienced by CRP upon the binding of cyclic AMP is suspected to result in the observed loss of the bioluminescent signal from aequorin. The "on/off" bioluminescence can be modulated by cyclic AMP over a range of several orders of magnitude in a linear fashion in addition to the capacity to detect changes in cellular cyclic AMP of intact cells exposed to different external stimuli without the need to lyse the cells. We envision that the molecular switch could find applications in vitro as well as In Vivo cyclic AMP detection and/or imaging.

  16. Allosteric modulation of G-protein coupled receptors

    DEFF Research Database (Denmark)

    Jensen, Anders A.; Spalding, Tracy A

    2004-01-01

    The superfamily of G-protein coupled receptors (GPCRs) has more than 1000 members and is the largest family of proteins in the body. GPCRs mediate signalling of stimuli as diverse as light, ions, small molecules, peptides and proteins and are the targets for many pharmaceuticals. Most GPCR ligands....... 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....

  17. Characterization of G-protein coupled receptor kinase interaction with the neurokinin-1 receptor using bioluminescence resonance energy transfer

    DEFF Research Database (Denmark)

    Jorgensen, Rasmus; Holliday, Nicholas D; Hansen, Jakob L

    2007-01-01

    To analyze the interaction between the neurokinin-1 (NK-1) receptor and G-protein coupled receptor kinases (GRKs), we performed bioluminescence resonance energy transfer(2) (BRET(2)) measurements between the family A NK-1 receptor and GRK2 and GRK5 as well as their respective kinase-inactive muta......To analyze the interaction between the neurokinin-1 (NK-1) receptor and G-protein coupled receptor kinases (GRKs), we performed bioluminescence resonance energy transfer(2) (BRET(2)) measurements between the family A NK-1 receptor and GRK2 and GRK5 as well as their respective kinase...

  18. A receptor for infectious and cellular prion protein

    Directory of Open Access Journals (Sweden)

    V.R. Martins

    1999-07-01

    Full Text Available Prions are an unconventional form of infectious agents composed only of protein and involved in transmissible spongiform encephalopathies in humans and animals. The infectious particle is composed by PrPsc which is an isoform of a normal cellular glycosyl-phosphatidylinositol (GPI anchored protein, PrPc, of unknown function. The two proteins differ only in conformation, PrPc is composed of 40% a helix while PrPsc has 60% ß-sheet and 20% a helix structure. The infection mechanism is trigged by interaction of PrPsc with cellular prion protein causing conversion of the latter's conformation. Therefore, the infection spreads because new PrPsc molecules are generated exponentially from the normal PrPc. The accumulation of insoluble PrPsc is probably one of the events that lead to neuronal death. Conflicting data in the literature showed that PrPc internalization is mediated either by clathrin-coated pits or by caveolae-like membranous domains. However, both pathways seem to require a third protein (a receptor or a prion-binding protein either to make the connection between the GPI-anchored molecule to clathrin or to convert PrPc into PrPsc. We have recently characterized a 66-kDa membrane receptor which binds PrPc in vitro and in vivo and mediates the neurotoxicity of a human prion peptide. Therefore, the receptor should have a role in the pathogenesis of prion-related diseases and in the normal cellular process. Further work is necessary to clarify the events triggered by the association of PrPc/PrPsc with the receptor.

  19. 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.

  20. Persistent signaling by thyrotropin-releasing hormone receptors correlates with G-protein and receptor levels

    Science.gov (United States)

    Boutin, Alisa; Allen, Michael D.; Neumann, Susanne; Gershengorn, Marvin C.

    2012-01-01

    G-protein-coupled receptors with dissociable agonists for thyrotropin, parathyroid hormone, and sphingosine-1-phosphate were found to signal persistently hours after agonist withdrawal. Here we show that mouse thyrotropin-releasing hormone (TRH) receptors, subtypes 2 and 1(TRH-R2 and TRH-R1), can signal persistently in HEK-EM293 cells under appropriate conditions, but TRH-R2 exhibits higher persistent signaling activity. Both receptors couple primarily to Gαq/11. To gain insight into the mechanism of persistent signaling, we compared proximal steps of inositolmonophosphate (IP1) signaling by TRH-Rs. Persistent signaling was not caused by slower dissociation of TRH from TRH-R2 (t1/2=77±8.1 min) compared with TRH-R1 (t1/2=82±12 min) and was independent of internalization, as inhibition of internalization did not affect persistent signaling (115% of control), but required continuously activated receptors, as an inverse agonist decreased persistent signaling by 60%. Gαq/11 knockdown decreased persistent signaling by TRH-R2 by 82%, and overexpression of Gαq/11 induced persistent signaling in cells expressing TRH-R1. Lastly, persistent signaling was induced in cells expressing high levels of TRH-R1. We suggest that persistent signaling by TRHRs is exhibited when sufficient levels of agonist/receptor/G-protein complexes are established and maintained and that TRH-R2 forms and maintains these complexes more efficiently than TRH-R1.—Boutin, A., Allen, M. D., Neumann, S., Gershengorn, M. C. Persistent signaling by thyrotropin-releasing hormone receptors correlates with G-protein and receptor levels. PMID:22593547

  1. cAMP inhibits CSF-1-stimulated tyrosine phosphorylation but augments CSF-1R-mediated macrophage differentiation and ERK activation.

    Science.gov (United States)

    Wilson, Nicholas J; Cross, Maddalena; Nguyen, Thao; Hamilton, John A

    2005-08-01

    Macrophage colony stimulating factor (M-CSF) or CSF-1 controls the development of the macrophage lineage through its receptor tyrosine kinase, c-Fms. cAMP has been shown to influence proliferation and differentiation in many cell types, including macrophages. In addition, modulation of cellular ERK activity often occurs when cAMP levels are raised. We have shown previously that agents that increase cellular cAMP inhibited CSF-1-dependent proliferation in murine bone marrow-derived macrophages (BMM) which was associated with an enhanced extracellular signal-regulated kinase (ERK) activity. We report here that increasing cAMP levels, by addition of either 8-bromo cAMP (8BrcAMP) or prostaglandin E(1) (PGE1), can induce macrophage differentiation in M1 myeloid cells engineered to express the CSF-1 receptor (M1/WT cells) and can potentiate CSF-1-induced differentiation in the same cells. The enhanced CSF-1-dependent differentiation induced by raising cAMP levels correlated with enhanced ERK activity. Thus, elevated cAMP can promote either CSF-1-induced differentiation or inhibit CSF-1-induced proliferation depending on the cellular context. The mitogen-activated protein kinase/extracellular signal-related protein kinase kinase (MEK) inhibitor, PD98059, inhibited both the cAMP- and the CSF-1R-dependent macrophage differentiation of M1/WT cells suggesting that ERK activity might be important for differentiation in the M1/WT cells. Surprisingly, addition of 8BrcAMP or PGE1 to either CSF-1-treated M1/WT or BMM cells suppressed the CSF-1R-dependent tyrosine phosphorylation of cellular substrates, including that of the CSF-1R itself. It appears that there are at least two CSF-1-dependent pathway(s), one MEK/ERK dependent pathway and another controlling the bulk of the tyrosine phosphorylation, and that cAMP can modulate signalling through both of these pathways.

  2. G-protein-coupled receptors and localized signaling in the primary cilium during ventral neural tube patterning.

    Science.gov (United States)

    Hwang, Sun-Hee; Mukhopadhyay, Saikat

    2015-01-01

    The primary cilium is critical in sonic hedgehog (Shh)-dependent ventral patterning of the vertebrate neural tube. Most mutants that cause disruption of the cilium result in decreased Shh signaling in the neural tube. In contrast, mutations in the intraflagellar complex A (IFT-A) and the tubby family protein, Tulp3, result in increased Shh signaling in the neural tube. Proteomic analysis of Tulp3-binding proteins first pointed to the role of the IFT-A complex in trafficking Tulp3 into the cilia. Tulp3 directs trafficking of rhodopsin family G-protein-coupled receptors (GPCRs) to the cilia, suggesting the role of a GPCR in mediating the paradoxical effects of the Tulp3/IFT-A complex in causing increased Shh signaling. Gpr161 has recently been identified as a Tulp3/IFT-A-regulated GPCR that localizes to the primary cilium. A null knock-out mouse model of Gpr161 phenocopies Tulp3 and IFT-A mutants, and causes increased Shh signaling throughout the neural tube. In the absence of Shh, the bifunctional Gli transcription factors are proteolytically processed into repressor forms in a protein kinase A (PKA) -dependent and cilium-dependent manner. Gpr161 activity results in increased cAMP levels in a Gαs -coupled manner, and determines processing of Gli3. Shh signaling also results in removal of Gpr161 from the cilia, suggesting that Gpr161 functions in a positive feedback loop in the Shh pathway. As PKA-null and Gαs mutant embryos also exhibit increased Shh signaling in the neural tube, Gpr161 is a strong candidate for a GPCR that regulates ciliary cAMP levels, and activates PKA in close proximity to the cilia. © 2014 Wiley Periodicals, Inc.

  3. G protein stoichiometry dictates biased agonism through distinct receptor-G protein partitioning.

    Science.gov (United States)

    Onfroy, Lauriane; Galandrin, Ségolène; Pontier, Stéphanie M; Seguelas, Marie-Hélène; N'Guyen, Du; Sénard, Jean-Michel; Galés, Céline

    2017-08-11

    Biased agonism at G protein coupled receptors emerges as an opportunity for development of drugs with enhanced benefit/risk balance making biased ligand identification a priority. However, ligand biased signature, classically inferred from ligand activity across multiple pathways, displays high variability in recombinant systems. Functional assays usually necessity receptor/effector overexpression that should be controlled among assays to allow comparison but this calibration currently fails. Herein, we demonstrate that Gα expression level dictates the biased profiling of agonists and, to a lesser extent of β-blockers, in a Gα isoform- and receptor-specific way, depending on specific G protein activity in different membrane territories. These results have major therapeutic implications since they suggest that the ligand bias phenotype is not necessarily maintained in pathological cell background characterized by fluctuations in G protein expression. Thus, we recommend implementation of G protein stoichiometry as a new parameter in biased ligand screening programs.

  4. Molecular basis for amino acid sensing by family C G-protein-coupled receptors

    DEFF Research Database (Denmark)

    Wellendorph, Petrine; Bräuner-Osborne, Hans

    2009-01-01

    Family C of human G-protein-coupled receptors (GPCRs) is constituted by eight metabotropic glutamate receptors, two gamma-aminobutyric acid type B (GABA(B1-2)) subunits forming the heterodimeric GABA(B) receptor, the calcium-sensing receptor, three taste1 receptors (T1R1-3), a promiscuous L...

  5. Structural Basis for Receptor Activity-Modifying Protein-Dependent Selective Peptide Recognition by a G Protein-Coupled Receptor.

    Science.gov (United States)

    Booe, Jason M; Walker, Christopher S; Barwell, James; Kuteyi, Gabriel; Simms, John; Jamaluddin, Muhammad A; Warner, Margaret L; Bill, Roslyn M; Harris, Paul W; Brimble, Margaret A; Poyner, David R; Hay, Debbie L; Pioszak, Augen A

    2015-06-18

    Association of receptor activity-modifying proteins (RAMP1-3) with the G protein-coupled receptor (GPCR) calcitonin receptor-like receptor (CLR) enables selective recognition of the peptides calcitonin gene-related peptide (CGRP) and adrenomedullin (AM) that have diverse functions in the cardiovascular and lymphatic systems. How peptides selectively bind GPCR:RAMP complexes is unknown. We report crystal structures of CGRP analog-bound CLR:RAMP1 and AM-bound CLR:RAMP2 extracellular domain heterodimers at 2.5 and 1.8 Å resolutions, respectively. The peptides similarly occupy a shared binding site on CLR with conformations characterized by a β-turn structure near their C termini rather than the α-helical structure common to peptides that bind related GPCRs. The RAMPs augment the binding site with distinct contacts to the variable C-terminal peptide residues and elicit subtly different CLR conformations. The structures and accompanying pharmacology data reveal how a class of accessory membrane proteins modulate ligand binding of a GPCR and may inform drug development targeting CLR:RAMP complexes. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

  6. DMPD: LPS-binding proteins and receptors. [Dynamic Macrophage Pathway CSML Database

    Lifescience Database Archive (English)

    Full Text Available 9665271 LPS-binding proteins and receptors. Fenton MJ, Golenbock DT. J Leukoc Biol.... 1998 Jul;64(1):25-32. (.png) (.svg) (.html) (.csml) Show LPS-binding proteins and receptors. PubmedID 9665271 Title LPS-binding prot...eins and receptors. Authors Fenton MJ, Golenbock DT. Publication J Leukoc Biol. 199

  7. Portraying G Protein-Coupled Receptors with Fluorescent Ligands

    Science.gov (United States)

    2015-01-01

    The thermodynamics of ligand–receptor interactions at the surface of living cells represents a fundamental aspect of G protein-coupled receptor (GPCR) biology; thus, its detailed elucidation constitutes a challenge for modern pharmacology. Interestingly, fluorescent ligands have been developed for a variety of GPCRs in order to monitor ligand–receptor binding in living cells. Accordingly, new methodological strategies derived from noninvasive fluorescence-based approaches, especially fluorescence resonance energy transfer (FRET), have been successfully developed to characterize ligand–receptor interactions. Importantly, these technologies are supplanting more hazardous and expensive radioactive binding assays. In addition, FRET-based tools have also become extremely powerful approaches for visualizing receptor–receptor interactions (i.e., GPCR oligomerization) in living cells. Thus, by means of the synthesis of compatible fluorescent ligands these novel techniques can be implemented to demonstrate the existence of GPCR oligomerization not only in heterologous systems but also in native tissues. Finally, there is no doubt that these methodologies would also be relevant in drug discovery in order to develop new high-throughput screening approaches or to identify new therapeutic targets. Overall, herein, we provide a thorough assessment of all technical and biological aspects, including strengths and weaknesses, of these fluorescence-based methodologies when applied to the study of GPCR biology at the plasma membrane of living cells. PMID:25010291

  8. G-Protein Coupled Receptors Targeted by Analgesic Venom Peptides

    OpenAIRE

    James T. Daniel; Clark, Richard J.

    2017-01-01

    Chronic pain is a complex and debilitating condition associated with a large personal and socioeconomic burden. Current pharmacological approaches to treating chronic pain such as opioids, antidepressants and anticonvulsants exhibit limited efficacy in many patients and are associated with dose-limiting side effects that hinder their clinical use. Therefore, improved strategies for the pharmacological treatment of pathological pain are urgently needed. G-protein coupled receptors (GPCRs) are ...

  9. Mining flexible-receptor docking experiments to select promising protein receptor snapshots.

    Science.gov (United States)

    Machado, Karina S; Winck, Ana T; Ruiz, Duncan D A; de Souza, Osmar Norberto

    2010-12-22

    Molecular docking simulation is the Rational Drug Design (RDD) step that investigates the affinity between protein receptors and ligands. Typically, molecular docking algorithms consider receptors as rigid bodies. Receptors are, however, intrinsically flexible in the cellular environment. The use of a time series of receptor conformations is an approach to explore its flexibility in molecular docking computer simulations, but it is extensively time-consuming. Hence, selection of the most promising conformations can accelerate docking experiments and, consequently, the RDD efforts. We previously docked four ligands (NADH, TCL, PIF and ETH) to 3,100 conformations of the InhA receptor from M. tuberculosis. Based on the receptor residues-ligand distances we preprocessed all docking results to generate appropriate input to mine data. Data preprocessing was done by calculating the shortest interatomic distances between the ligand and the receptor's residues for each docking result. They were the predictive attributes. The target attribute was the estimated free-energy of binding (FEB) value calculated by the AutodDock3.0.5 software. The mining inputs were submitted to the M5P model tree algorithm. It resulted in short and understandable trees. On the basis of the correlation values, for NADH, TCL and PIF we obtained more than 95% correlation while for ETH, only about 60%. Post processing the generated model trees for each of its linear models (LMs), we calculated the average FEB for their associated instances. From these values we considered a LM as representative if its average FEB was smaller than or equal the average FEB of the test set. The instances in the selected LMs were considered the most promising snapshots. It totalized 1,521, 1,780, 2,085 and 902 snapshots, for NADH, TCL, PIF and ETH respectively. By post processing the generated model trees we were able to propose a criterion of selection of linear models which, in turn, is capable of selecting a set of

  10. 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 structure and biology. Great progress has been made over the past three decades in understanding diverse GPCRs, from pharmacology to functional characterization in vivo. Recent high-resolution structural studies have provided insights into the molecular mechanisms of GPCR activation and constitutive...

  11. Treponema pallidum receptor binding proteins interact with fibronectin

    Energy Technology Data Exchange (ETDEWEB)

    Peterson, K.M.; Baseman, J.B.; Alderete, J.F.

    1983-06-01

    Analysis of plasma proteins avidly bound to T. pallidum surfaces revealed the ability of T. pallidum to acquire numerous host macromolecules. No acquisition was evident by the avirulent spirochete, T. phagedenis biotype Reiter. Western blotting technology using hyperimmune antifibronectin serum as a probe revealed the ability of virulent treponemes to avidly bind fibronectin from a complex medium such as plasma. The specificity of the tiplike adherence of motile T. pallidum to fibronectin-coated glass surfaces and to fibronectin on HEp-2 cells was reinforced by the observation that pretreatment of coverslips or cell monolayers with monospecific antiserum against fibronectin substantially reduced T. pallidum attachment. The stoichiometric binding of T. pallidum to fibronectin-coated coverslips and the inability of unlabeled or /sup 35/S-radiolabeled treponemes to interact with glass surfaces treated with other plasma proteins further established the specific nature of the interaction between virulent T. pallidum and fibronectin. The avid association between three outer envelope proteins of T. pallidum and fibronectin was also demonstrated. These treponemal surface proteins have been previously identified as putative receptor-binding proteins responsible for T. pallidum parasitism of host cells. The data suggest that surface fibronectin mediates tip-oriented attachment of T. pallidum to host cells via a receptor-ligand mechanism of recognition.

  12. Carboxyl-terminal receptor domains control the differential dephosphorylation of somatostatin receptors by protein phosphatase 1 isoforms.

    Directory of Open Access Journals (Sweden)

    Andreas Lehmann

    Full Text Available We have recently identified protein phosphatase 1β (PP1β as G protein-coupled receptor (GPCR phosphatase for the sst2 somatostatin receptor using siRNA knockdown screening. By contrast, for the sst5 somatostatin receptor we identified protein phosphatase 1γ (PP1γ as GPCR phosphatase using the same approach. We have also shown that sst2 and sst5 receptors differ substantially in the temporal dynamics of their dephosphorylation and trafficking patterns. Whereas dephosphorylation and recycling of the sst2 receptor requires extended time periods of ∼30 min, dephosphorylation and recycling of the sst5 receptor is completed in less than 10 min. Here, we examined which receptor domains determine the selection of phosphatases for receptor dephosphorylation. We found that generation of tail-swap mutants between sst2 and sst5 was required and sufficient to reverse the patterns of dephosphorylation and trafficking of these two receptors. In fact, siRNA knockdown confirmed that the sst5 receptor carrying the sst2 tail is predominantly dephosphorylated by PP1β, whereas the sst2 receptor carrying the sst5 tail is predominantly dephosphorylated by PP1γ. Thus, the GPCR phosphatase responsible for dephosphorylation of individual somatostatin receptor subtypes is primarily determined by their different carboxyl-terminal receptor domains. This phosphatase specificity has in turn profound consequences for the dephosphorylation dynamics and trafficking patterns of GPCRs.

  13. Engineered Context-Sensitive Agonism: Tissue-Selective Drug Signaling through a G Protein-Coupled Receptor.

    Science.gov (United States)

    Seemann, Wiebke K; Wenzel, Daniela; Schrage, Ramona; Etscheid, Justine; Bödefeld, Theresa; Bartol, Anna; Warnken, Mareille; Sasse, Philipp; Klöckner, Jessica; Holzgrabe, Ulrike; DeAmici, Marco; Schlicker, Eberhard; Racké, Kurt; Kostenis, Evi; Meyer, Rainer; Fleischmann, Bernd K; Mohr, Klaus

    2017-02-01

    Drug discovery strives for selective ligands to achieve targeted modulation of tissue function. Here we introduce engineered context-sensitive agonism as a postreceptor mechanism for tissue-selective drug action through a G protein-coupled receptor. Acetylcholine M2-receptor activation is known to mediate, among other actions, potentially dangerous slowing of the heart rate. This unwanted side effect is one of the main reasons that limit clinical application of muscarinic agonists. Herein we show that dualsteric (orthosteric/allosteric) agonists induce less cardiac depression ex vivo and in vivo than conventional full agonists. Exploration of the underlying mechanism in living cells employing cellular dynamic mass redistribution identified context-sensitive agonism of these dualsteric agonists. They translate elevation of intracellular cAMP into a switch from full to partial agonism. Designed context-sensitive agonism opens an avenue toward postreceptor pharmacologic selectivity, which even works in target tissues operated by the same subtype of pharmacologic receptor. Copyright © 2017 by The American Society for Pharmacology and Experimental Therapeutics.

  14. Alterations in phosphorylated cAMP response element-binding protein (pCREB) signaling: an endophenotype of lithium-responsive bipolar disorder?

    Science.gov (United States)

    Alda, Martin; Shao, Li; Wang, Jun-Feng; Lopez de Lara, Catalina; Jaitovich-Groisman, Iris; Lebel, Veronique; Sun, Xiujun; Duffy, Anne; Grof, Paul; Rouleau, Guy A; Turecki, Gustavo; Young, L Trevor

    2013-12-01

    Abnormalities of signal transduction are considered among the susceptibility factors for bipolar disorder (BD). These include changes in G-protein-mediated signaling and subsequent modification of gene expression via transcription factors such as cAMP response element-binding protein (CREB). We investigated levels of CREB in lymphoblasts from patients with BD, all responders to lithium prophylaxis (n = 13), and healthy control subjects (n = 15). Phosphorylated CREB (pCREB) was measured by immunoblotting in subjects with BD (n = 15) as well as in their affected (n = 17) and unaffected (n = 18) relatives, and healthy controls (n = 16). Basal CREB levels were comparable in patients and control subjects and were not changed by lithium treatment. pCREB levels were increased in both patients and their relatives compared to controls (p = 0.003). Forskolin stimulation led to a 24% increase in pCREB levels in cells from healthy subjects (p = 0.002) but not in the other three groups. When using basal and stimulated pCREB levels as a biochemical phenotype in a preliminary linkage study, we found the strongest support for linkage in regions largely overlapping with those showing linkage with the clinical phenotype (3p, 6p, 16p, 17q, 19q, and 21q). Abnormal pCREB signaling could be considered a biochemical phenotype for lithium-responsive BD. © 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  15. Localization of somatostatin receptors at the light and electron microscopical level by using antibodies raised against fusion proteins

    DEFF Research Database (Denmark)

    Helboe, Lone; Møller, Morten

    2000-01-01

    Somatostatin, antibodies against somatostatin receptors, hypothalamus, Müller cells, fusion protein technique......Somatostatin, antibodies against somatostatin receptors, hypothalamus, Müller cells, fusion protein technique...

  16. Cyclic AMP receptor protein regulates pheromone-mediated bioluminescence at multiple levels in Vibrio fischeri ES114.

    Science.gov (United States)

    Lyell, Noreen L; Colton, Deanna M; Bose, Jeffrey L; Tumen-Velasquez, Melissa P; Kimbrough, John H; Stabb, Eric V

    2013-11-01

    Bioluminescence in Vibrio fischeri ES114 is activated by autoinducer pheromones, and this regulation serves as a model for bacterial cell-cell signaling. As in other bacteria, pheromone concentration increases with cell density; however, pheromone synthesis and perception are also modulated in response to environmental stimuli. Previous studies suggested that expression of the pheromone-dependent bioluminescence activator LuxR is regulated in response to glucose by cyclic AMP (cAMP) receptor protein (CRP) (P. V. Dunlap and E. P. Greenberg, J. Bacteriol. 164:45-50, 1985; P. V. Dunlap and E. P. Greenberg, J. Bacteriol. 170:4040-4046, 1988; P. V. Dunlap, J. Bacteriol. 171:1199-1202, 1989; and W. F. Friedrich and E. P. Greenberg, Arch. Microbiol. 134:87-91, 1983). Consistent with this model, we found that bioluminescence in V. fischeri ES114 is modulated by glucose and stimulated by cAMP. In addition, a Δcrp mutant was ∼100-fold dimmer than ES114 and did not increase luminescence in response to added cAMP, even though cells lacking crp were still metabolically capable of producing luminescence. We further discovered that CRP regulates not only luxR but also the alternative pheromone synthase gene ainS. We found that His-tagged V. fischeri CRP could bind sequences upstream of both luxR and ainS, supporting bioinformatic predictions of direct regulation at both promoters. Luminescence increased in response to cAMP if either the ainS or luxR system was under native regulation, suggesting cAMP-CRP significantly increases luminescence through both systems. Finally, using transcriptional reporters in transgenic Escherichia coli, we elucidated two additional regulatory connections. First, LuxR-independent basal transcription of the luxI promoter was enhanced by CRP. Second, the effect of CRP on the ainS promoter depended on whether the V. fischeri regulatory gene litR was also introduced. These results suggest an integral role for CRP in pheromone signaling that goes

  17. Angiopoietin-like protein 4 is an exercise-induced hepatokine in humans, regulated by glucagon and cAMP

    DEFF Research Database (Denmark)

    Ingerslev, Bodil; Hansen, Jakob S; Hoffmann, Christoph

    2017-01-01

    OBJECTIVE: Angiopoietin-like protein-4 (ANGPTL4) is a circulating protein that is highly expressed in liver and implicated in regulation of plasma triglyceride levels. Systemic ANGPTL4 increases during prolonged fasting and is suggested to be secreted from skeletal muscle following exercise. METH...

  18. Cell death sensitization of leukemia cells by opioid receptor activation.

    Science.gov (United States)

    Friesen, Claudia; Roscher, Mareike; Hormann, Inis; Fichtner, Iduna; Alt, Andreas; Hilger, Ralf A; Debatin, Klaus-Michael; Miltner, Erich

    2013-05-01

    Cyclic AMP (cAMP) regulates a number of cellular processes and modulates cell death induction. cAMP levels are altered upon stimulation of specific G-protein-coupled receptors inhibiting or activating adenylyl cyclases. Opioid receptor stimulation can activate inhibitory Gi-proteins which in turn block adenylyl cyclase activity reducing cAMP. Opioids such as D,L-methadone induce cell death in leukemia cells. However, the mechanism how opioids trigger apoptosis and activate caspases in leukemia cells is not understood. In this study, we demonstrate that downregulation of cAMP induced by opioid receptor activation using the opioid D,L-methadone kills and sensitizes leukemia cells for doxorubicin treatment. Enhancing cAMP levels by blocking opioid-receptor signaling strongly reduced D,L-methadone-induced apoptosis, caspase activation and doxorubicin-sensitivity. Induction of cell death in leukemia cells by activation of opioid receptors using the opioid D,L-methadone depends on critical levels of opioid receptor expression on the cell surface. Doxorubicin increased opioid receptor expression in leukemia cells. In addition, the opioid D,L-methadone increased doxorubicin uptake and decreased doxorubicin efflux in leukemia cells, suggesting that the opioid D,L-methadone as well as doxorubicin mutually increase their cytotoxic potential. Furthermore, we found that opioid receptor activation using D,L-methadone alone or in addition to doxorubicin inhibits tumor growth significantly in vivo. These results demonstrate that opioid receptor activation via triggering the downregulation of cAMP induces apoptosis, activates caspases and sensitizes leukemia cells for doxorubicin treatment. Hence, opioid receptor activation seems to be a promising strategy to improve anticancer therapies.

  19. Recreation Summer Camps

    Data.gov (United States)

    Montgomery County of Maryland — List of all Camps (Register here:https://apm.activecommunities.com/montgomerycounty/Home) to include Aquatics, Basketball, Soccer, Special Interest, General Sports,...

  20. Registration Summer Camp 2016

    CERN Multimedia

    2016-01-01

    Reminder: registration for the CERN Staff Association Summer Camp is now open for children from 4 to 6 years old.   More information on the website: http://nurseryschool.web.cern.ch/. The summer camp is open to all children. The proposed cost is 480.-CHF/week, lunch included. The camp will be open weeks 27, 28, 29 and 30, from 8:30 a.m. to 5:30 p.m. For further questions, you are welcome to contact us by email at Summer.Camp@cern.ch. CERN Staff Association

  1. Receptor recruitment: A mechanism for interactions between G protein-coupled receptors

    OpenAIRE

    Holtbäck, Ulla; Brismar, Hjalmar; DiBona, Gerald F.; Fu, Michael; Greengard, Paul; Aperia, Anita

    1999-01-01

    There is a great deal of evidence for synergistic interactions between G protein-coupled signal transduction pathways in various tissues. As two specific examples, the potent effects of the biogenic amines norepinephrine and dopamine on sodium transporters and natriuresis can be modulated by neuropeptide Y and atrial natriuretic peptide, respectively. Here, we report, using a renal epithelial cell line, that both types of modulation involve recruitment of receptors from the interior of the ce...

  2. Noradrenaline represses PPAR (peroxisome-proliferator-activated receptor) gamma2 gene expression in brown adipocytes: intracellular signalling and effects on PPARgamma2 and PPARgamma1 protein levels

    DEFF Research Database (Denmark)

    Lindgren, Eva M; Nielsen, Ronni; Petrovic, Natasa

    2004-01-01

    gradually recovered. The down-regulation was beta-adrenoceptor-induced and intracellularly mediated via cAMP and protein kinase A; the signalling pathway did not involve phosphoinositide 3-kinase, Src, p38 mitogen-activated protein kinase or extracellular-signal-regulated kinases 1 and 2. Treatment......PPAR (peroxisome-proliferator-activated receptor) gamma is expressed in brown and white adipose tissues and is involved in the control of differentiation and proliferation. Noradrenaline stimulates brown pre-adipocyte proliferation and brown adipocyte differentiation. The aim of the present study...... of the cells with the protein synthesis inhibitor cycloheximide not only abolished the noradrenaline-induced down-regulation of PPARgamma2 mRNA, but also in itself induced PPARgamma2 hyperexpression. The down-regulation was probably the result of suppression of transcription. The down-regulation of PPARgamma2...

  3. 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.

  4. The TRPM8 Protein Is a Testosterone Receptor

    Science.gov (United States)

    Asuthkar, Swapna; Demirkhanyan, Lusine; Sun, Xiaohui; Elustondo, Pia A.; Krishnan, Vivek; Baskaran, Padmamalini; Velpula, Kiran Kumar; Thyagarajan, Baskaran; Pavlov, Evgeny V.; Zakharian, Eleonora

    2015-01-01

    Testosterone is a key steroid hormone in the development of male reproductive tissues and the regulation of the central nervous system. The rapid signaling mechanism induced by testosterone affects numerous behavioral traits, including sexual drive, aggressiveness, and fear conditioning. However, the currently identified testosterone receptor(s) is not believed to underlie the fast signaling, suggesting an orphan pathway. Here we report that an ion channel from the transient receptor potential family, TRPM8, commonly known as the cold and menthol receptor is the major component of testosterone-induced rapid actions. Using cultured and primary cell lines along with the purified TRPM8 protein, we demonstrate that testosterone directly activates TRPM8 channel at low picomolar range. Specifically, testosterone induced TRPM8 responses in primary human prostate cells, PC3 prostate cancer cells, dorsal root ganglion neurons, and hippocampal neurons. Picomolar concentrations of testosterone resulted in full openings of the purified TRPM8 channel in planar lipid bilayers. Furthermore, acute applications of testosterone on human skin elicited a cooling sensation. Our data conclusively demonstrate that testosterone is an endogenous and highly potent agonist of TRPM8, suggesting a role of TRPM8 channels well beyond their well established function in somatosensory neurons. This discovery may further imply TRPM8 channel function in testosterone-dependent behavioral traits. PMID:25480785

  5. G protein-coupled receptor modulation with pepducins

    DEFF Research Database (Denmark)

    Dimond, Patricia; Carlson, Kenneth; Bouvier, Michel

    2011-01-01

    At the 2nd Pepducin Science Symposium held in Cambridge, Massachusetts, on November 4-5, 2010, investigators working in G protein-coupled receptor (GPCR) research convened to discuss progress since last year's inaugural conference. This year's symposium focused on increasing knowledge of the stru......At the 2nd Pepducin Science Symposium held in Cambridge, Massachusetts, on November 4-5, 2010, investigators working in G protein-coupled receptor (GPCR) research convened to discuss progress since last year's inaugural conference. This year's symposium focused on increasing knowledge...... of the structure and function of this ubiquitous superfamily of membrane receptors and their potential modulation for disease treatment. Presentations also focused on how GPCR mechanisms might be exploited to treat diseases with pepducins, novel synthetic lipopeptide pharmacophores that modulate heptahelical GPCR...... activity. While the multiple roles of GPCRs in physiological and pathophysiological processes offer significant opportunities for novel drug development, the global nature of their activity challenges drug-specific and validated target identification. This year's conference highlighted advances...

  6. Regulation of transferrin receptor 2 protein levels by transferrin.

    Science.gov (United States)

    Robb, Aeisha; Wessling-Resnick, Marianne

    2004-12-15

    Transferrin receptor 2 (TfR2) plays a critical role in iron homeostasis because patients carrying disabling mutations in the TFR2 gene suffer from hemochromatosis. In this study, iron-responsive regulation of TfR2 at the protein level was examined in vitro and in vivo. HepG2 cell TfR2 protein levels were up-regulated after exposure to holotransferrin (holoTf) in a time- and dose-responsive manner. ApoTf or high-iron treatment with non-Tf-bound iron failed to elicit similar effects, suggesting that TfR2 regulation reflects interactions of the iron-bound ligand. Hepatic TfR2 protein levels also reflected an adaptive response to changing iron status in vivo. Liver TfR2 protein levels were down- and up-regulated in rats fed an iron-deficient and a high-iron diet, respectively. TfR2 was also up-regulated in Hfe(-/-) mice, an animal model that displays liver iron loading. In contrast, TfR2 levels were reduced in hypotransferrinemic mice despite liver iron overload, supporting the idea that regulation of the receptor is dependent on Tf. This idea is confirmed by up-regulation of TfR2 in beta-thalassemic mice, which, like hypotransferrinemic mice, are anemic and incur iron loading, but have functional Tf. Based on these combined results, we hypothesize that TfR2 acts as a sensor of iron status such that receptor levels reflect Tf saturation.

  7. Sphingolipids in the function of G protein-coupled receptors.

    Science.gov (United States)

    Jafurulla, Mohammad; Chattopadhyay, Amitabha

    2015-09-15

    G protein-coupled receptors (GPCRs) constitute the largest and most diverse protein family in mammals and are involved in information transfer across cellular membranes. GPCRs are known to regulate multiple physiological functions and therefore represent major drug targets in all clinical areas. The fact that GPCRs are integral membrane proteins raises the possibility of their interaction with functionally important membrane lipids such as sphingolipids. Sphingolipids are essential membrane components and are recognized as diverse and dynamic regulators of a multitude of cellular processes. Interaction with sphingolipids could lead to modulation of GPCR structure and function. In this review, we highlight the role of sphingolipids in the function of GPCRs with specific examples. A comprehensive understanding of molecular events involved in GPCR-lipid interaction would provide better insight into GPCR function in health and disease. Copyright © 2015 Elsevier B.V. All rights reserved.

  8. Curcumin induces human cathelicidin antimicrobial peptide gene expression through a vitamin D receptor-independent pathway

    DEFF Research Database (Denmark)

    Guo, Chunxiao; Rosoha, Elena; Lowry, Malcolm B

    2013-01-01

    The vitamin D receptor (VDR) mediates the pleiotropic biologic effects of 1α,25 dihydroxy-vitamin D(3). Recent in vitro studies suggested that curcumin and polyunsaturated fatty acids (PUFAs) also bind to VDR with low affinity. As potential ligands for the VDR, we hypothesized that curcumin...... cancer cell line HT-29 and keratinocyte cell line HaCaT. We demonstrated that PUFAs failed to induce CAMP or CYP24A1 mRNA expression in all three cell lines, but curcumin up-regulated CAMP mRNA and protein levels in U937 cells. Curcumin treatment induced CAMP promoter activity from a luciferase reporter...... construct lacking the VDR binding site and did not increase binding of the VDR to the CAMP promoter as determined by chromatin immunoprecipitation assays. These findings indicate that induction of CAMP by curcumin occurs through a vitamin D receptor-independent manner. We conclude that PUFAs and curcumin do...

  9. Structural predictions of neurobiologically relevant G-protein coupled receptors and intrinsically disordered proteins.

    Science.gov (United States)

    Rossetti, Giulia; Dibenedetto, Domenica; Calandrini, Vania; Giorgetti, Alejandro; Carloni, Paolo

    2015-09-15

    G protein coupled receptors (GPCRs) and intrinsic disordered proteins (IDPs) are key players for neuronal function and dysfunction. Unfortunately, their structural characterization is lacking in most cases. From one hand, no experimental structure has been determined for the two largest GPCRs subfamilies, both key proteins in neuronal pathways. These are the odorant (450 members out of 900 human GPCRs) and the bitter taste receptors (25 members) subfamilies. On the other hand, also IDPs structural characterization is highly non-trivial. They exist as dynamic, highly flexible structural ensembles that undergo conformational conversions on a wide range of timescales, spanning from picoseconds to milliseconds. Computational methods may be of great help to characterize these neuronal proteins. Here we review recent progress from our lab and other groups to develop and apply in silico methods for structural predictions of these highly relevant, fascinating and challenging systems. Copyright © 2015 Elsevier Inc. All rights reserved.

  10. Protein receptor-independent plasma membrane remodeling by HAMLET

    DEFF Research Database (Denmark)

    Nadeem, Aftab; Sanborn, Jeremy; Gettel, Douglas L.

    2015-01-01

    A central tenet of signal transduction in eukaryotic cells is that extra-cellular ligands activate specific cell surface receptors, which orchestrate downstream responses. This "protein-centric" view is increasingly challenged by evidence for the involvement of specialized membrane domains...... in signal transduction. Here, we propose that membrane perturbation may serve as an alternative mechanism to activate a conserved cell-death program in cancer cells. This view emerges from the extraordinary manner in which HAMLET (Human Alpha-lactalbumin Made LEthal to Tumor cells) kills a wide range...... of tumor cells in vitro and demonstrates therapeutic efficacy and selectivity in cancer models and clinical studies. We identify a "receptor independent" transformation of vesicular motifs in model membranes, which is paralleled by gross remodeling of tumor cell membranes. Furthermore, we find that HAMLET...

  11. Role of amyloid β protein receptors in mediating synaptic plasticity.

    Science.gov (United States)

    Li, Yu; Sun, Zhongqing; Cao, Qiaoyu; Chen, Meiwan; Luo, Huanmin; Lin, Xi; Xiao, Fei

    2017-04-01

    There are few diseases in modern biomedicine that have garnered as much scientific interest and public concern as Alzheimer's disease (AD). The amyloid hypothesis has become the dominant model of AD pathogenesis; however, the details of the hypothesis are changing over time. Recently, given the increasing recognition, subtle effects of amyloid β protein (Aβ) on synaptic efficacy may be critical to AD progression. Synaptic plasticity is the important neurochemical foundation of learning and memory. Recent studies have identified that soluble Aβ oligomers combine with certain receptors to impair synaptic plasticity in AD, which advanced the amyloid hypothesis. The aim of the present review was to summarize the role of Aβ-relevant receptors in regulating synaptic plasticity and their downstream signaling cascades, which may provide novel insights into the understanding of the pathogenesis of AD and the development of therapeutic strategies to slow down the progression of AD-associated memory decline in the early stages.

  12. Self-organized criticality in proteins: Hydropathic roughening profiles of G-protein-coupled receptors

    Science.gov (United States)

    Phillips, J. C.

    2013-03-01

    Proteins appear to be the most dramatic natural example of self-organized criticality (SOC), a concept that explains many otherwise apparently unlikely phenomena. Protein conformational functionality is often dominated by long-range hydrophobic or hydrophilic interactions which both drive protein compaction and mediate protein-protein interactions. Superfamily transmembrane G-protein-coupled receptors (GPCRs) are the largest family of proteins in the human genome; their amino acid sequences form the largest database for protein-membrane interactions. While there are now structural data on the heptad transmembrane structures of representatives of several heptad families, here we show how fresh insights into global and some local chemical trends in GPCR properties can be obtained accurately from sequences alone, especially by algebraically separating the extracellular and cytoplasmic loops from transmembrane segments. The global mediation of long-range water-protein interactions occurs in conjunction with modulation of these interactions by roughened interfaces. Hydropathic roughening profiles are defined here solely in terms of amino acid sequences, and knowledge of protein coordinates is not required. Roughening profiles both for GPCR and some simpler protein families display accurate and transparent connections to protein functionality, and identify natural length scales for protein functionality.

  13. Analysis of odorant receptor protein function in the yellow fever mosquito, aedes aegypti

    Science.gov (United States)

    Odorant receptors (ORs) in insects are ligand-gated ion channels comprised of two subunits: a variable receptor and an obligatory co-receptor (Orco). This protein receptor complex of unknown stoichiometry interacts with an odor molecule leading to changes in permeability of the sensory dendrite, th...

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

    NARCIS (Netherlands)

    Araç, Demet; Aust, Gabriela; Calebiro, Davide; Engel, Felix B.; Formstone, Caroline; Goffinet, André; Hamann, Jörg; Kittel, Robert J.; Liebscher, Ines; Lin, Hsi-Hsien; Monk, Kelly R.; Petrenko, Alexander; Piao, Xianhua; Prömel, Simone; Schiöth, Helgi B.; Schwartz, Thue W.; Stacey, Martin; Ushkaryov, Yuri A.; Wobus, Manja; Wolfrum, Uwe; Xu, Lei; Langenhan, Tobias

    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

  15. Scrum Code Camps

    DEFF Research Database (Denmark)

    Pries-Heje, Jan; Pries-Heje, Lene; Dahlgaard, Bente

    2013-01-01

    is required. In this paper we present the design of such a new approach, the Scrum Code Camp, which can be used to assess agile team capability in a transparent and consistent way. A design science research approach is used to analyze properties of two instances of the Scrum Code Camp where seven agile teams...

  16. Friends' Discovery Camp

    Science.gov (United States)

    Seymour, Seth

    2008-01-01

    This article features Friends' Discovery Camp, a program that allows children with and without autism spectrum disorder to learn and play together. In Friends' Discovery Camp, campers take part in sensory-rich experiences, ranging from hands-on activities and performing arts to science experiments and stories teaching social skills. Now in its 7th…

  17. Molecular characterization of a novel human hybrid-type receptor that binds the alpha2-macroglobulin receptor-associated protein

    DEFF Research Database (Denmark)

    Jacobsen, Linda; Madsen, P; Moestrup, S K

    1996-01-01

    The 39-40-kDa receptor-associated protein (RAP) binds to the members of the low density lipoprotein receptor gene family and functions as a specialized endoplasmic reticulum/Golgi chaperone. Using RAP affinity chromatography, we have purified a novel approximately 250-kDa brain protein and isolated...... the corresponding cDNA. The gene, designated SORL1, maps to chromosome 11q 23/24 and encodes a 2214-residue type 1 receptor containing a furin cleavage site immediately preceding the N terminus determined in the purified protein. The receptor, designated sorLA-1, has a short cytoplasmic tail containing a tyrosine......-based internalization signal and a large external part containing (from the N-terminal): 1) a segment homologous to domains in the yeast vacuolar protein sorting 10 protein, Vps10p, that binds carboxypeptidase Y, 2) five tandemly arranged YWTD repeats and a cluster of 11 class A repeats characteristic of the low...

  18. 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.

  19. G Protein-Coupled Receptor Multimers : A Question Still Open Despite the Use of Novel Approaches

    NARCIS (Netherlands)

    Vischer, Henry F; Castro, Marián; Pin, Jean Philippe

    Heteromerization of G protein-coupled receptors (GPCRs) can significantly change the functional properties of involved receptors. Various biochemical and biophysical methodologies have been developed in the last two decades to identify and functionally evaluate GPCR heteromers in heterologous cells,

  20. Angiotensin-(1-7) Is an Endogenous Ligand for the G Protein-Coupled Receptor Mas

    National Research Council Canada - National Science Library

    Robson A. S. Santos; Ana C. Simoes e Silva; Christine Maric; Denise M. R. Silva; Raquel Pillar Machado; Insa de Buhr; Silvia Heringer-Walther; Sergio Veloso B. Pinheiro; Myriam Teresa Lopes; Michael Bader; Elizabeth P. Mendes; Virgina Soares Lemos; Maria Jose Campagnole-Santos; Heinz-Peter Schultheiss; Robert Speth; Thomas Walther

    2003-01-01

    ...) antagonist indicated the existence of a distinct Ang-(1-7) receptor. We demonstrate that genetic deletion of the G protein-coupled receptor encoded by the Mas protooncogene abolishes the binding of Ang-(1-7) to mouse kidneys...

  1. G-Protein-Coupled Receptors in Adult Neurogenesis

    Science.gov (United States)

    Doze, Van A.

    2012-01-01

    The importance of adult neurogenesis has only recently been accepted, resulting in a completely new field of investigation within stem cell biology. The regulation and functional significance of adult neurogenesis is currently an area of highly active research. G-protein-coupled receptors (GPCRs) have emerged as potential modulators of adult neurogenesis. GPCRs represent a class of proteins with significant clinical importance, because approximately 30% of all modern therapeutic treatments target these receptors. GPCRs bind to a large class of neurotransmitters and neuromodulators such as norepinephrine, dopamine, and serotonin. Besides their typical role in cellular communication, GPCRs are expressed on adult neural stem cells and their progenitors that relay specific signals to regulate the neurogenic process. This review summarizes the field of adult neurogenesis and its methods and specifies the roles of various GPCRs and their signal transduction pathways that are involved in the regulation of adult neural stem cells and their progenitors. Current evidence supporting adult neurogenesis as a model for self-repair in neuropathologic conditions, adult neural stem cell therapeutic strategies, and potential avenues for GPCR-based therapeutics are also discussed. PMID:22611178

  2. Inverse agonism of cannabinoid CB1 receptor blocks the adhesion of encephalitogenic T cells in inflamed brain venules by a protein kinase A-dependent mechanism.

    Science.gov (United States)

    Rossi, Barbara; Zenaro, Elena; Angiari, Stefano; Ottoboni, Linda; Bach, Simona; Piccio, Laura; Pietronigro, Enrica C; Scarpini, Elio; Fusco, Mariella; Leon, Alberta; Constantin, Gabriela

    2011-04-01

    It is well known that the cannabinoid system has a significant role in the regulation of the immune responses. Cannabinoid receptors CB1 and CB2 are expressed on T lymphocytes and mediate the immunomodulatory effects of cannabinoids on T cell functions. Here we show that the treatment of proteolipid protein (PLP)139-151-specific T cells with SR141716A, a CB1 inverse agonist and prototype of the diarylpyrazoles series, induced a strong inhibition of firm adhesion in inflamed brain venules in intravital microscopy experiments. In contrast, SR144528, a potent CB2 inverse agonist, had no significant effect on both rolling and arrest of activated T cells. In addition, two analogs of SR141716A and CB1 inverse agonists, AM251 and AM281 inhibited encephalitogenic T cell adhesion suggesting that selective CB1 inverse agonism interfere with lymphocyte trafficking in the CNS. Flow cytometry experiments showed that CB1 inverse agonists have no effect on adhesion molecule expression suggesting that CB1 blockade interferes with signal transduction pathways controlling T cell adhesion in inflamed brain venules. In addition, integrin clustering was not altered after treatment with CB1 inverse agonists suggesting that adhesion blockade is not due to the modulation of integrin valency. Notably, the inhibitory effect exerted by AM251 and AM281 on the adhesive interactions was completely reverted in the presence of protein kinase A (PKA) inhibitor H89, suggesting that cAMP and PKA activation play a key role in the adhesion blockade mediated by CB1 inverse agonists. To further strengthen these results and unveil a previously unknown inhibitory role of cAMP on activated T cell adhesion in vivo in the context of CNS inflammation, we showed that intracellular increase of cAMP induced by treatment with Bt2cAMP, a permeable analog of cAMP, and phosphodiesterase (PDE) inhibitor theophylline efficiently blocked the arrest of encephalitogenic T cells in inflamed brain venules. Our data show

  3. Cannabinoid receptor-interacting protein Crip1a modulates CB1 receptor signaling in mouse hippocampus.

    Science.gov (United States)

    Guggenhuber, Stephan; Alpar, Alan; Chen, Rongqing; Schmitz, Nina; Wickert, Melanie; Mattheus, Tobias; Harasta, Anne E; Purrio, Martin; Kaiser, Nadine; Elphick, Maurice R; Monory, Krisztina; Kilb, Werner; Luhmann, Heiko J; Harkany, Tibor; Lutz, Beat; Klugmann, Matthias

    2016-05-01

    The cannabinoid type 1 receptor (Cnr1, CB1R) mediates a plethora of physiological functions in the central nervous system as a presynaptic modulator of neurotransmitter release. The recently identified cannabinoid receptor-interacting protein 1a (Cnrip1a, CRIP1a) binds to the C-terminal domain of CB1R, a region known to be important for receptor desensitization and internalization. Evidence that CRIP1a and CB1R interact in vivo has been reported, but the neuroanatomical distribution of CRIP1a is unknown. Moreover, while alterations of hippocampal CRIP1a levels following limbic seizures indicate a role in controlling excessive neuronal activity, the physiological function of CRIP1a in vivo has not been investigated. In this study, we analyzed the spatial distribution of CRIP1a in the hippocampus and examined CRIP1a as a potential modulator of CB1R signaling. We found that Cnrip1a mRNA is co-expressed with Cnr1 mRNA in pyramidal neurons and interneurons of the hippocampal formation. CRIP1a protein profiles were largely segregated from CB1R profiles in mossy cell terminals but not in hippocampal CA1 region. CB1R activation induced relocalization to close proximity with CRIP1a. Adeno-associated virus-mediated overexpression of CRIP1a specifically in the hippocampus revealed that CRIP1a modulates CB1R activity by enhancing cannabinoid-induced G protein activation. CRIP1a overexpression extended the depression of excitatory currents by cannabinoids in pyramidal neurons of the hippocampus and diminished the severity of chemically induced acute epileptiform seizures. Collectively, our data indicate that CRIP1a enhances hippocampal CB1R signaling in vivo.

  4. G protein-coupled receptor kinase 2 promotes cardiac hypertrophy

    Science.gov (United States)

    Tscheschner, Henrike; Gao, Erhe; Schumacher, Sarah M.; Yuan, Ancai; Backs, Johannes; Most, Patrick; Wieland, Thomas; Koch, Walter J.; Katus, Hugo A.; Raake, Philip W.

    2017-01-01

    The increase in protein activity and upregulation of G-protein coupled receptor kinase 2 (GRK2) is a hallmark of cardiac stress and heart failure. Inhibition of GRK2 improved cardiac function and survival and diminished cardiac remodeling in various animal heart failure models. The aim of the present study was to investigate the effects of GRK2 on cardiac hypertrophy and dissect potential molecular mechanisms. In mice we observed increased GRK2 mRNA and protein levels following transverse aortic constriction (TAC). Conditional GRK2 knockout mice showed attenuated hypertrophic response with preserved ventricular geometry 6 weeks after TAC operation compared to wild-type animals. In isolated neonatal rat ventricular cardiac myocytes stimulation with angiotensin II and phenylephrine enhanced GRK2 expression leading to enhanced signaling via protein kinase B (PKB or Akt), consecutively inhibiting glycogen synthase kinase 3 beta (GSK3β), such promoting nuclear accumulation and activation of nuclear factor of activated T-cells (NFAT). Cardiac myocyte hypertrophy induced by in vitro GRK2 overexpression increased the cytosolic interaction of GRK2 and phosphoinositide 3-kinase γ (PI3Kγ). Moreover, inhibition of PI3Kγ as well as GRK2 knock down prevented Akt activation resulting in halted NFAT activity and reduced cardiac myocyte hypertrophy. Our data show that enhanced GRK2 expression triggers cardiac hypertrophy by GRK2-PI3Kγ mediated Akt phosphorylation and subsequent inactivation of GSK3β, resulting in enhanced NFAT activity. PMID:28759639

  5. Alpha-Bulges in G Protein-Coupled Receptors

    Directory of Open Access Journals (Sweden)

    Rob van der Kant

    2014-05-01

    Full Text Available Agonist binding is related to a series of motions in G protein-coupled receptors (GPCRs that result in the separation of transmembrane helices III and VI at their cytosolic ends and subsequent G protein binding. A large number of smaller motions also seem to be associated with activation. Most helices in GPCRs are highly irregular and often contain kinks, with extensive literature already available about the role of prolines in kink formation and the precise function of these kinks. GPCR transmembrane helices also contain many α-bulges. In this article we aim to draw attention to the role of these α-bulges in ligand and G-protein binding, as well as their role in several aspects of the mobility associated with GPCR activation. This mobility includes regularization and translation of helix III in the extracellular direction, a rotation of the entire helix VI, an inward movement of the helices near the extracellular side, and a concerted motion of the cytosolic ends of the helices that makes their orientation appear more circular and that opens up space for the G protein to bind. In several cases, α-bulges either appear or disappear as part of the activation process.

  6. Melanocortin Receptor Accessory Proteins (MRAPs): Functions in the melanocortin system and beyond.

    Science.gov (United States)

    Rouault, Alix A J; Srinivasan, Dinesh K; Yin, Terry C; Lee, Abigail A; Sebag, Julien A

    2017-10-01

    G-protein coupled receptors (GPCRs) are regulated by numerous proteins including kinases, G-proteins, β-arrestins and accessory proteins. Several families of GPCR accessory proteins like Receptor Activity Modifying Proteins, Receptor Transporting Proteins and Melanocortin Receptor Accessory Proteins (MRAPs) have been identified as regulator of receptor trafficking, signaling and ligand specificity. The MRAP family contains two members, MRAP1 and MRAP2, responsible for the formation of a functional ACTH receptor and for the regulation of energy homeostasis respectively. Like all known GPCR accessory proteins, MRAPs are single transmembrane proteins, however, they form a unique structure since they assemble as an anti-parallel homodimer. Moreover, the accepted idea that MRAPs are specific regulators of melanocortin receptors was recently challenged by the discovery that MRAP2 inhibits the activity of prokineticin receptors. Recent studies are starting to explain the role of the unusual structure of MRAPs and to illustrate the importance of MRAP2 for the maintenance of both energy and glucose homeostasis. This article is part of a Special Issue entitled: Melanocortin Receptors - edited by Ya-Xiong Tao. Copyright © 2017 Elsevier B.V. All rights reserved.

  7. Stimulation of G protein-coupled bile acid receptor enhances vascular endothelial barrier function via activation of protein kinase A and Rac1.

    Science.gov (United States)

    Kida, Taiki; Omori, Keisuke; Hori, Masatoshi; Ozaki, Hiroshi; Murata, Takahisa

    2014-01-01

    Bile acids are end products of cholesterol metabolism, and they constantly exist at high concentrations in the blood. Since vascular endothelial cells express G protein-coupled bile acid receptor (GPBAR), bile acids potentially modulate endothelial function. Here, we investigated whether and how GPBAR agonism affects endothelial barrier function. In bovine aortic endothelial cells (BAECs), treatment with a GPBAR agonist, taurolithocholic acid (TLCA) increased the transendothelial electrical resistance. In addition, TLCA suppressed the thrombin-induced dextran infiltration through the endothelial monolayer. Knockdown of GPBAR abolished the inhibitory effect of TLCA on hyperpermeability. These results indicate that stimulation of GPBAR enhances endothelial barrier function. TLCA increased intracellular cAMP production in BAECs. Inhibition of protein kinase A (PKA) or Rac1 significantly attenuated the TLCA-induced endothelial barrier protection. TLCA induced cortical actin polymerization, which was attenuated by a Rac1 inhibitor. In vivo, local administration of TLCA into the mouse ear significantly inhibited vascular leakage and edema formation induced by croton oil or vascular endothelial growth factor. These results indicate that stimulation of GPBAR enhances endothelial barrier function by cAMP/PKA/Rac1-dependent cytoskeletal rearrangement.

  8. Summer camp nurtures student

    OpenAIRE

    Earl Anderson

    2017-01-01

    Summer camp is a coordinated program for youths or teenagers driven in the midst of the late spring months in a couple of countries. Adolescents and young people who go to summer camp are known as campers. It is each parent's stress: What is the perfect way for your adolescent to contribute his or her free vitality in the midst of summer and school breaks? Research Paper Help. To a couple, it is a period for youths to play and have an incredible time. By joining the late spring camp, yout...

  9. Amide H/2H exchange reveals communication between the cAMP and catalytic subunit-binding sites in the R(I)alpha subunit of protein kinase A.

    Science.gov (United States)

    Anand, Ganesh S; Hughes, Carrie A; Jones, John M; Taylor, Susan S; Komives, Elizabeth A

    2002-10-18

    The changes in backbone hydrogen/deuterium (H/2H) exchange in the regulatory subunit (R(I)alpha(94-244)) of cyclic AMP-dependent protein kinase A (PKA) were probed by MALDI-TOF mass spectrometry. The three naturally occurring states of the regulatory subunit were studied: (1) free R(I)alpha(94-244), which likely represents newly synthesized protein, (2) R(I)alpha(94-244) bound to the catalytic (C) subunit, or holoenzyme, and (3) R(I)alpha(94-244) bound to cAMP. Protection from amide exchange upon C-subunit binding was observed for the helical subdomain, including the A-helix and B-helix, pointing to regions adjacent to those shown to be important by mutagenesis. In addition, C-subunit binding caused changes in observed amide exchange in the distal cAMP-binding pocket. Conversely, cAMP binding caused protection in the cAMP-binding pocket and increased exchange in the helical subdomain. These results suggest that the mutually exclusive binding of either cAMP or C-subunit is controlled by binding at one site transmitting long distance changes to the other site.

  10. Identification of Toxoplasma gondii cAMP dependent protein kinase and its role in the tachyzoite growth.

    Directory of Open Access Journals (Sweden)

    Hitomi Kurokawa

    Full Text Available cAMP-dependent protein kinase (PKA has been implicated in the asexual stage of the Toxoplasma gondii life cycle through assaying the effect of a PKA-specific inhibitor on its growth rate. Since inhibition of the host cell PKA cannot be ruled out, a more precise evaluation of the role of PKA, as well as characterization of the kinase itself, is necessary.The inhibitory effects of two PKA inhibitors, H89, an ATP-competitive chemical inhibitor, and PKI, a substrate-competitive mammalian natural peptide inhibitor, were estimated. In the in vitro kinase assay, the inhibitory effect of PKI on a recombinant T. gondii PKA catalytic subunit (TgPKA-C was weaker compared to that on mammalian PKA-C. In a tachyzoite growth assay, PKI had little effect on the growth of tachyzoites, whereas H89 strongly inhibited it. Moreover, T. gondii PKA regulatory subunit (TgPKA-R-overexpressing tachyzoites showed a significant growth defect.Our data suggest that PKA plays an important role in the growth of tachyzoites, and the inhibitory effect of substrate-competitive inhibitor PKI on T. gondii PKA was low compared to that of the ATP competitive inhibitor H89.

  11. Erythropoietin activates the phosporylated cAMP [adenosine 3'5' cyclic monophosphate] response element-binding protein pathway and attenuates delayed paraplegia after ischemia-reperfusion injury.

    Science.gov (United States)

    Mares, Joshua M; Foley, Lisa S; Bell, Marshall T; Bennett, Daine T; Freeman, Kirsten A; Meng, Xianzhong; Weyant, Michael J; Cleveland, Joseph C; Fullerton, David A; Puskas, Ferenc; Reece, Thomas Brett

    2015-03-01

    Paraplegia remains a devastating complication of complex aortic surgery. Erythropoietin (EPO) has been shown to prevent paraplegia after ischemia reperfusion, but the protective mechanism remains poorly described in the spinal cord. We hypothesized that EPO induces the CREB (cAMP [adenosine 3'5' cyclic monophosphate] response element-binding protein) pathway and neurotrophin production in the murine spinal cord, attenuating functional and cellular injury. Adult male mice were subjected to 4 minutes of spinal cord ischemia via an aortic and left subclavian cross-clamp. Experimental groups included EPO treatment 4 hours before incision (n = 7), ischemic control (n = 7), and shams (n = 4). Hind-limb function was assessed using the Basso motor score for 48 hours after reperfusion. Spinal cords were harvested and analyzed for neuronal viability using histology and staining with a fluorescein derivative. Expression of phosphorylated (p)AKT (a serine/threonine-specific kinase), pCREB, B-cell lymphoma 2, and brain-derived neurotrophic factor were determined using immunoblotting. By 36 hours of reperfusion, EPO significantly preserved hind-limb function after ischemia-reperfusion injury (P < .01). Histology demonstrated preserved cytoarchitecture in the EPO treatment group. Cords treated with EPO expressed significant increases in pAKT (P = .021) and pCREB (P = .038). Treatment with EPO induced expression of both of the neurotrophins, B-cell lymphoma 2, and brain-derived neurotrophic factor, beginning at 12 hours. Erythropoietin-mediated induction of the CREB pathway and production of neurotrophins is associated with improved neurologic function and increased neuronal viability following spinal cord ischemia reperfusion. Further elucidation of EPO-derived neuroprotection will allow for expansion of adjunct mechanisms for spinal cord protection in high-risk thoracoabdominal aortic intervention. Copyright © 2015 The American Association for Thoracic Surgery. Published by

  12. S-Nitrosothiols modulate G protein-coupled receptor signaling in a reversible and highly receptor-specific manner

    Directory of Open Access Journals (Sweden)

    Mönkkönen Kati S

    2005-04-01

    Full Text Available Abstract Background Recent studies indicate that the G protein-coupled receptor (GPCR signaling machinery can serve as a direct target of reactive oxygen species, including nitric oxide (NO and S-nitrosothiols (RSNOs. To gain a broader view into the way that receptor-dependent G protein activation – an early step in signal transduction – might be affected by RSNOs, we have studied several receptors coupling to the Gi family of G proteins in their native cellular environment using the powerful functional approach of [35S]GTPγS autoradiography with brain cryostat sections in combination with classical G protein activation assays. Results We demonstrate that RSNOs, like S-nitrosoglutathione (GSNO and S-nitrosocysteine (CysNO, can modulate GPCR signaling via reversible, thiol-sensitive mechanisms probably involving S-nitrosylation. RSNOs are capable of very targeted regulation, as they potentiate the signaling of some receptors (exemplified by the M2/M4 muscarinic cholinergic receptors, inhibit others (P2Y12 purinergic, LPA1lysophosphatidic acid, and cannabinoid CB1 receptors, but may only marginally affect signaling of others, such as adenosine A1, μ-opioid, and opiate related receptors. Amplification of M2/M4 muscarinic responses is explained by an accelerated rate of guanine nucleotide exchange, as well as an increased number of high-affinity [35S]GTPγS binding sites available for the agonist-activated receptor. GSNO amplified human M4 receptor signaling also under heterologous expression in CHO cells, but the effect diminished with increasing constitutive receptor activity. RSNOs markedly inhibited P2Y12 receptor signaling in native tissues (rat brain and human platelets, but failed to affect human P2Y12 receptor signaling under heterologous expression in CHO cells, indicating that the native cellular signaling partners, rather than the P2Y12 receptor protein, act as a molecular target for this action. Conclusion These in vitro studies

  13. Gαs signalling of the CB1 receptor and the influence of receptor number.

    Science.gov (United States)

    Finlay, David B; Cawston, Erin E; Grimsey, Natasha L; Hunter, Morag R; Korde, Anisha; Vemuri, V Kiran; Makriyannis, Alexandros; Glass, Michelle

    2017-08-01

    CB1 receptor signalling is canonically mediated through inhibitory Gαi proteins, but occurs through other G proteins under some circumstances, Gαs being the most characterized secondary pathway. Determinants of this signalling switch identified to date include Gαi blockade, CB1 /D2 receptor co-stimulation, CB1 agonist class and cell background. Hence, we examined the effects of receptor number and different ligands on CB1 receptor signalling. CB1 receptors were expressed in HEK cells at different levels, and signalling characterized for cAMP by real-time BRET biosensor -CAMYEL - and for phospho-ERK by AlphaScreen. Homogenate and whole cell radioligand binding assays were performed to characterize AM6544, a novel irreversible CB1 receptor antagonist. In HEK cells expressing high levels of CB1 receptors, agonist treatment stimulated cAMP, a response not known to be mediated by receptor number. Δ(9) -THC and BAY59-3074 increased cAMP only in high-expressing cells pretreated with pertussis toxin, and agonists demonstrated more diverse signalling profiles in the stimulatory pathway than the canonical inhibitory pathway. Pharmacological CB1 receptor knockdown and Gαi 1 supplementation restored canonical Gαi signalling to high-expressing cells. Constitutive signalling in both low- and high-expressing cells was Gαi -mediated. CB1 receptor coupling to opposing G proteins is determined by both receptor and G protein expression levels, which underpins a mechanism for non-canonical signalling in a fashion consistent with Gαs signalling. CB1 receptors mediate opposite consequences in endpoints such as tumour viability depending on expression levels; our results may help to explain such effects at the level of G protein coupling. © 2017 The British Pharmacological Society.

  14. PDF and cAMP enhance PER stability in Drosophila clock neurons.

    Science.gov (United States)

    Li, Yue; Guo, Fang; Shen, James; Rosbash, Michael

    2014-04-01

    The neuropeptide PDF is important for Drosophila circadian rhythms: pdf(01) (pdf-null) animals are mostly arrhythmic or short period in constant darkness and have an advanced activity peak in light-dark conditions. PDF contributes to the amplitude, synchrony, as well as the pace of circadian rhythms within clock neurons. PDF is known to increase cAMP levels in PDR receptor (PDFR)-containing neurons. However, there is no known connection of PDF or of cAMP with the Drosophila molecular clockworks. We discovered that the mutant period gene per(S) ameliorates the phenotypes of pdf-null flies. The period protein (PER) is a well-studied repressor of clock gene transcription, and the per(S) protein (PERS) has a markedly short half-life. The result therefore suggests that the PDF-mediated increase in cAMP might lengthen circadian period by directly enhancing PER stability. Indeed, increasing cAMP levels and cAMP-mediated protein kinase A (PKA) activity stabilizes PER, in S2 tissue culture cells and in fly circadian neurons. Adding PDF to fly brains in vitro has a similar effect. Consistent with these relationships, a light pulse causes more prominent PER degradation in pdf(01) circadian neurons than in wild-type neurons. The results indicate that PDF contributes to clock neuron synchrony by increasing cAMP and PKA, which enhance PER stability and decrease clock speed in intrinsically fast-paced PDFR-containing clock neurons. We further suggest that the more rapid degradation of PERS bypasses PKA regulation and makes the pace of clock neurons more uniform, allowing them to avoid much of the asynchrony caused by the absence of PDF.

  15. 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...

  16. Hitler's Death Camps.

    Science.gov (United States)

    Wieser, Paul

    1995-01-01

    Presents a high school lesson on Hitler's death camps and the widespread policy of brutality and oppression against European Jews. Includes student objectives, instructional procedures, and a chart listing the value of used clothing taken from the Jews. (CFR)

  17. Growth hormone receptor/binding protein: physiology and function.

    Science.gov (United States)

    Herington, A C; Ymer, S I; Stevenson, J L; Roupas, P

    1994-07-01

    Soluble truncated forms of the growth hormone receptor (GHR) are present in the circulation of many species and are also produced by many tissues/cell types. The major high-affinity forms of these GH-binding proteins (GHBP) are derived by alternative splicing of GHR mRNA in rodents, but probably by proteolytic cleavage in other species. Questions still remain with respect to the origins, native molecular form(s), physiology, and function of the GHBPs, however. The observation that GH induces dimerization of the soluble GHBP and membrane GHR, and that dimerization of GHR appears to be critical for GH bioactivity suggests that the presentation of GH to target cells, in an unbound form or as a monomeric or dimeric complex with GHBP, may have significant implications for the ability of GH to activate specific postreceptor signaling pathways (tyrosine kinase, protein kinase C, G-protein pathways) known to be utilized by GH for its diverse biological effects. This minireview addresses some of these aspects and highlights several new questions which have arisen as a result of recent advances in our understanding of the structure, function, and signaling mechanisms of the membrane bound GHR.

  18. Growth hormone receptor/binding protein: Physiology and function

    Energy Technology Data Exchange (ETDEWEB)

    Herington, A.C.; Ymer, S.I.; Stevenson, J.L.; Roupas, P. [Royal Children`s Hospital, Melbourne (Australia)

    1994-12-31

    Soluble truncated forms of the growth hormone receptor (GHR) are present in the circulation of many species and are also produced by many tissues/cell types. The major high-affinity forms of these GH-binding proteins (GHBP) are derived by alternative splicing of GHR mRNA in rodents, but probably by proteolytic cleavage in other species. Questions still remain with respect to the origins, native molecular forms(s), physiology, and function of the GHBPs, however. The observation that GH induces dimerization of the soluble GHBP and a membrane GHR, and that dimerization of GHR appears to be critical for GH bioactivity suggests that the presentation of GH to target cells, in an unbound form or as a monomeric or dimeric complex with GHBP, may have significant implications for the ability of GH to activate specific postreceptor signaling pathways (tyrosine kinase, protein kinase C, G-protein pathways) known to be utilized by GH for its diverse biological effects. This minireview addresses some of these aspects and highlights several new questions which have arisen as a result of recent advances in our understanding of the structure, function, and signaling mechanisms of the membrane bound GHR. 43 refs.

  19. Involvement of cyclic AMP receptor protein in regulation of the rmf gene encoding the ribosome modulation factor in Escherichia coli.

    Science.gov (United States)

    Shimada, Tomohiro; Yoshida, Hideji; Ishihama, Akira

    2013-05-01

    The decrease in overall translation in stationary-phase Escherichia coli is accompanied with the formation of functionally inactive 100S ribosomes mediated by the ribosome modulation factor (RMF). At present, however, little is known regarding the regulation of stationary-phase-coupled RMF expression. In the course of a systematic screening of regulation targets of DNA-binding transcription factors from E. coli, we realized that CRP (cyclic AMP [cAMP] receptor protein), the global regulator for carbon source utilization, participates in regulation of some ribosomal protein genes, including the rmf gene. In this study, we carried out detailed analysis of the regulation of the RMF gene by cAMP-CRP. The cAMP-dependent binding of CRP to the rmf gene promoter was confirmed by gel shift and DNase I footprinting assays. By using a reporter assay system, the expression level of RMF was found to decrease in the crp knockout mutant, indicating the involvement of CRP as an activator of the rmf promoter. In good agreement with the reduction of rmf promoter activity, we observed decreases in RMF production and 100S ribosome dimerization in the absence of CRP. Taken together, we propose that CRP regulates transcription activation of the rmf gene for formation of 100S ribosome dimers. Physiological roles of CRP involvement in RMF production are discussed.

  20. CDC Disease Detective Camp

    Centers for Disease Control (CDC) Podcasts

    2010-08-02

    The CDC Disease Detective Camp gives rising high school juniors and seniors exposure to key aspects of the CDC, including basic epidemiology, infectious and chronic disease tracking, public health law, and outbreak investigations. The camp also helps students explore careers in public health.  Created: 8/2/2010 by Centers for Disease Control and Prevention (CDC).   Date Released: 8/2/2010.

  1. Uncovering Molecular Bases Underlying Bone Morphogenetic Protein Receptor Inhibitor Selectivity.

    Directory of Open Access Journals (Sweden)

    Abdelaziz Alsamarah

    Full Text Available Abnormal alteration of bone morphogenetic protein (BMP signaling is implicated in many types of diseases including cancer and heterotopic ossifications. Hence, small molecules targeting BMP type I receptors (BMPRI to interrupt BMP signaling are believed to be an effective approach to treat these diseases. However, lack of understanding of the molecular determinants responsible for the binding selectivity of current BMP inhibitors has been a big hindrance to the development of BMP inhibitors for clinical use. To address this issue, we carried out in silico experiments to test whether computational methods can reproduce and explain the high selectivity of a small molecule BMP inhibitor DMH1 on BMPRI kinase ALK2 vs. the closely related TGF-β type I receptor kinase ALK5 and vascular endothelial growth factor receptor type 2 (VEGFR2 tyrosine kinase. We found that, while the rigid docking method used here gave nearly identical binding affinity scores among the three kinases; free energy perturbation coupled with Hamiltonian replica-exchange molecular dynamics (FEP/H-REMD simulations reproduced the absolute binding free energies in excellent agreement with experimental data. Furthermore, the binding poses identified by FEP/H-REMD led to a quantitative analysis of physical/chemical determinants governing DMH1 selectivity. The current work illustrates that small changes in the binding site residue type (e.g. pre-hinge region in ALK2 vs. ALK5 or side chain orientation (e.g. Tyr219 in caALK2 vs. wtALK2, as well as a subtle structural modification on the ligand (e.g. DMH1 vs. LDN193189 will cause distinct binding profiles and selectivity among BMP inhibitors. Therefore, the current computational approach represents a new way of investigating BMP inhibitors. Our results provide critical information for designing exclusively selective BMP inhibitors for the development of effective pharmacotherapy for diseases caused by aberrant BMP signaling.

  2. Recruitment of SHP-1 protein tyrosine phosphatase and signalling by a chimeric T-cell receptor-killer inhibitory receptor

    DEFF Research Database (Denmark)

    Christensen, M D; Geisler, C

    2000-01-01

    recognize MHC class I molecules. Following coligation of KIR with an activating receptor, the tyrosine in the ITIM is phosphorylated and the cytoplasmic protein tyrosine phosphatase SHP-1 is recruited to the ITIM via its SH2 domains. It is still not clear how SHP-1 affects T-cell receptor (TCR) signalling....... In this study, we constructed a chimeric TCR-KIR receptor. We demonstrated that SHP-1 is recruited to the chimeric TCR-KIR receptor following T-cell stimulation with either anti-TCR monoclonal antibody (MoAb) or superantigen. However, in spite of this we could not detect any effect of SHP-1 on TCR signalling...

  3. Identification of Human P2X1 Receptor-interacting Proteins Reveals a Role of the Cytoskeleton in Receptor Regulation*

    Science.gov (United States)

    Lalo, Ulyana; Roberts, Jonathan A.; Evans, Richard J.

    2011-01-01

    P2X1 receptors are ATP-gated ion channels expressed by smooth muscle and blood cells. Carboxyl-terminally His-FLAG-tagged human P2X1 receptors were stably expressed in HEK293 cells and co-purified with cytoskeletal proteins including actin. Disruption of the actin cytoskeleton with cytochalasin D inhibited P2X1 receptor currents with no effect on the time course of the response or surface expression of the receptor. Stabilization of the cytoskeleton with jasplakinolide had no effect on P2X1 receptor currents but decreased receptor mobility. P2X2 receptor currents were unaffected by cytochalasin, and P2X1/2 receptor chimeras were used to identify the molecular basis of actin sensitivity. These studies showed that the intracellular amino terminus accounts for the inhibitory effects of cytoskeletal disruption similar to that shown for lipid raft/cholesterol sensitivity. Stabilization of the cytoskeleton with jasplakinolide abolished the inhibitory effects of cholesterol depletion on P2X1 receptor currents, suggesting that lipid rafts may regulate the receptor through stabilization of the cytoskeleton. These studies show that the cytoskeleton plays an important role in P2X1 receptor regulation. PMID:21757694

  4. The repertoire of olfactory C family G protein-coupled receptors in zebrafish: candidate chemosensory receptors for amino acids

    Directory of Open Access Journals (Sweden)

    Ngai John

    2006-12-01

    Full Text Available Abstract Background Vertebrate odorant receptors comprise at least three types of G protein-coupled receptors (GPCRs: the OR, V1R, and V2R/V2R-like receptors, the latter group belonging to the C family of GPCRs. These receptor families are thought to receive chemosensory information from a wide spectrum of odorant and pheromonal cues that influence critical animal behaviors such as feeding, reproduction and other social interactions. Results Using genome database mining and other informatics approaches, we identified and characterized the repertoire of 54 intact "V2R-like" olfactory C family GPCRs in the zebrafish. Phylogenetic analysis – which also included a set of 34 C family GPCRs from fugu – places the fish olfactory receptors in three major groups, which are related to but clearly distinct from other C family GPCRs, including the calcium sensing receptor, metabotropic glutamate receptors, GABA-B receptor, T1R taste receptors, and the major group of V2R vomeronasal receptor families. Interestingly, an analysis of sequence conservation and selective pressure in the zebrafish receptors revealed the retention of a conserved sequence motif previously shown to be required for ligand binding in other amino acid receptors. Conclusion Based on our findings, we propose that the repertoire of zebrafish olfactory C family GPCRs has evolved to allow the detection and discrimination of a spectrum of amino acid and/or amino acid-based compounds, which are potent olfactory cues in fish. Furthermore, as the major groups of fish receptors and mammalian V2R receptors appear to have diverged significantly from a common ancestral gene(s, these receptors likely mediate chemosensation of different classes of chemical structures by their respective organisms.

  5. Degradation of pro-insulin-receptor proteins by proteasomes.

    Science.gov (United States)

    Cruz, Miguel; Velasco, Eduardo; Kumate, Jesús

    2004-01-01

    Type-2 diabetes is characterized by hyperinsulinemia, peripheral insulin resistance, and diminished tyrosine phosphorylation activity. It has been recently shown that proteasomes are implicated in the degradation of the insulin receptor substrate-1 (IRS-1) but not in that of the insulin receptor (IR). However, it is unknown whether proteasomes are involved in pro-IR degradation. We used CHO-IR and the 3T3-L1 cells treated with insulin at different concentrations and compared the proteasome activity of IRS-1, IR, and pro-IR degradation either in presence or in absence of lactacystin. A total of 100 nM of insulin allowed degradation of IRS-1 after 6 h of incubation. At 1,000 nM of insulin, pro-IR degradation began at 1 h of incubation, similar to IRS-1 degradation. Surprisingly, at a higher concentration (10 microM) of insulin, a drastic decrease of proteins was observed from the first minute of incubation. This activity was blocked by lactacystin, a specific proteasome inhibitor. According to these results, we propose that pro-IR is degraded by proteasomes.

  6. G-protein-coupled receptors as fat sensors.

    Science.gov (United States)

    Vinolo, Marco A R; Hirabara, Sandro M; Curi, Rui

    2012-03-01

    It has been demonstrated that fatty acids (FAs) are physiological ligands of G-protein-coupled receptors (GPRs). Activation of the GPRs (40, 41, 43, 84, 119 and 120) by FAs or synthetic agonists modulates several responses. In this review, we discuss the current knowledge on the actions of FA-activated GPRs and their relevance in normal and pathological conditions. Studies have shown that FA-activated GPRs modulate hormone secretion (incretin, insulin and glucagon), activation of leukocytes and several aspects of metabolism. Understanding GPR actions and their involvement in the development of insulin-resistance, β-cell failure, dyslipidemia and inflammation associated with obesity, type 2 diabetes, metabolic syndrome and cardiovascular diseases is important for the comprehension of the mechanisms underlying these pathological conditions and for the establishment of new and effective interventions.

  7. G protein-coupled receptors as regulators of energy homeostasis.

    Science.gov (United States)

    Tao, Ya-Xiong; Yuan, Zong-Hui; Xie, Jun

    2013-01-01

    G protein-coupled receptors (GPCRs) are versatile regulators of physiological processes. They are also important drug targets. Many of the molecules controlling energy homeostasis act through GPCRs. This article summarizes the regulators of energy homeostasis in the central nervous system: those secreted by the gastrointestinal peptides and those secreted by the endocrine pancreas. Some examples of orphan GPCRs are also given. The regulation of energy homeostasis is conserved in other mammals, including those species relevant in veterinary medicine, and fish. Finally, the genetics of human obesity is briefly summarized. Genetic susceptibility in the current obesogenic environment is likely causing the obesity pandemic. A better understanding of the regulation of energy homeostasis will lead to novel pharmacotherapy for obesity treatment. Copyright © 2013 Elsevier Inc. All rights reserved.

  8. cAMP receptor protein regulates mouse colonization, motility, fimbria-mediated adhesion, and stress tolerance in uropathogenic Proteus mirabilis

    National Research Council Canada - National Science Library

    Yi-Lin Tsai; Hsiung-Fei Chien; Kuo-Tong Huang; Wen-Yuan Lin; Shwu-Jen Liaw

    2017-01-01

    .... This study demonstrated that Crp affects numerous virulence-related phenotypes, including colonization of mice, motility, fimbria-mediated adhesion, and glucose stress tolerance in uropathogenic Proteus mirabilis...

  9. REEPs Are Membrane Shaping Adapter Proteins That Modulate Specific G Protein-Coupled Receptor Trafficking by Affecting ER Cargo Capacity

    OpenAIRE

    Susann Björk; Hurt, Carl M.; Ho, Vincent K.; Timothy Angelotti

    2013-01-01

    Receptor expression enhancing proteins (REEPs) were identified by their ability to enhance cell surface expression of a subset of G protein-coupled receptors (GPCRs), specifically GPCRs that have proven difficult to express in heterologous cell systems. Further analysis revealed that they belong to the Yip (Ypt-interacting protein) family and that some REEP subtypes affect ER structure. Yip family comparisons have established other potential roles for REEPs, including regulation of ER-Golgi t...

  10. The octopamine receptor OAMB mediates ovulation via Ca2+/calmodulin-dependent protein kinase II in the Drosophila oviduct epithelium.

    Directory of Open Access Journals (Sweden)

    Hyun-Gwan Lee

    Full Text Available Ovulation is an essential physiological process in sexual reproduction; however, the underlying cellular mechanisms are poorly understood. We have previously shown that OAMB, a Drosophila G-protein-coupled receptor for octopamine (the insect counterpart of mammalian norepinephrine, is required for ovulation induced upon mating. OAMB is expressed in the nervous and reproductive systems and has two isoforms (OAMB-AS and OAMB-K3 with distinct capacities to increase intracellular Ca2+ or intracellular Ca2+ and cAMP in vitro. Here, we investigated tissue specificity and intracellular signals required for OAMB's function in ovulation. Restricted OAMB expression in the adult oviduct epithelium, but not the nervous system, reinstated ovulation in oamb mutant females, in which either OAMB isoform was sufficient for the rescue. Consistently, strong immunoreactivities for both isoforms were observed in the wild-type oviduct epithelium. To delineate the cellular mechanism by which OAMB regulates ovulation, we explored protein kinases functionally interacting with OAMB by employing a new GAL4 driver with restricted expression in the oviduct epithelium. Conditional inhibition of Ca2+/Calmodulin-dependent protein kinase II (CaMKII, but not protein kinase A or C, in the oviduct epithelium inhibited ovulation. Moreover, constitutively active CaMKII, but not protein kinase A, expressed only in the adult oviduct epithelium fully rescued the oamb female's phenotype, demonstrating CaMKII as a major downstream molecule conveying the OAMB's ovulation signal. This is consistent with the ability of both OAMB isoforms, whose common intracellular signal in vitro is Ca2+, to reinstate ovulation in oamb females. These observations reveal the critical roles of the oviduct epithelium and its cellular components OAMB and CaMKII in ovulation. It is conceivable that the OAMB-mediated cellular activities stimulated upon mating are crucial for secretory activities suitable for egg

  11. Requirements and ontology for a G protein-coupled receptor oligomerization knowledge base

    NARCIS (Netherlands)

    Skrabanek, L.; Murcia, M.; Bouvier, M.; Devi, L.; George, S.R.; Lohse, M.J.; Milligan, G.; Neubig, R.; Palczewski, K.; Parmentier, M.; Pin, J.P.; Vriend, G.; Javitch, J.A.; Campagne, F.; Filizola, M.

    2007-01-01

    BACKGROUND: G Protein-Coupled Receptors (GPCRs) are a large and diverse family of membrane proteins whose members participate in the regulation of most cellular and physiological processes and therefore represent key pharmacological targets. Although several bioinformatics resources support research

  12. Endocytosis and membrane receptor internalization: implication of F-BAR protein Carom.

    Science.gov (United States)

    Xu, Yanjie; Xia, Jixiang; Liu, Suxuan; Stein, Sam; Ramon, Cueto; Xi, Hang; Wang, Luqiao; Xiong, Xinyu; Zhang, Lixiao; He, Dingwen; Yang, William; Zhao, Xianxian; Cheng, Xiaoshu; Yang, Xiaofeng; Wang, Hong

    2017-03-01

    Endocytosis is a cellular process mostly responsible for membrane receptor internalization. Cell membrane receptors bind to their ligands and form a complex which can be internalized. We previously proposed that F-BAR protein initiates membrane curvature and mediates endocytosis via its binding partners. However, F-BAR protein partners involved in membrane receptor endocytosis and the regulatory mechanism remain unknown. In this study, we established database mining strategies to explore mechanisms underlying receptor-related endocytosis. We identified 34 endocytic membrane receptors and 10 regulating proteins in clathrin-dependent endocytosis (CDE), a major process of membrane receptor internalization. We found that F-BAR protein FCHSD2 (Carom) may facilitate endocytosis via 9 endocytic partners. Carom is highly expressed, along with highly expressed endocytic membrane receptors and partners, in endothelial cells and macrophages. We established 3 models of Carom-receptor complexes and their intracellular trafficking based on protein interaction and subcellular localization. We conclude that Carom may mediate receptor endocytosis and transport endocytic receptors to the cytoplasm for receptor signaling and lysosome/proteasome degradation, or to the nucleus for RNA processing, gene transcription and DNA repair.

  13. Deletion of G-protein-coupled receptor 55 promotes obesity by reducing physical activity

    Science.gov (United States)

    Cannabinoid receptor 1 (CB1) is the best-characterized cannabinoid receptor, and CB1 antagonists are used in clinical trials to treat obesity. Because of the wide range of CB1 functions, the side effects of CB1 antagonists pose serious concerns. G-protein-coupled receptor 55 (GPR55) is an atypical c...

  14. Tachykinin-Related Peptides Share a G Protein-Coupled Receptor with Ion Transport Peptide-Like in the Silkworm Bombyx mori.

    Directory of Open Access Journals (Sweden)

    Chiaki Nagai-Okatani

    Full Text Available Recently, we identified an orphan Bombyx mori neuropeptide G protein-coupled receptor (BNGR-A24 as an ion transport peptide-like (ITPL receptor. BNGR-A24 belongs to the same clade as BNGR-A32 and -A33, which were recently identified as natalisin receptors. Since these three BNGRs share high similarities with known receptors for tachykinin-related peptides (TRPs, we examined whether these BNGRs can function as physiological receptors for five endogenous B. mori TRPs (TK-1-5. In a heterologous expression system, BNGR-A24 acted as a receptor for all five TRPs. In contrast, BNGR-A32 responded only to TK-5, and BNGR-A33 did not respond to any of the TRPs. These findings are consistent with recent studies on the ligand preferences for B. mori natalisins. Furthermore, we evaluated whether the binding of ITPL and TRPs to BNGR-A24 is competitive by using a Ca2+ imaging assay. Concomitant addition of a TRP receptor antagonist, spantide I, reduced the responses of BNGR-A24 not only to TK-4 but also to ITPL. The results of a binding assay using fluorescent-labeled BNGR-A24 and ligands demonstrated that the binding of ITPL to BNGR-A24 was inhibited by TK-4 as well as by spantide I, and vice versa. In addition, the ITPL-induced increase in cGMP levels of BNGR-A24-expressing BmN cells was suppressed by the addition of excess TK-4 or spantide I. The intracellular levels of cAMP and cGMP, as second messenger candidates of the TRP signaling, were not altered by the five TRPs, suggesting that these peptides act via different signaling pathways from cAMP and cGMP signaling at least in BmN cells. Taken together, the present findings suggest that ITPL and TRPs are endogenous orthosteric ligands of BNGR-A24 that may activate discrete signaling pathways. This receptor, which shares orthosteric ligands, may constitute an important model for studying ligand-biased signaling.

  15. Regulation of Toll-like receptor 4-mediated immune responses through Pasteurella multocida toxin-induced G protein signalling

    Directory of Open Access Journals (Sweden)

    Hildebrand Dagmar

    2012-08-01

    Full Text Available Abstract Background Lipopolysaccharide (LPS-triggered Toll-like receptor (TLR 4-signalling belongs to the key innate defence mechanisms upon infection with Gram-negative bacteria and triggers the subsequent activation of adaptive immunity. There is an active crosstalk between TLR4-mediated and other signalling cascades to secure an effective immune response, but also to prevent excessive inflammation. Many pathogens induce signalling cascades via secreted factors that interfere with TLR signalling to modify and presumably escape the host response. In this context heterotrimeric G proteins and their coupled receptors have been recognized as major cellular targets. Toxigenic strains of Gram-negative Pasteurella multocida produce a toxin (PMT that constitutively activates the heterotrimeric G proteins Gαq, Gα13 and Gαi independently of G protein-coupled receptors through deamidation. PMT is known to induce signalling events involved in cell proliferation, cell survival and cytoskeleton rearrangement. Results Here we show that the activation of heterotrimeric G proteins through PMT suppresses LPS-stimulated IL-12p40 production and eventually impairs the T cell-activating ability of LPS-treated monocytes. This inhibition of TLR4-induced IL-12p40 expression is mediated by Gαi-triggered signalling as well as by Gβγ-dependent activation of PI3kinase and JNK. Taken together we propose the following model: LPS stimulates TLR4-mediated activation of the NFĸB-pathway and thereby the production of TNF-α, IL-6 and IL-12p40. PMT inhibits the production of IL-12p40 by Gαi-mediated inhibition of adenylate cyclase and cAMP accumulation and by Gβγ-mediated activation of PI3kinase and JNK activation. Conclusions On the basis of the experiments with PMT this study gives an example of a pathogen-induced interaction between G protein-mediated and TLR4-triggered signalling and illustrates how a bacterial toxin is able to interfere with the host’s immune

  16. Cannabinoid receptor-interacting protein 1a modulates CB1 receptor signaling and regulation.

    Science.gov (United States)

    Smith, Tricia H; Blume, Lawrence C; Straiker, Alex; Cox, Jordan O; David, Bethany G; McVoy, Julie R Secor; Sayers, Katherine W; Poklis, Justin L; Abdullah, Rehab A; Egertová, Michaela; Chen, Ching-Kang; Mackie, Ken; Elphick, Maurice R; Howlett, Allyn C; Selley, Dana E

    2015-04-01

    Cannabinoid CB1 receptors (CB1Rs) mediate the presynaptic effects of endocannabinoids in the central nervous system (CNS) and most behavioral effects of exogenous cannabinoids. Cannabinoid receptor-interacting protein 1a (CRIP1a) binds to the CB1R C-terminus and can attenuate constitutive CB1R-mediated inhibition of Ca(2+) channel activity. We now demonstrate cellular colocalization of CRIP1a at neuronal elements in the CNS and show that CRIP1a inhibits both constitutive and agonist-stimulated CB1R-mediated guanine nucleotide-binding regulatory protein (G-protein) activity. Stable overexpression of CRIP1a in human embryonic kidney (HEK)-293 cells stably expressing CB1Rs (CB1-HEK), or in N18TG2 cells endogenously expressing CB1Rs, decreased CB1R-mediated G-protein activation (measured by agonist-stimulated [(35)S]GTPγS (guanylyl-5'-[O-thio]-triphosphate) binding) in both cell lines and attenuated inverse agonism by rimonabant in CB1-HEK cells. Conversely, small-interfering RNA-mediated knockdown of CRIP1a in N18TG2 cells enhanced CB1R-mediated G-protein activation. These effects were not attributable to differences in CB1R expression or endocannabinoid tone because CB1R levels did not differ between cell lines varying in CRIP1a expression, and endocannabinoid levels were undetectable (CB1-HEK) or unchanged (N18TG2) by CRIP1a overexpression. In CB1-HEK cells, 4-hour pretreatment with cannabinoid agonists downregulated CB1Rs and desensitized agonist-stimulated [(35)S]GTPγS binding. CRIP1a overexpression attenuated CB1R downregulation without altering CB1R desensitization. Finally, in cultured autaptic hippocampal neurons, CRIP1a overexpression attenuated both depolarization-induced suppression of excitation and inhibition of excitatory synaptic activity induced by exogenous application of cannabinoid but not by adenosine A1 agonists. These results confirm that CRIP1a inhibits constitutive CB1R activity and demonstrate that CRIP1a can also inhibit agonist

  17. Abnormalities in osteoclastogenesis and decreased tumorigenesis in mice deficient for ovarian cancer G protein-coupled receptor 1.

    Directory of Open Access Journals (Sweden)

    Hui Li

    2009-05-01

    Full Text Available Ovarian cancer G protein-coupled receptor 1 (OGR1 has been shown to be a proton sensing receptor in vitro. We have shown that OGR1 functions as a tumor metastasis suppressor gene when it is over-expressed in human prostate cancer cells in vivo. To examine the physiological functions of OGR1, we generated conditional OGR1 deficient mice by homologous recombination. OGR1 deficient mice were viable and upon gross-inspection appeared normal. Consistent with in vitro studies showing that OGR1 is involved in osteoclastogenesis, reduced osteoclasts were detected in OGR1 deficient mice. A pH-dependent osteoclasts survival effect was also observed. However, overall abnormality in the bones of these animals was not observed. In addition, melanoma cell tumorigenesis was significantly inhibited in OGR1 deficient mice. OGR1 deficient mice in the mixed background produced significantly less peritoneal macrophages when stimulated with thioglycolate. These macrophages also showed altered extracellular signal-regulated kinases (ERK activation and nitric oxide (NO production in response to lipopolysaccharide. OGR1-dependent pH responses assessed by cAMP production and cell survival in macrophages or brown fat cells were not observed, presumably due to the presence of other proton sensing receptors in these cells. Our results indicate that OGR1's role in osteoclastogenesis is not strong enough to affect overall bone development and its role in tumorigenesis warrants further investigation. The mice generated can be potentially used for several disease models, including cancers or osteoclast-related diseases.

  18. Bile Acids Trigger GLP-1 Release Predominantly by Accessing Basolaterally Located G Protein-Coupled Bile Acid Receptors.

    Science.gov (United States)

    Brighton, Cheryl A; Rievaj, Juraj; Kuhre, Rune E; Glass, Leslie L; Schoonjans, Kristina; Holst, Jens J; Gribble, Fiona M; Reimann, Frank

    2015-11-01

    Bile acids are well-recognized stimuli of glucagon-like peptide-1 (GLP-1) secretion. This action has been attributed to activation of the G protein-coupled bile acid receptor GPBAR1 (TGR5), although other potential bile acid sensors include the nuclear farnesoid receptor and the apical sodium-coupled bile acid transporter ASBT. The aim of this study was to identify pathways important for GLP-1 release and to determine whether bile acids target their receptors on GLP-1-secreting L-cells from the apical or basolateral compartment. Using transgenic mice expressing fluorescent sensors specifically in L-cells, we observed that taurodeoxycholate (TDCA) and taurolithocholate (TLCA) increased intracellular cAMP and Ca(2+). In primary intestinal cultures, TDCA was a more potent GLP-1 secretagogue than taurocholate (TCA) and TLCA, correlating with a stronger Ca(2+) response to TDCA. Using small-volume Ussing chambers optimized for measuring GLP-1 secretion, we found that both a GPBAR1 agonist and TDCA stimulated GLP-1 release better when applied from the basolateral than from the luminal direction and that luminal TDCA was ineffective when intestinal tissue was pretreated with an ASBT inhibitor. ASBT inhibition had no significant effect in nonpolarized primary cultures. Studies in the perfused rat gut confirmed that vascularly administered TDCA was more effective than luminal TDCA. Intestinal primary cultures and Ussing chamber-mounted tissues from GPBAR1-knockout mice did not secrete GLP-1 in response to either TLCA or TDCA. We conclude that the action of bile acids on GLP-1 secretion is predominantly mediated by GPBAR1 located on the basolateral L-cell membrane, suggesting that stimulation of gut hormone secretion may include postabsorptive mechanisms.

  19. Ligand binding to G protein-coupled receptors in tethered cell membranes

    DEFF Research Database (Denmark)

    Martinez, Karen L.; Meyer, Bruno H.; Hovius, Ruud

    2003-01-01

    for the surface immobilization of membrane proteins was developed using the prototypic seven transmembrane neurokinin-1 receptor. The receptor was expressed as a biotinylated protein in mammalian cells. Membranes from cell homogenates were selectively immobilized on glass surfaces covered with streptavidin. TIRF...... measurements showed that a fluorescent agonist binds to the receptor on the sensor surface with similar affinity as to the receptor in live cells. This approach offers the possibility to investigate minute amounts of membrane protein in an active form and in its native environment without purification....

  20. G-protein-coupled receptors and islet function-implications for treatment of type 2 diabetes.

    Science.gov (United States)

    Winzell, Maria Sörhede; Ahrén, Bo

    2007-12-01

    Islet function is regulated by a number of different signals. A main signal is generated by glucose, which stimulates insulin secretion and inhibits glucagon secretion. The glucose effects are modulated by many factors, including hormones, neurotransmitters and nutrients. Several of these factors signal through guanine nucleotide-binding protein (G protein)-coupled receptors (GPCR). Examples of islet GPCR are GPR40 and GPR119, which are GPCR with fatty acids as ligands, the receptors for the incretin hormones glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP), the receptors for the islet hormones glucagon and somatostatin, the receptors for the classical neurotransmittors acetylcholine (ACh; M(3) muscarinic receptors) and noradrenaline (beta(2)- and alpha(2)-adrenoceptors) and for the neuropeptides pituitary adenylate cyclase-activating polypeptide (PACAP) and vasoactive intestinal polypeptide (VIP; PAC(1) and VPAC(2) receptors), cholecystokinin (CCK(A) receptors) and neuropeptide Y (NPY Y1 receptors). Other islet GPCR are the cannabinoid receptor (CB(1) receptors), the vasopressin receptors (V1(B) receptors) and the purinergic receptors (P(2Y) receptors). The islet GPCR couple mainly to adenylate cyclase and to phospholipase C (PLC). Since important pharmacological strategies for treatment of type 2 diabetes are stimulation of insulin secretion and inhibition of glucagon secretion, islet GPCR are potential drug targets. This review summarizes knowledge on islet GPCR.

  1. Protein receptor-independent plasma membrane remodeling by HAMLET: a tumoricidal protein-lipid complex.

    Science.gov (United States)

    Nadeem, Aftab; Sanborn, Jeremy; Gettel, Douglas L; James, Ho C S; Rydström, Anna; Ngassam, Viviane N; Klausen, Thomas Kjær; Pedersen, Stine Falsig; Lam, Matti; Parikh, Atul N; Svanborg, Catharina

    2015-11-12

    A central tenet of signal transduction in eukaryotic cells is that extra-cellular ligands activate specific cell surface receptors, which orchestrate downstream responses. This ''protein-centric" view is increasingly challenged by evidence for the involvement of specialized membrane domains in signal transduction. Here, we propose that membrane perturbation may serve as an alternative mechanism to activate a conserved cell-death program in cancer cells. This view emerges from the extraordinary manner in which HAMLET (Human Alpha-lactalbumin Made LEthal to Tumor cells) kills a wide range of tumor cells in vitro and demonstrates therapeutic efficacy and selectivity in cancer models and clinical studies. We identify a ''receptor independent" transformation of vesicular motifs in model membranes, which is paralleled by gross remodeling of tumor cell membranes. Furthermore, we find that HAMLET accumulates within these de novo membrane conformations and define membrane blebs as cellular compartments for direct interactions of HAMLET with essential target proteins such as the Ras family of GTPases. Finally, we demonstrate lower sensitivity of healthy cell membranes to HAMLET challenge. These features suggest that HAMLET-induced curvature-dependent membrane conformations serve as surrogate receptors for initiating signal transduction cascades, ultimately leading to cell death.

  2. Severe malaria is associated with parasite binding to endothelial protein C receptor

    DEFF Research Database (Denmark)

    Turner, Louise; Lavstsen, Thomas; Berger, Sanne S

    2013-01-01

    . falciparum erythrocyte membrane protein 1 (PfEMP1) family and receptors on the endothelial lining. Severe childhood malaria is associated with expression of specific PfEMP1 subtypes containing domain cassettes (DCs) 8 and 13 (ref. 3), but the endothelial receptor for parasites expressing these proteins...

  3. 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.

  4. In silico interaction analysis of cannabinoid receptor interacting protein 1b (CRIP1b) - CB1 cannabinoid receptor.

    Science.gov (United States)

    Singh, Pratishtha; Ganjiwale, Anjali; Howlett, Allyn C; Cowsik, Sudha M

    2017-10-01

    Cannabinoid Receptor Interacting Protein isoform 1b (CRIP1b) is known to interact with the CB1 receptor. Alternative splicing of the CNRIP1 gene produces CRIP1a and CRIP1b with a difference in the third exon only. Exons 1 and 2 encode for a functional domain in both proteins. CRIP1a is involved in regulating CB1 receptor internalization, but the function of CRIP1b is not very well characterized. Since there are significant identities in functional domains of these proteins, CRIP1b is a potential target for drug discovery. We report here predicted structure of CRIP1b followed by its interaction analysis with CB1 receptor by in-silico methods A number of complementary computational techniques, including, homology modeling, ab-initio and protein threading, were applied to generate three-dimensional molecular models for CRIP1b. The computed model of CRIP1b was refined, followed by docking with C terminus of CB1 receptor to generate a model for the CRIP1b- CB1 receptor interaction. The structure of CRIP1b obtained by homology modelling using RHO_GDI-2 as template is a sandwich fold structure having beta sheets connected by loops, similar to predicted CRIP1a structure. The best scoring refined model of CRIP1b in complex with the CB1 receptor C terminus peptide showed favourable polar interactions. The overall binding pocket of CRIP1b was found to be overlapping to that of CRIP1a. The Arg82 and Cys126 of CRIP1b are involved in the majority of hydrogen bond interactions with the CB1 receptor and are possible key residues required for interactions between the CB1 receptor and CRIP1b. Copyright © 2017 Elsevier Inc. All rights reserved.

  5. Impact of protein binding on receptor occupancy: A two-compartment model

    OpenAIRE

    Peletier, Lambertus A.; Benson, Neil; Van Der Graaf, Piet H.

    2010-01-01

    Abstract In this paper we analyse the impact of protein- and lipid- and receptor-binding on receptor occupancy in a two-compartment system, with proteins in both compartments and lipids and receptors in the peripheral compartment only. We do this for two manners of drug administration: a bolus administration and a constant rate infusion, both into the central compartment. We derive explicit approximations for the time-curves of the different compounds valid for a wide range of real...

  6. Effect of neoadjuvant chemotherapy on low-density lipoprotein (LDL) receptor and LDL receptor-related protein 1 (LRP-1) receptor in locally advanced breast cancer

    Energy Technology Data Exchange (ETDEWEB)

    Pires, L.A. [Laboratório de Metabolismo de Lípides, Instituto do Coração, Faculdade de Medicina, Hospital das Clínicas, Universidade de São Paulo, São Paulo, SP (Brazil); Departamento de Ginecologia, Faculdade de Medicina, Hospital das Clínicas, Universidade de São Paulo, São Paulo, SP (Brazil); Hegg, R. [Departamento de Ginecologia, Faculdade de Medicina, Hospital das Clínicas, Universidade de São Paulo, São Paulo, SP (Brazil); Freitas, F.R.; Tavares, E.R.; Almeida, C.P. [Laboratório de Metabolismo de Lípides, Instituto do Coração, Faculdade de Medicina, Hospital das Clínicas, Universidade de São Paulo, São Paulo, SP (Brazil); Baracat, E.C. [Departamento de Ginecologia, Faculdade de Medicina, Hospital das Clínicas, Universidade de São Paulo, São Paulo, SP (Brazil); Maranhão, R.C. [Laboratório de Metabolismo de Lípides, Instituto do Coração, Faculdade de Medicina, Hospital das Clínicas, Universidade de São Paulo, São Paulo, SP (Brazil); Faculdade de Ciências Farmacêuticas, Universidade de São Paulo, São Paulo, SP (Brazil)

    2012-05-04

    Low-density lipoprotein (LDL) receptors are overexpressed in most neoplastic cell lines and provide a mechanism for the internalization and concentration of drug-laden nanoemulsions that bind to these receptors. The aim of the present study was to determine whether the administration of standard chemotherapeutic schemes can alter the expression of LDL and LDL receptor-related protein 1 (LRP-1) receptors in breast carcinoma. Fragments of tumoral and normal breast tissue from 16 consecutive volunteer women with breast cancer in stage II or III were obtained from biopsies before the beginning of neoadjuvant chemotherapy and after chemotherapy, from fragments excised during mastectomy. Tissues were analyzed by immunohistochemistry for both receptors. Because complete response to treatment was achieved in 4 patients, only the tumors from 12 were analyzed. Before chemotherapy, there was overexpression of LDL receptor in the tumoral tissue compared to normal breast tissue in 8 of these patients. LRP-1 receptor overexpression was observed in tumors of 4 patients. After chemotherapy, expression of both receptors decreased in the tumors of 6 patients, increased in 4 and was unchanged in 2. Nonetheless, even when chemotherapy reduced receptors expression, the expression was still above normal. The fact that chemotherapy does not impair LDL receptors expression supports the use of drug carrier systems that target neoplastic cells by the LDL receptor endocytic pathway in patients on conventional chemotherapy.

  7. Effect of neoadjuvant chemotherapy on low-density lipoprotein (LDL receptor and LDL receptor-related protein 1 (LRP-1 receptor in locally advanced breast cancer

    Directory of Open Access Journals (Sweden)

    L.A. Pires

    2012-06-01

    Full Text Available Low-density lipoprotein (LDL receptors are overexpressed in most neoplastic cell lines and provide a mechanism for the internalization and concentration of drug-laden nanoemulsions that bind to these receptors. The aim of the present study was to determine whether the administration of standard chemotherapeutic schemes can alter the expression of LDL and LDL receptor-related protein 1 (LRP-1 receptors in breast carcinoma. Fragments of tumoral and normal breast tissue from 16 consecutive volunteer women with breast cancer in stage II or III were obtained from biopsies before the beginning of neoadjuvant chemotherapy and after chemotherapy, from fragments excised during mastectomy. Tissues were analyzed by immunohistochemistry for both receptors. Because complete response to treatment was achieved in 4 patients, only the tumors from 12 were analyzed. Before chemotherapy, there was overexpression of LDL receptor in the tumoral tissue compared to normal breast tissue in 8 of these patients. LRP-1 receptor overexpression was observed in tumors of 4 patients. After chemotherapy, expression of both receptors decreased in the tumors of 6 patients, increased in 4 and was unchanged in 2. Nonetheless, even when chemotherapy reduced receptors expression, the expression was still above normal. The fact that chemotherapy does not impair LDL receptors expression supports the use of drug carrier systems that target neoplastic cells by the LDL receptor endocytic pathway in patients on conventional chemotherapy.

  8. G-protein-coupled receptors for free fatty acids

    DEFF Research Database (Denmark)

    Milligan, Graeme; Ulven, Trond; Murdoch, Hannah

    2014-01-01

    that communicate cellular signals initiated by hormones and neurotransmitters. Recently, based on tissue expression patterns of these receptors and the concept that they may elicit the production of a range of appetite- and hunger-regulating peptides, such nutrient sensing GPCRs are attracting considerable...... of these receptors. However, ongoing clinical trials of agonists of free fatty acid receptor 1 suggest that this receptor and other receptors for free fatty acids may provide a successful strategy for controlling hyperglycaemia and providing novel approaches to treat diabetes. Receptors responsive to free fatty acid...

  9. Purification of family B G protein-coupled receptors using nanodiscs: Application to human glucagon-like peptide-1 receptor.

    Directory of Open Access Journals (Sweden)

    Yingying Cai

    Full Text Available Family B G protein-coupled receptors (GPCRs play vital roles in hormone-regulated homeostasis. They are drug targets for metabolic diseases, including type 2 diabetes and osteoporosis. Despite their importance, the signaling mechanisms for family B GPCRs at the molecular level remain largely unexplored due to the challenges in purification of functional receptors in sufficient amount for biophysical characterization. Here, we purified the family B GPCR human glucagon-like peptide-1 (GLP-1 receptor (GLP1R, whose agonists, e.g. exendin-4, are used for the treatment of type 2 diabetes mellitus. The receptor was expressed in HEK293S GnTl- cells using our recently developed protocol. The protocol incorporates the receptor into the native-like lipid environment of reconstituted high density lipoprotein (rHDL particles, also known as nanodiscs, immediately after the membrane solubilization step followed by chromatographic purification, minimizing detergent contact with the target receptor to reduce denaturation and prolonging stabilization of receptor in lipid bilayers without extra steps of reconstitution. This method yielded purified GLP1R in nanodiscs that could bind to GLP-1 and exendin-4 and activate Gs protein. This nanodisc purification method can potentially be a general strategy to routinely obtain purified family B GPCRs in the 10s of microgram amounts useful for spectroscopic analysis of receptor functions and activation mechanisms.

  10. 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 ...... kinase activation, gene induction and cell growth. From these data, we conclude that Shc represents a crucial point of convergence between signaling pathways activated by receptor tyrosine kinases and G protein-coupled receptors.......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...... recently been elucidated biochemically and genetically. The present study was undertaken to determine whether common signaling components are used by these two distinct classes of receptors. Here we report that the adaptor protein Shc, is phosphorylated on tyrosine residues following stimulation...

  11. Identification of a preassembled TRH receptor-G(q/11) protein complex in HEK293 cells.

    Science.gov (United States)

    Drastichova, Zdenka; Novotny, Jiri

    2012-01-01

    Protein-protein interactions define specificity in signal transduction and these interactions are central to transmembrane signaling by G-protein-coupled receptors (GPCRs). It is not quite clear, however, whether GPCRs and the regulatory trimeric G-proteins behave as freely and independently diffusible molecules in the plasma membrane or whether they form some preassociated complexes. Here we used clear-native polyacrylamide gel electrophoresis (CN-PAGE) to investigate the presumed coupling between thyrotropin-releasing hormone (TRH) receptor and its cognate G(q/11) protein in HEK293 cells expressing high levels of these proteins. Under different solubilization conditions, the TRH receptor (TRH-R) was identified to form a putative pentameric complex composed of TRH-R homodimer and G(q/11) protein. The presumed association of TRH-R with G(q/11)α or Gβ proteins in plasma membranes was verified by RNAi experiments. After 10- or 30-min hormone treatment, TRH-R signaling complexes gradually dissociated with a concomitant release of receptor homodimers. These observations support the model in which GPCRs can be coupled to trimeric G-proteins in preassembled signaling complexes, which might be dynamically regulated upon receptor activation. The precoupling of receptors with their cognate G-proteins can contribute to faster G-protein activation and subsequent signal transfer into the cell interior.

  12. G protein-coupled receptors not currently in the spotlight: free fatty acid receptor 2 and GPR35.

    Science.gov (United States)

    Milligan, Graeme

    2017-09-21

    It is widely appreciated that G protein-coupled receptors have been the most successfully exploited class of targets for the development of small molecule medicines. Despite this, to date, less than 15% of the non-olfactory G protein-coupled receptors in the human genome are the targets of a clinically used medicine. In many cases, this is likely to reflect a lack of understanding of the basic underpinning biology of many G protein-coupled receptors that are not currently in the spotlight, as well as a paucity of pharmacological tool compounds and appropriate animal models to test in vivo function of such G protein-coupled receptors in both normal physiology and in the context of disease. 'Open Innovation' arrangements, in which pharmaceutical companies and public-private partnerships provide wider access to tool compounds identified from ligand screening programmes, alongside enhanced medicinal chemistry support to convert such screening 'hits' into useful 'tool' compounds will provide important routes to improved understanding. However, in parallel, novel approaches to define and fully appreciate the selectivity and mode of action of such tool compounds, as well as better understanding of potential species orthologue variability in the pharmacology and/or signalling profile of a wide range of currently poorly understood and understudied G protein-coupled receptors, will be vital to fully exploit the therapeutic potential of this large target class. I consider these themes using as exemplars two G protein-coupled receptors, free fatty acid receptor 2 and GPR35. © 2017 The Authors. British Journal of Pharmacology published by John Wiley & Sons Ltd on behalf of British Pharmacological Society.

  13. The antimicrobial peptide derived from insulin-like growth factor-binding protein 5, AMP-IBP5, regulates keratinocyte functions through Mas-related gene X receptors.

    Science.gov (United States)

    Chieosilapatham, Panjit; Niyonsaba, François; Kiatsurayanon, Chanisa; Okumura, Ko; Ikeda, Shigaku; Ogawa, Hideoki

    2017-10-01

    In addition to their microbicidal properties, host defense peptides (HDPs) display various immunomodulatory functions, including keratinocyte production of cytokines/chemokines, proliferation, migration and wound healing. Recently, a novel HDP named AMP-IBP5 (antimicrobial peptide derived from insulin-like growth factor-binding protein 5) was shown to exhibit antimicrobial activity against numerous pathogens, even at concentrations comparable to those of human β-defensins and LL-37. However, the immunomodulatory role of AMP-IBP5 in cutaneous tissue remains unknown. To investigate whether AMP-IBP5 triggers keratinocyte activation and to clarify its mechanism. Production of cytokines/chemokines and growth factors was determined by appropriate ELISA kits. Cell migration was assessed by in vitro wound closure assay, whereas cell proliferation was analyzed using BrdU incorporation assay complimented with XTT assay. MAPK and NF-κB activation was determined by Western blotting. Intracellular cAMP levels were assessed using cAMP enzyme immunoassay kit. Among various cytokines/chemokines and growth factors tested, AMP-IBP5 selectively increased the production of IL-8 and VEGF. Moreover, AMP-IBP5 markedly enhanced keratinocyte migration and proliferation. AMP-IBP5-induced keratinocyte activation was mediated by Mrg X1-X4 receptors with MAPK and NF-κB pathways working downstream, as evidenced by the inhibitory effects of MrgX1-X4 siRNAs and ERK-, JNK-, p38- and NF-κB-specific inhibitors. We confirmed that AMP-IBP5 indeed induced MAPK and NF-κB activation. Furthermore, AMP-IBP5-induced VEGF but not IL-8 production correlated with an increase in intracellular cAMP. Our findings suggest that in addition to its antimicrobial function, AMP-IBP5 might contribute to wound healing process through activation of keratinocytes. Copyright © 2017 Japanese Society for Investigative Dermatology. Published by Elsevier B.V. All rights reserved.

  14. Antibody-protein A conjugated quantum dots for multiplexed imaging of surface receptors in living cells.

    Science.gov (United States)

    Jin, Takashi; Tiwari, Dhermendra K; Tanaka, Shin-Ichi; Inouye, Yasushi; Yoshizawa, Keiko; Watanabe, Tomonobu M

    2010-11-01

    To use quantum dots (QDs) as fluorescent probes for receptor imaging, QD surface should be modified with biomolecules such as antibodies, peptides, carbohydrates, and small-molecule ligands for receptors. Among these QDs, antibody conjugated QDs are the most promising fluorescent probes. There are many kinds of coupling reactions that can be used for preparing antibody conjugated QDs. Most of the antibody coupling reactions, however, are non-selective and time-consuming. In this paper, we report a facile method for preparing antibody conjugated QDs for surface receptor imaging. We used ProteinA as an adaptor protein for binding of antibody to QDs. By using ProteinA conjugated QDs, various types of antibodies are easily attached to the surface of the QDs via non-covalent binding between the F(c) (fragment crystallization) region of antibody and ProteinA. To show the utility of ProteinA conjugated QDs, HER2 (anti-human epidermal growth factor receptor 2) in KPL-4 human breast cancer cells were stained by using anti-HER2 antibody conjugated ProteinA-QDs. In addition, multiplexed imaging of HER2 and CXCR4 (chemokine receptor) in the KPL-4 cells was performed. The result showed that CXCR4 receptors coexist with HER2 receptors in the membrane surface of KPL-4 cells. ProteinA mediated antibody conjugation to QDs is very useful to prepare fluorescent probes for multiplexed imaging of surface receptors in living cells.

  15. Identifying neuropeptide and protein hormone receptors in Drosophila melanogaster by exploiting genomic data

    DEFF Research Database (Denmark)

    Hauser, Frank; Williamson, Michael; Cazzamali, Giuseppe

    2006-01-01

    Most neuropeptide and protein hormone receptors belong to the large superfamily of G-protein-coupled receptors (GPCRs). These cell membrane proteins steer many important processes such as development, reproduction, homeostasis and behaviour when activated by their corresponding ligands. The first...... insect genome, that of the fruitfly Drosophila melanogaster, was sequenced in 2000, and about 200 GPCRs have been annnotated in this model insect. About 50 of these receptors were predicted to have neuropeptides or protein hormones as their ligands. Since 2000, the cDNAs of most of these candidate...

  16. Crystal structure of the β2 adrenergic receptor-Gs protein complex

    DEFF Research Database (Denmark)

    Rasmussen, Søren Gøgsig Faarup; DeVree, Brian T; Zou, Yaozhong

    2011-01-01

    G protein-coupled receptors (GPCRs) are responsible for the majority of cellular responses to hormones and neurotransmitters as well as the senses of sight, olfaction and taste. The paradigm of GPCR signalling is the activation of a heterotrimeric GTP binding protein (G protein) by an agonist......-occupied receptor. The β(2) adrenergic receptor (β(2)AR) activation of Gs, the stimulatory G protein for adenylyl cyclase, has long been a model system for GPCR signalling. Here we present the crystal structure of the active state ternary complex composed of agonist-occupied monomeric β(2)AR and nucleotide-free Gs...... by a GPCR....

  17. 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

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

    DEFF Research Database (Denmark)

    Steen, Anne; Larsen, Olav; Thiele, Stefanie

    2014-01-01

    Biased signaling or functional selectivity occurs when a 7TM-receptor preferentially activates one of several available pathways. It can be divided into three distinct forms: ligand bias, receptor bias, and tissue or cell bias, where it is mediated by different ligands (on the same receptor), dif...

  19. Cannabinoid CB1 receptor-interacting proteins: novel targets for central nervous system drug discovery?

    Science.gov (United States)

    Smith, Tricia H; Sim-Selley, Laura J; Selley, Dana E

    2010-01-01

    The main pharmacological effects of marijuana, as well as synthetic and endogenous cannabinoids, are mediated through G-protein-coupled receptors (GPCRs), including CB1 and CB2 receptors. The CB1 receptor is the major cannabinoid receptor in the central nervous system and has gained increasing interest as a target for drug discovery for treatment of nausea, cachexia, obesity, pain, spasticity, neurodegenerative diseases and mood and substance abuse disorders. Evidence has accumulated to suggest that CB1 receptors, like other GPCRs, interact with and are regulated by several other proteins beyond the established role of heterotrimeric G-proteins. These proteins, which include the GPCR kinases, β-arrestins, GPCR-associated sorting proteins, factor associated with neutral sphingomyelinase, other GPCRs (heterodimerization) and the novel cannabinoid receptor-interacting proteins: CRIP1a/b, are thought to play important roles in the regulation of intracellular trafficking, desensitization, down-regulation, signal transduction and constitutive activity of CB1 receptors. This review examines CB1 receptor-interacting proteins, including heterotrimeric G-proteins, but with particular emphasis on non-G-protein entities, that might comprise the CB1 receptosomal complex. The evidence for direct interaction with CB1 receptors and potential functional roles of these interacting proteins is discussed, as are future directions and challenges in this field with an emphasis on the possibility of eventually targeting these proteins for drug discovery. This article is part of a themed issue on Cannabinoids. To view the editorial for this themed issue visit http://dx.doi.org/10.1111/j.1476-5381.2010.00831.x PMID:20590557

  20. BIOLUMINISCENCE RESONANCE ENERGY TRANSFER (BRET) METHODS TO STUDY G PROTEIN-COUPLED RECEPTOR - RECEPTOR TYROSINE KINASE HETERORECEPTOR COMPLEXES

    OpenAIRE

    Borroto-Escuela, Dasiel O.; Flajolet, Marc; Agnati, Luigi F.; Greengard, Paul; Fuxe, Kjell

    2013-01-01

    A large body of evidence indicates that G protein-coupled receptors (GPCRs) and Receptor tyrosine kinases (RTKs) can form heteroreceptor complexes. In these complexes, the signalling from each interacting protomer is modulated to produce an integrated and therefore novel response upon agonist(s) activation. In the GPCR-RTK heteroreceptor complexes, GPCRs can activate RTK in the absence of added growth factor through the use of RTK signalling molecules. This integrative phenomenon is reciproca...

  1. An RBCC protein implicated in maintenance of steady-state neuregulin receptor levels.

    Science.gov (United States)

    Diamonti, A John; Guy, Pamela M; Ivanof, Caryn; Wong, Karen; Sweeney, Colleen; Carraway, Kermit L

    2002-03-05

    Despite numerous recent advances in our understanding of the molecular mechanisms underlying receptor tyrosine kinase down-regulation and degradation in response to growth factor binding, relatively little is known about ligand-independent receptor tyrosine kinase degradation mechanisms. In a screen for proteins that might regulate the trafficking or localization of the ErbB3 receptor, we have identified a tripartite or RBCC (RING, B-box, coiled-coil) protein that interacts with the cytoplasmic tail of the receptor in an activation-independent manner. We have named this protein Nrdp1 for neuregulin receptor degradation protein-1. Northern blotting reveals ubiquitous distribution of Nrdp1 in human adult tissues, but message is particularly prominent in heart, brain, and skeletal muscle. Nrdp1 interacts specifically with the neuregulin receptors ErbB3 and ErbB4 and not with epidermal growth factor receptor or ErbB2. When coexpressed in COS7 cells, Nrdp1 mediates the redistribution of ErbB3 from the cell surface to intracellular compartments and induces the suppression of ErbB3 and ErbB4 receptor levels but not epidermal growth factor receptor or ErbB2 levels. A putative dominant-negative form of Nrdp1 potentiates neuregulin-stimulated Erk1/2 activity in transfected MCF7 breast tumor cells. Our observations suggest that Nrdp1 may act to regulate steady-state cell surface neuregulin receptor levels, thereby influencing the efficiency of neuregulin signaling.

  2. Dimers of G-Protein Coupled Receptors as Versatile Storage and Response Units

    Directory of Open Access Journals (Sweden)

    Michael S. Parker

    2014-03-01

    Full Text Available The status and use of transmembrane, extracellular and intracellular domains in oligomerization of heptahelical G-protein coupled receptors (GPCRs are reviewed and for transmembrane assemblies also supplemented by new experimental evidence. The transmembrane-linked GPCR oligomers typically have as the minimal unit an asymmetric ~180 kDa pentamer consisting of receptor homodimer or heterodimer and a G-protein αβγ subunit heterotrimer. With neuropeptide Y (NPY receptors, this assembly is converted to ~90 kDa receptor monomer-Gα complex by receptor and Gα agonists, and dimers/heteropentamers are depleted by neutralization of Gαi subunits by pertussis toxin. Employing gradient centrifugation, quantification and other characterization of GPCR dimers at the level of physically isolated and identified heteropentamers is feasible with labeled agonists that do not dissociate upon solubilization. This is demonstrated with three neuropeptide Y (NPY receptors and could apply to many receptors that use large peptidic agonists.

  3. Lighting up G protein-coupled purinergic receptors with engineered fluorescent ligands

    Science.gov (United States)

    Ciruela, Francisco; Fernández-Dueñas, Víctor; Jacobson, Kenneth A.

    2015-01-01

    The use of G protein-coupled receptors fluorescent ligands is undergoing continuous expansion. In line with this, fluorescent agonists and antagonists of high affinity for G protein-coupled adenosine and P2Y receptors have been shown to be useful pharmacological probe compounds. Fluorescent ligands for A1R, A2AR, and A3R (adenosine receptors) and P2Y2R, P2Y4R, P2Y6R, and P2Y14R (nucleotide receptors) have been reported. Such ligands have been successfully applied to drug discovery and to GPCR characterization by flow cytometry, fluorescence correlation spectroscopy, fluorescence microscopy, fluorescence polarization, fluorescence resonance energy transfer and scanning confocal microscopy. Here we summarize recently reported and readily available representative fluorescent ligands of purinergic receptors. In addition, we pay special attention on the use of this family of fluorescent ligands revealing two main aspects of purinergic receptor biology, namely ligand binding and receptor oligomerization. PMID:25890205

  4. BDNF downregulates 5-HT(2A) receptor protein levels in hippocampal cultures

    DEFF Research Database (Denmark)

    Trajkovska, V; Santini, M A; Marcussen, Anders Bue

    2009-01-01

    Both brain-derived neurotrophic factor (BDNF) and the serotonin receptor 2A (5-HT(2A)) have been related to depression pathology. Specific 5-HT(2A) receptor changes seen in BDNF conditional mutant mice suggest that BDNF regulates the 5-HT(2A) receptor level. Here we show a direct effect of BDNF...... on 5-HT(2A) receptor protein levels in primary hippocampal neuronal and mature hippocampal organotypic cultures exposed to different BDNF concentrations for either 1, 3, 5 or 7 days. In vivo effects of BDNF on hippocampal 5-HT(2A) receptor levels were further corroborated in (BDNF +/-) mice...... with reduced BDNF levels. In primary neuronal cultures, 7 days exposure to 25 and 50ng/mL BDNF resulted in downregulation of 5-HT(2A), but not of 5-HT(1A), receptor protein levels. The BDNF-associated downregulation of 5-HT(2A) receptor levels was also observed in mature hippocampal organotypic cultures...

  5. The camp model for entrepreneurship teaching

    DEFF Research Database (Denmark)

    Bager, Torben

    2011-01-01

    Artiklen omhandler brugen af camps i entrepreneurship undervising - illustreret med danske camp eksempler Udgivelsesdato: online 31.03.2010......Artiklen omhandler brugen af camps i entrepreneurship undervising - illustreret med danske camp eksempler Udgivelsesdato: online 31.03.2010...

  6. Human odontoblasts express transient receptor protein and acid-sensing ion channel mechanosensor proteins.

    Science.gov (United States)

    Solé-Magdalena, Antonio; Revuelta, Enrique G; Menénez-Díaz, Ivan; Calavia, Marta G; Cobo, Teresa; García-Suárez, Olivia; Pérez-Piñera, Pablo; De Carlos, Felix; Cobo, Juan; Vega, Jose A

    2011-05-01

    Diverse proteins of the denegerin/epithelial sodium channel (DEG/ENa(+) C) superfamily, in particular those belonging to the acid-sensing ion channel (ASIC) family, as well as some members of the transient receptor protein (TRP) channel, function as mechanosensors or may be required for mechanosensation in a diverse range of species and cell types. Therefore, we investigated the putative mechanosensitive function of human odontoblasts using immunohistochemistry to detect ENa(+) C subunits (α, β, and γ) and ASIC (1, 2, 3, and 4) proteins, as well as TRPV4, in these cells. Positive and specific immunoreactivity in the odontoblast soma and/or processes was detected for all proteins studied except α-ENa(+) C. The intensity of immunostaining was high for β-ENa(+) C and ASIC2, whereas it was low for ASIC1, ASIC3, γ-ENa(+) C, and TRPV4, being absent for α-ENa(+) C and ASIC4. These results suggest that human odontoblasts in situ express proteins related to mechanosensitive channels that probably participate in the mechanisms involved in teeth sensory transmission. Copyright © 2010 Wiley-Liss, Inc.

  7. The E92K melanocortin 1 receptor mutant induces cAMP production and arrestin recruitment but not ERK activity indicating biased constitutive signaling

    DEFF Research Database (Denmark)

    Benned-Jensen, Tau; Mokrosinski, Jacek; Rosenkilde, Mette M

    2011-01-01

    The melanocortin 1 receptor (MC1R) constitutes a key regulator of melanism. Consequently, many naturally-occurring MC1R mutations are associated with a change in color. An example is the Glu-to-Lys substitution found at position II:20/2.60 in the top of transmembrane helix II which has been ident...

  8. G-protein coupled receptor expression patterns delineate medulloblastoma subgroups

    Science.gov (United States)

    2013-01-01

    Background Medulloblastoma is the most common malignant brain tumor in children. Genetic profiling has identified four principle tumor subgroups; each subgroup is characterized by different initiating mutations, genetic and clinical profiles, and prognoses. The two most well-defined subgroups are caused by overactive signaling in the WNT and SHH mitogenic pathways; less is understood about Groups 3 and 4 medulloblastoma. Identification of tumor subgroup using molecular classification is set to become an important component of medulloblastoma diagnosis and staging, and will likely guide therapeutic options. However, thus far, few druggable targets have emerged. G-protein coupled receptors (GPCRs) possess characteristics that make them ideal targets for molecular imaging and therapeutics; drugs targeting GPCRs account for 30-40% of all current pharmaceuticals. While expression patterns of many proteins in human medulloblastoma subgroups have been discerned, the expression pattern of GPCRs in medulloblastoma has not been investigated. We hypothesized that analysis of GPCR expression would identify clear subsets of medulloblastoma and suggest distinct GPCRs that might serve as molecular targets for both imaging and therapy. Results Our study found that medulloblastoma tumors fall into distinct clusters based solely on GPCR expression patterns. Normal cerebellum clustered separately from the tumor samples. Further, two of the tumor clusters correspond with high fidelity to the WNT and SHH subgroups of medulloblastoma. Distinct over-expressed GPCRs emerge; for example, LGR5 and GPR64 are significantly and uniquely over-expressed in the WNT subgroup of tumors, while PTGER4 is over-expressed in the SHH subgroup. Uniquely under-expressed GPCRs were also observed. Our key findings were independently validated using a large international dataset. Conclusions Our results identify GPCRs with potential to act as imaging and therapeutic targets. Elucidating tumorigenic pathways

  9. Diferric transferrin regulates transferrin receptor 2 protein stability.

    Science.gov (United States)

    Johnson, Martha B; Enns, Caroline A

    2004-12-15

    Transferrin receptor 2 (TfR2) is a type 2 transmembrane protein expressed in hepatocytes that binds iron-bound transferrin (Tf). Mutations in TfR2 cause one form of hereditary hemochromatosis, a disease in which excessive absorption of dietary iron can lead to liver cirrhosis, diabetes, arthritis, and heart failure. The function of TfR2 in iron homeostasis is unknown. We have studied the regulation of TfR2 in HepG2 cells. Western blot analysis shows that TfR2 increases in a time- and dose-dependent manner after diferric Tf is added to the culture medium. In cells exposed to diferric Tf, the amount of TfR2 returns to control levels within 8 hours after the removal of diferric Tf from the medium. However, TfR2 does not increase when non-Tf-bound iron (FeNTA) or apo Tf is added to the medium. The response to diferric Tf appears to be hepatocyte specific. Real-time quantitative reverse transcription-polymerase chain reaction (qRT-PCR) analysis shows that TfR2 mRNA levels do not change in cells exposed to diferric Tf. Rather, the increase in TfR2 is attributed to an increase in the half-life of TfR2 protein in cells exposed to diferric Tf. Our results support a role for TfR2 in monitoring iron levels by sensing changes in the concentration of diferric Tf.

  10. G Protein-Coupled Receptors: Extranuclear Mediators for the Non-Genomic Actions of Steroids

    Directory of Open Access Journals (Sweden)

    Chen Wang

    2014-09-01

    Full Text Available Steroids hormones possess two distinct actions, a delayed genomic effect and a rapid non-genomic effect. Rapid steroid-triggered signaling is mediated by specific receptors localized most often to the plasma membrane. The nature of these receptors is of great interest and accumulated data suggest that G protein-coupled receptors (GPCRs are appealing candidates. Increasing evidence regarding the interaction between steroids and specific membrane proteins, as well as the involvement of G protein and corresponding downstream signaling, have led to identification of physiologically relevant GPCRs as steroid extranuclear receptors. Examples include G protein-coupled receptor 30 (GPR30 for estrogen, membrane progestin receptor for progesterone, G protein-coupled receptor family C group 6 member A (GPRC6A and zinc transporter member 9 (ZIP9 for androgen, and trace amine associated receptor 1 (TAAR1 for thyroid hormone. These receptor-mediated biological effects have been extended to reproductive development, cardiovascular function, neuroendocrinology and cancer pathophysiology. However, although great progress have been achieved, there are still important questions that need to be answered, including the identities of GPCRs responsible for the remaining steroids (e.g., glucocorticoid, the structural basis of steroids and GPCRs’ interaction and the integration of extranuclear and nuclear signaling to the final physiological function. Here, we reviewed the several significant developments in this field and highlighted a hypothesis that attempts to explain the general interaction between steroids and GPCRs.

  11. Definition of the G protein-coupled receptor transmembrane bundle binding pocket and calculation of receptor similarities for drug design

    DEFF Research Database (Denmark)

    Gloriam, David Erik Immanuel; Foord, Steven M; Blaney, Frank E

    2009-01-01

    Recent advances in structural biology for G-protein-coupled receptors (GPCRs) have provided new opportunities to improve the definition of the transmembrane binding pocket. Here a reference set of 44 residue positions accessible for ligand binding was defined through detailed analysis of all...... currently available crystal structures. This was used to characterize pharmacological relationships of Family A/Rhodopsin family GPCRs, minimizing evolutionary influence from parts of the receptor that do not generally affect ligand binding. The resultant dendogram tended to group receptors according...... the pharmacology/selectivity profile of ligands at Family A GPCRs. This has wide applicability to GPCR drug design problems across many disease areas....

  12. Evidence for a vasopressin receptor-GTP binding protein complex

    Energy Technology Data Exchange (ETDEWEB)

    Fitzgerald, T.J.; Uhing, R.J.; Exton, J.H.

    1986-05-01

    Plasma membranes from the livers of rats were able to hydrolyze the ..gamma..-phosphate from guanosine-5'-triphosphate (GTP). The rate of GTP hydrolysis could be decreased to 10% of its initial rate by the addition of adenosine-5'-triphosphate with a concomitant decrease in the K/sub m/ for GTP from approx. 10/sup -3/ M to 10/sup -6/ M. The low K/sub m/ GTPase activity was inhibited by the addition of nonhydrolyzable analogs of GTP. In addition, the GTPase activity was stimulated from 10 to 30% over basal by the addition of vasopressin. A dose dependency curve showed that the maximum stimulation was obtained with 10/sup -8/ M vasopressin. Identical results were obtained from plasma membranes that had been solubilized with 1% digitonin. When membranes that had been solubilized in the presence of (Phenylalanyl-3,4,5-/sup 3/H(N))vasopressin were subjected to sucrose gradient centrifugation, the majority of bound (/sup 3/H)vasopressin migrated with an approximate molecular weight of 300,000. Moreover, there was a GTPase activity that migrated with the bound (/sup 3/H)vasopressin. This peak of bound (/sup 3/H)vasopressin was decreased by 90% when the sucrose gradient centrifugation was run in the presence of 10/sup -5/ M guanosine-5'-O-(3-thiotriphosphate). These results support the conclusion that liver plasma membranes contain a GTP-binding protein that can complex with the vasopressin receptor.

  13. G-Protein Coupled Receptors Targeted by Analgesic Venom Peptides

    Directory of Open Access Journals (Sweden)

    James T. Daniel

    2017-11-01

    Full Text Available Chronic pain is a complex and debilitating condition associated with a large personal and socioeconomic burden. Current pharmacological approaches to treating chronic pain such as opioids, antidepressants and anticonvulsants exhibit limited efficacy in many patients and are associated with dose-limiting side effects that hinder their clinical use. Therefore, improved strategies for the pharmacological treatment of pathological pain are urgently needed. G-protein coupled receptors (GPCRs are ubiquitously expressed on the surface of cells and act to transduce extracellular signals and regulate physiological processes. In the context of pain, numerous and diverse families of GPCRs expressed in pain pathways regulate most aspects of physiological and pathological pain and are thus implicated as potential targets for therapy of chronic pain. In the search for novel compounds that produce analgesia via GPCR modulation, animal venoms offer an enormous and virtually untapped source of potent and selective peptide molecules. While many venom peptides target voltage-gated and ligand-gated ion channels to inhibit neuronal excitability and blunt synaptic transmission of pain signals, only a small proportion are known to interact with GPCRs. Of these, only a few have shown analgesic potential in vivo. Here we review the current state of knowledge regarding venom peptides that target GPCRs to produce analgesia, and their development as therapeutic compounds.

  14. GATA Factor-G-Protein-Coupled Receptor Circuit Suppresses Hematopoiesis

    Directory of Open Access Journals (Sweden)

    Xin Gao

    2016-03-01

    Full Text Available Hematopoietic stem cells (HSCs originate from hemogenic endothelium within the aorta-gonad-mesonephros (AGM region of the mammalian embryo. The relationship between genetic circuits controlling stem cell genesis and multi-potency is not understood. A Gata2 cis element (+9.5 enhances Gata2 expression in the AGM and induces the endothelial to HSC transition. We demonstrated that GATA-2 rescued hematopoiesis in +9.5−/− AGMs. As G-protein-coupled receptors (GPCRs are the most common targets for FDA-approved drugs, we analyzed the GPCR gene ensemble to identify GATA-2-regulated GPCRs. Of the 20 GATA-2-activated GPCR genes, four were GATA-1-activated, and only Gpr65 expression resembled Gata2. Contrasting with the paradigm in which GATA-2-activated genes promote hematopoietic stem and progenitor cell genesis/function, our mouse and zebrafish studies indicated that GPR65 suppressed hematopoiesis. GPR65 established repressive chromatin at the +9.5 site, restricted occupancy by the activator Scl/TAL1, and repressed Gata2 transcription. Thus, a Gata2 cis element creates a GATA-2-GPCR circuit that limits positive regulators that promote hematopoiesis.

  15. Triggering of the Newcastle Disease Virus Fusion Protein by a Chimeric Attachment Protein That Binds to Nipah Virus Receptors*

    Science.gov (United States)

    Mirza, Anne M.; Aguilar, Hector C.; Zhu, Qiyun; Mahon, Paul J.; Rota, Paul A.; Lee, Benhur; Iorio, Ronald M.

    2011-01-01

    The fusion (F) proteins of Newcastle disease virus (NDV) and Nipah virus (NiV) are both triggered by binding to receptors, mediated in both viruses by a second protein, the attachment protein. However, the hemagglutinin-neuraminidase (HN) attachment protein of NDV recognizes sialic acid receptors, whereas the NiV G attachment protein recognizes ephrinB2/B3 as receptors. Chimeric proteins composed of domains from the two attachment proteins have been evaluated for fusion-promoting activity with each F protein. Chimeras having NiV G-derived globular domains and NDV HN-derived stalks, transmembranes, and cytoplasmic tails are efficiently expressed, bind ephrinB2, and trigger NDV F to promote fusion in Vero cells. Thus, the NDV F protein can be triggered by binding to the NiV receptor, indicating that an aspect of the triggering cascade induced by the binding of HN to sialic acid is conserved in the binding of NiV G to ephrinB2. However, the fusion cascade for triggering NiV F by the G protein and that of triggering NDV F by the chimeras can be distinguished by differential exposure of a receptor-induced conformational epitope. The enhanced exposure of this epitope marks the triggering of NiV F by NiV G but not the triggering of NDV F by the chimeras. Thus, the triggering cascade for NiV G-F fusion may be more complex than that of NDV HN and F. This is consistent with the finding that reciprocal chimeras having NDV HN-derived heads and NiV G-derived stalks, transmembranes, and tails do not trigger either F protein for fusion, despite efficient cell surface expression and receptor binding. PMID:21460213

  16. Triggering of the newcastle disease virus fusion protein by a chimeric attachment protein that binds to Nipah virus receptors.

    Science.gov (United States)

    Mirza, Anne M; Aguilar, Hector C; Zhu, Qiyun; Mahon, Paul J; Rota, Paul A; Lee, Benhur; Iorio, Ronald M

    2011-05-20

    The fusion (F) proteins of Newcastle disease virus (NDV) and Nipah virus (NiV) are both triggered by binding to receptors, mediated in both viruses by a second protein, the attachment protein. However, the hemagglutinin-neuraminidase (HN) attachment protein of NDV recognizes sialic acid receptors, whereas the NiV G attachment protein recognizes ephrinB2/B3 as receptors. Chimeric proteins composed of domains from the two attachment proteins have been evaluated for fusion-promoting activity with each F protein. Chimeras having NiV G-derived globular domains and NDV HN-derived stalks, transmembranes, and cytoplasmic tails are efficiently expressed, bind ephrinB2, and trigger NDV F to promote fusion in Vero cells. Thus, the NDV F protein can be triggered by binding to the NiV receptor, indicating that an aspect of the triggering cascade induced by the binding of HN to sialic acid is conserved in the binding of NiV G to ephrinB2. However, the fusion cascade for triggering NiV F by the G protein and that of triggering NDV F by the chimeras can be distinguished by differential exposure of a receptor-induced conformational epitope. The enhanced exposure of this epitope marks the triggering of NiV F by NiV G but not the triggering of NDV F by the chimeras. Thus, the triggering cascade for NiV G-F fusion may be more complex than that of NDV HN and F. This is consistent with the finding that reciprocal chimeras having NDV HN-derived heads and NiV G-derived stalks, transmembranes, and tails do not trigger either F protein for fusion, despite efficient cell surface expression and receptor binding. © 2011 by The American Society for Biochemistry and Molecular Biology, Inc.

  17. Role of post-translational modifications on structure, function and pharmacology of class C G protein-coupled receptors

    DEFF Research Database (Denmark)

    Nørskov-Lauritsen, Lenea; Bräuner-Osborne, Hans

    2015-01-01

    taste receptors (T1R1-3), one calcium-sensing (CaS) receptor, one GPCR, class C, group 6, subtype A (GPRC6) receptor, and seven orphan receptors. G protein-coupled receptors undergo a number of post-translational modifications, which regulate their structure, function and/or pharmacology. Here, we...

  18. Complete Reversible Refolding of a G-Protein Coupled Receptor on a Solid Support.

    Directory of Open Access Journals (Sweden)

    Natalie Di Bartolo

    Full Text Available The factors defining the correct folding and stability of integral membrane proteins are poorly understood. Folding of only a few select membrane proteins has been scrutinised, leaving considerable deficiencies in knowledge for large protein families, such as G protein coupled receptors (GPCRs. Complete reversible folding, which is problematic for any membrane protein, has eluded this dominant receptor family. Moreover, attempts to recover receptors from denatured states are inefficient, yielding at best 40-70% functional protein. We present a method for the reversible unfolding of an archetypal family member, the β1-adrenergic receptor, and attain 100% recovery of the folded, functional state, in terms of ligand binding, compared to receptor which has not been subject to any unfolding and retains its original, folded structure. We exploit refolding on a solid support, which could avoid unwanted interactions and aggregation that occur in bulk solution. We determine the changes in structure and function upon unfolding and refolding. Additionally, we employ a method that is relatively new to membrane protein folding; pulse proteolysis. Complete refolding of β1-adrenergic receptor occurs in n-decyl-β-D-maltoside (DM micelles from a urea-denatured state, as shown by regain of its original helical structure, ligand binding and protein fluorescence. The successful refolding strategy on a solid support offers a defined method for the controlled refolding and recovery of functional GPCRs and other membrane proteins that suffer from instability and irreversible denaturation once isolated from their native membranes.

  19. A Novel GLP1 Receptor Interacting Protein ATP6ap2 Regulates Insulin Secretion in Pancreatic Beta Cells.

    Science.gov (United States)

    Dai, Feihan F; Bhattacharjee, Alpana; Liu, Ying; Batchuluun, Battsetseg; Zhang, Ming; Wang, Xinye Serena; Huang, Xinyi; Luu, Lemieux; Zhu, Dan; Gaisano, Herbert; Wheeler, Michael B

    2015-10-09

    GLP1 activates its receptor, GLP1R, to enhance insulin secretion. The activation and transduction of GLP1R requires complex interactions with a host of accessory proteins, most of which remain largely unknown. In this study, we used membrane-based split ubiquitin yeast two-hybrid assays to identify novel GLP1R interactors in both mouse and human islets. Among these, ATP6ap2 (ATPase H(+)-transporting lysosomal accessory protein 2) was identified in both mouse and human islet screens. ATP6ap2 was shown to be abundant in islets including both alpha and beta cells. When GLP1R and ATP6ap2 were co-expressed in beta cells, GLP1R was shown to directly interact with ATP6ap2, as assessed by co-immunoprecipitation. In INS-1 cells, overexpression of ATP6ap2 did not affect insulin secretion; however, siRNA knockdown decreased both glucose-stimulated and GLP1-induced insulin secretion. Decreases in GLP1-induced insulin secretion were accompanied by attenuated GLP1 stimulated cAMP accumulation. Because ATP6ap2 is a subunit required for V-ATPase assembly of insulin granules, it has been reported to be involved in granule acidification. In accordance with this, we observed impaired insulin granule acidification upon ATP6ap2 knockdown but paradoxically increased proinsulin secretion. Importantly, as a GLP1R interactor, ATP6ap2 was required for GLP1-induced Ca(2+) influx, in part explaining decreased insulin secretion in ATP6ap2 knockdown cells. Taken together, our findings identify a group of proteins that interact with the GLP1R. We further show that one interactor, ATP6ap2, plays a novel dual role in beta cells, modulating both GLP1R signaling and insulin processing to affect insulin secretion. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  20. Involvement of G proteins and cAMP in the production of chitinolytic enzymes by Trichoderma harzianum Envolvimento de proteínas G e cAMP na produção de enzimas quitinolíticas por Trichoderma harzianum

    Directory of Open Access Journals (Sweden)

    Alexandre A.P. Firmino

    2002-06-01

    Full Text Available The effect of G protein modulators and cyclic AMP (cAMP on N-acetylglucosaminidase (NAGase production was investigated during 84 h of growth of a Trichoderma harzianum strain in chitin-containing medium. Caffeine (5 mM, N6--2'-O-dibutyryladenosine 3'5'-cyclic monophosphate sodium salt (dBcAMP (1 mM and 3-isobutyl-1-methylxanthine (IBMX (2 mM decreased extracellular NAGase activity by 80%, 77% and 37%, respectively. AlCl3/KF (100 µM/10 mM and 200 µM/ 20 mM decreased the activity by 85% and 95%, respectively. Cholera (10 µ/mL and pertussis (20 µ/mL toxins also affected NAGase activity, causing a decrease of approximately 75%. Upon all treatments, protein bands of approximately 73 kDa, 68 kDa and 45 kDa had their signals diminished whilst a 50 kDa band was enhanced only by treatment with cholera and pertussis toxins. N-terminal sequencing analysis identified the 73 kDa and 68 kDa proteins as being T. harzianum NAGase in two different truncated forms whereas the 45 kDa band comprised a T. harzianum endochitinase. The 50 kDa protein showed sequence similarity to Coriolus vesicolor cellobiohydrolase. The above results suggest that a signaling pathway comprising G-proteins, adenylate cyclase and cAMP may be involved in the synthesis of T. harzianum chitinases.O efeito de cAMP e de moduladores de proteínas G sobre a produção de N-acetilglicosaminidase (NAGase foi investigado durante o crescimento de Trichoderma harzianum em meio contendo quitina. Cafeína (5 mM, dBcAMP (1mM e IBMX (2 mM provocaram diminuições na atividade extracelular de NAGase em 80%, 77% e 37%, respectivamente. Por outro lado, a presença de AlCl3/KF nas concentrações de 100 µM/10 mM e 200 µM/ 20 mM causou decréscimo na atividade em 85% e 95%, respectivamente. A toxina do cólera (10 µ/mL e a toxina pertussis (20 µ/mL também afetaram a atividade de NAGase, causando um decréscimo de aproximadamente 75%. Análises eletroforéticas mostraram que todos os tratamentos

  1. Structural–Functional Features of the Thyrotropin Receptor: A Class A G-Protein-Coupled Receptor at Work

    Science.gov (United States)

    Kleinau, Gunnar; Worth, Catherine L.; Kreuchwig, Annika; Biebermann, Heike; Marcinkowski, Patrick; Scheerer, Patrick; Krause, Gerd

    2017-01-01

    The thyroid-stimulating hormone receptor (TSHR) is a member of the glycoprotein hormone receptors, a sub-group of class A G-protein-coupled receptors (GPCRs). TSHR and its endogenous ligand thyrotropin (TSH) are of essential importance for growth and function of the thyroid gland and proper function of the TSH/TSHR system is pivotal for production and release of thyroid hormones. This receptor is also important with respect to pathophysiology, such as autoimmune (including ophthalmopathy) or non-autoimmune thyroid dysfunctions and cancer development. Pharmacological interventions directly targeting the TSHR should provide benefits to disease treatment compared to currently available therapies of dysfunctions associated with the TSHR or the thyroid gland. Upon TSHR activation, the molecular events conveying conformational changes from the extra- to the intracellular side of the cell across the membrane comprise reception, conversion, and amplification of the signal. These steps are highly dependent on structural features of this receptor and its intermolecular interaction partners, e.g., TSH, antibodies, small molecules, G-proteins, or arrestin. For better understanding of signal transduction, pathogenic mechanisms such as autoantibody action and mutational modifications or for developing new pharmacological strategies, it is essential to combine available structural data with functional information to generate homology models of the entire receptor. Although so far these insights are fragmental, in the past few decades essential contributions have been made to investigate in-depth the involved determinants, such as by structure determination via X-ray crystallography. This review summarizes available knowledge (as of December 2016) concerning the TSHR protein structure, associated functional aspects, and based on these insights we suggest several receptor complex models. Moreover, distinct TSHR properties will be highlighted in comparison to other class A GPCRs to

  2. Transcriptional and Functional Characterization of the G Protein-Coupled Receptor Repertoire of Gastric Somatostatin Cells

    DEFF Research Database (Denmark)

    Egerod, Kristoffer L; Engelstoft, Maja S; Lund, Mari L

    2015-01-01

    In the stomach, somatostatin (SST) acts as a general paracrine negative regulator of exocrine secretion of gastric acid and pepsinogen and endocrine secretion of gastrin, ghrelin, and histamine. Using reporter mice expressing red fluorescent protein (RFP) under control of the SST promotor, we have...... expressed and/or enriched. 1) The metabolite receptors calcium-sensing receptor and free fatty acid receptor 4 (GPR120) functioned as positive and negative regulators, respectively. 2) Among the neurotransmitter receptors, adrenergic receptors α1a, α2a, α2b, and β1 were all highly expressed...

  3. Geographies of the camp

    NARCIS (Netherlands)

    Minca, C.

    2015-01-01

    Facing the current growing global archipelago of encampments – including concentration, detention, transit, identification, refugee, military and training camps, this article is a geographical reflection on ‘the camp’, as a modern institution and as a spatial bio-political technology. In particular,

  4. Camp Sea Lab Visit

    OpenAIRE

    2009-01-01

    Approved for public release; distribution is unlimited On Wednesday July 8th, CAVR hosted 32 eight to thirteen year olds from California State Monterey Bay’s summer Camp SEA Lab. The students had the opportunity to interact with robotic dogs, an autonomous underwater vehicle (AUV), remotely operated vehicle (ROV), and unmanned aerial vehicles (UAV).

  5. Does protein binding modulate the effect of angiotensin II receptor antagonists?

    Directory of Open Access Journals (Sweden)

    Marc P Maillard

    2001-03-01

    Full Text Available IntroductionAngiotensin II AT 1-receptor antagonists are highly bound to plasma proteins (≥ 99%. With some antagonists, such as DuP-532, the protein binding was such that no efficacy of the drug could be demonstrated clinically. Whether protein binding interferes with the efficacy of other antagonists is not known. We have therefore investigated in vitro how plasma proteins may affect the antagonistic effect of different AT1-receptor antagonists.MethodsA radio-receptor binding assay was used to analyse the interaction between proteins and the ability of various angiotensin II (Ang II antagonists to block AT1-receptors. In addition, the Biacore technology, a new technique which enables the real-time monitoring of binding events between two molecules, was used to evaluate the dissociation rate constants of five AT1-receptor antagonists from human serum albumin.ResultsThe in vitro AT 1-antagonistic effects of different Ang II receptor antagonists were differentially affected by the presence of human plasma, with rightward shifts of the IC50 ranging from one to several orders of magnitude. The importance of the shift correlates with the dissociation rate constants of these drugs from albumin. Our experiments also show that the way that AT1-receptor antagonists bind to proteins differs from one compound to another. These results suggest that the interaction with plasma proteins appears to modulate the efficacy of some Ang II antagonists.ConclusionAlthough the high binding level of Ang II receptor antagonist to plasma proteins appears to be a feature common to this class of compounds, the kinetics and characteristics of this binding is of great importance. With some antagonists, protein binding interferes markedly with their efficacy to block AT1-receptors.

  6. 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

  7. DMPD: The role of Toll-like receptors and Nod proteins in bacterial infection. [Dynamic Macrophage Pathway CSML Database

    Lifescience Database Archive (English)

    Full Text Available 15476921 The role of Toll-like receptors and Nod proteins in bacterial infection. P...of Toll-like receptors and Nod proteins in bacterial infection. PubmedID 15476921 Title The role of Toll-like receptors and Nod prote...ins in bacterial infection. Authors Philpott DJ, Girardi

  8. DMPD: Structure, function and regulation of the Toll/IL-1 receptor adaptor proteins. [Dynamic Macrophage Pathway CSML Database

    Lifescience Database Archive (English)

    Full Text Available 17667936 Structure, function and regulation of the Toll/IL-1 receptor adaptor proteins... (.svg) (.html) (.csml) Show Structure, function and regulation of the Toll/IL-1 receptor adaptor proteins. ...PubmedID 17667936 Title Structure, function and regulation of the Toll/IL-1 receptor adaptor proteins

  9. A robust and rapid method of producing soluble, stable, and functional G-protein coupled receptors.

    Directory of Open Access Journals (Sweden)

    Karolina Corin

    Full Text Available Membrane proteins, particularly G-protein coupled receptors (GPCRs, are notoriously difficult to express. Using commercial E. coli cell-free systems with the detergent Brij-35, we could rapidly produce milligram quantities of 13 unique GPCRs. Immunoaffinity purification yielded receptors at >90% purity. Secondary structure analysis using circular dichroism indicated that the purified receptors were properly folded. Microscale thermophoresis, a novel label-free and surface-free detection technique that uses thermal gradients, showed that these receptors bound their ligands. The secondary structure and ligand-binding results from cell-free produced proteins were comparable to those expressed and purified from HEK293 cells. Our study demonstrates that cell-free protein production using commercially available kits and optimal detergents is a robust technology that can be used to produce sufficient GPCRs for biochemical, structural, and functional analyses. This robust and simple method may further stimulate others to study the structure and function of membrane proteins.

  10. Protein interactions among Fe65, the low-density lipoprotein receptor-related protein, and the amyloid precursor protein.

    Science.gov (United States)

    Mulvihill, Melinda M; Guttman, Miklos; Komives, Elizabeth A

    2011-07-19

    The adapter protein Fe65 has been proposed to be the link between the intracellular domains of the amyloid precursor protein, APP (AICD), and the low-density lipoprotein receptor-related protein (LRP-CT). Functional linkage between these two proteins has been established, and mutations within LRP-CT affect the amount of Aβ produced from APP. Previous work showed that AICD binds to protein interaction domain 2 (PID2) of Fe65. Although the structure of PID1 was determined recently, all attempts to demonstrate LRP-CT binding to this domain failed. We used biophysical experiments and binding studies to investigate the binding among these three proteins. Full-length Fe65 bound more weakly to AICD than did N-terminally truncated forms; however, the intramolecular domain-domain interactions that had been proposed to inhibit binding could not be observed using amide H-D exchange. Surprisingly, when LRP-CT is phosphorylated at Tyr4507, it bound to Fe65 PID1 despite the fact that this domain belongs to the Dab-like subclass of PIDs that are not supposed to be phosphorylation-dependent. Mutation of a critical arginine abolished binding, providing further proof of the phosphorylation dependence. Fe65 PID1 thus provides a link between the Dab-like class and the IRS-like class of PIDs and is the first Dab-like family member to show phosphorylation-dependent binding.

  11. Minireview: Role of Intracellular Scaffolding Proteins in the Regulation of Endocrine G Protein-Coupled Receptor Signaling

    Science.gov (United States)

    Walther, Cornelia

    2015-01-01

    The majority of hormones stimulates and mediates their signal transduction via G protein-coupled receptors (GPCRs). The signal is transmitted into the cell due to the association of the GPCRs with heterotrimeric G proteins, which in turn activates an extensive array of signaling pathways to regulate cell physiology. However, GPCRs also function as scaffolds for the recruitment of a variety of cytoplasmic protein-interacting proteins that bind to both the intracellular face and protein interaction motifs encoded by GPCRs. The structural scaffolding of these proteins allows GPCRs to recruit large functional complexes that serve to modulate both G protein-dependent and -independent cellular signaling pathways and modulate GPCR intracellular trafficking. This review focuses on GPCR interacting PSD95-disc large-zona occludens domain containing scaffolds in the regulation of endocrine receptor signaling as well as their potential role as therapeutic targets for the treatment of endocrinopathies. PMID:25942107

  12. Ric-8A, a Gα protein guanine nucleotide exchange factor potentiates taste receptor signaling

    Directory of Open Access Journals (Sweden)

    Claire J Fenech

    2009-10-01

    Full Text Available Taste receptors for sweet, bitter and umami tastants are G-protein coupled receptors (GPCRs. While much effort has been devoted to understanding G-protein-receptor interactions and identifying the components of the signalling cascade downstream of these receptors, at the level of the G-protein the modulation of receptor signal transduction remains relatively unexplored. In this regard a taste-specific regulator of G-protein signaling (RGS, RGS21, has recently been identified. To study whether guanine nucleotide exchange factors (GEFs are involved in the transduction of the signal downstream of the taste GPCRs we investigated the expression of Ric-8A and Ric-8B in mouse taste cells and their interaction with G-protein subunits found in taste buds. Mammalian Ric-8 proteins were initially identified as potent GEFs for a range of Gα subunits and Ric-8B has recently been shown to amplify olfactory signal transduction. We find that both Ric-8A and Ric-8B are expressed in a large portion of taste bud cells and that most of these cells contain IP3R-3 a marker for sweet, umami and bitter taste receptor cells. Ric-8A interacts with Gα-gustducin and Gαi2 through which it amplifies the signal transduction of hTas2R16, a receptor for bitter compounds. Overall, these findings are consistent with a role for Ric-8 in mammalian taste signal transduction.

  13. Transcriptional activation of peroxisome proliferator-activated receptor-{gamma} requires activation of both protein kinase A and Akt during adipocyte differentiation

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Sang-pil [Department of Thoracic and Cardiovascular Surgery, Pusan National University School of Medicine (Korea, Republic of); Ha, Jung Min; Yun, Sung Ji; Kim, Eun Kyoung [MRC for Ischemic Tissue Regeneration, Medical Research Institute, and Department of Pharmacology, Pusan National University School of Medicine (Korea, Republic of); Chung, Sung Woon [Department of Thoracic and Cardiovascular Surgery, Pusan National University School of Medicine (Korea, Republic of); Hong, Ki Whan; Kim, Chi Dae [MRC for Ischemic Tissue Regeneration, Medical Research Institute, and Department of Pharmacology, Pusan National University School of Medicine (Korea, Republic of); Bae, Sun Sik, E-mail: sunsik@pusan.ac.kr [MRC for Ischemic Tissue Regeneration, Medical Research Institute, and Department of Pharmacology, Pusan National University School of Medicine (Korea, Republic of)

    2010-08-13

    Research highlights: {yields} Elevated cAMP activates both PKA and Epac. {yields} PKA activates CREB transcriptional factor and Epac activates PI3K/Akt pathway via Rap1. {yields} Akt modulates PPAR-{gamma} transcriptional activity in concert with CREB. -- Abstract: Peroxisome proliferator-activated receptor-{gamma} (PPAR-{gamma}) is required for the conversion of pre-adipocytes. However, the mechanism underlying activation of PPAR-{gamma} is unclear. Here we showed that cAMP-induced activation of protein kinase A (PKA) and Akt is essential for the transcriptional activation of PPAR-{gamma}. Hormonal induction of adipogenesis was blocked by a phosphatidylinositol 3-kinase (PI3K) inhibitor (LY294002), by a protein kinase A (PKA) inhibitor (H89), and by a Rap1 inhibitor (GGTI-298). Transcriptional activity of PPAR-{gamma} was markedly enhanced by 3-isobutyl-1-methylxanthine (IBMX), but not insulin and dexamethasone. In addition, IBMX-induced PPAR-{gamma} transcriptional activity was blocked by PI3K/Akt, PKA, or Rap1 inhibitors. 8-(4-Chlorophenylthio)-2'-O-methyl-cAMP (8-pCPT-2'-O-Me-cAMP) which is a specific agonist for exchanger protein directly activated by cAMP (Epac) significantly induced the activation of Akt. Furthermore, knock-down of Akt1 markedly attenuated PPAR-{gamma} transcriptional activity. These results indicate that both PKA and Akt signaling pathways are required for transcriptional activation of PPAR-{gamma}, suggesting post-translational activation of PPAR-{gamma} might be critical step for adipogenic gene expression.

  14. Regulation of Brain Muscarinic Receptors by Protein Kinase C

    Science.gov (United States)

    1991-06-21

    229, 1990. 25. Fryer, A.D., E.E. El-Fakahany and D.B. Jacoby: Parainfluenza Virus Type 1 Reduces the Affinity of Agonists for Muscarinic Receptors in...Abdallah, M. Evinger, C. Forray and E.E. El-Fakahany: The Presence of an M4 Subtype Muscarinic Receptor in the Bovine Adrenal Medulla Revealed by mRNA and

  15. Fucosylation and protein glycosylation create functional receptors for cholera toxin

    DEFF Research Database (Denmark)

    Wands, Amberlyn M; Fujita, Akiko; McCombs, Janet E

    2015-01-01

    Cholera toxin (CT) enters and intoxicates host cells after binding cell surface receptors using its B subunit (CTB). The ganglioside (glycolipid) GM1 is thought to be the sole CT receptor; however, the mechanism by which CTB binding to GM1 mediates internalization of CT remains enigmatic. Here we...... in normal human intestinal epithelia and could play a role in cholera....

  16. Reviews in molecular biology and biotechnology: transmembrane signaling by G protein-coupled receptors.

    Science.gov (United States)

    Luttrell, Louis M

    2008-07-01

    As the most diverse type of cell surface receptor, the importance heptahelical G protein-coupled receptors (GPCRs) to clinical medicine cannot be overestimated. Visual, olfactory and gustatory sensation, intermediary metabolism, cell growth and differentiation are all influenced by GPCR signals. The basic receptor-G protein-effector mechanism of GPCR signaling is tuned by a complex interplay of positive and negative regulatory events that amplify the effect of a hormone binding the receptor or that dampen cellular responsiveness. The association of heptahelical receptors with a variety of intracellular partners other than G proteins has led to the discovery of potential mechanisms of GPCR signaling that extend beyond the classical paradigms. While the physiologic relevance of many of these novel mechanisms of GPCR signaling remains to be established, their existence suggests that the mechanisms of GPCR signaling are even more diverse than previously imagined.

  17. Chapter Three - Ubiquitination and Protein Turnover of G-Protein-Coupled Receptor Kinases in GPCR Signaling and Cellular Regulation.

    Science.gov (United States)

    Penela, P

    2016-01-01

    G-protein-coupled receptors (GPCRs) are responsible for regulating a wide variety of physiological processes, and distinct mechanisms for GPCR inactivation exist to guarantee correct receptor functionality. One of the widely used mechanisms is receptor phosphorylation by specific G-protein-coupled receptor kinases (GRKs), leading to uncoupling from G proteins (desensitization) and receptor internalization. GRKs and β-arrestins also participate in the assembly of receptor-associated multimolecular complexes, thus initiating alternative G-protein-independent signaling events. In addition, the abundant GRK2 kinase has diverse "effector" functions in cellular migration, proliferation, and metabolism homeostasis by means of the phosphorylation or interaction with non-GPCR partners. Altered expression of GRKs (particularly of GRK2 and GRK5) occurs during pathological conditions characterized by impaired GPCR signaling including inflammatory syndromes, cardiovascular disease, and tumor contexts. It is increasingly appreciated that different pathways governing GRK protein stability play a role in the modulation of kinase levels in normal and pathological conditions. Thus, enhanced GRK2 degradation by the proteasome pathway occurs upon GPCR stimulation, what allows cellular adaptation to chronic stimulation in a physiological setting. β-arrestins participate in this process by facilitating GRK2 phosphorylation by different kinases and by recruiting diverse E3 ubiquitin ligase to the receptor complex. Different proteolytic systems (ubiquitin-proteasome, calpains), chaperone activities and signaling pathways influence the stability of GRKs in different ways, thus endowing specificity to GPCR regulation as protein turnover of GRKs can be differentially affected. Therefore, modulation of protein stability of GRKs emerges as a versatile mechanism for feedback regulation of GPCR signaling and basic cellular processes. Copyright © 2016 Elsevier Inc. All rights reserved.

  18. Neuronal low-density lipoprotein receptor-related protein 1 binds and endocytoses prion fibrils via receptor cluster 4

    DEFF Research Database (Denmark)

    Jen, Angela; Parkyn, Celia J; Mootoosamy, Roy C

    2010-01-01

    For infectious prion protein (designated PrP(Sc)) to act as a template to convert normal cellular protein (PrP(C)) to its distinctive pathogenic conformation, the two forms of prion protein (PrP) must interact closely. The neuronal receptor that rapidly endocytoses PrP(C) is the low-density lipop......For infectious prion protein (designated PrP(Sc)) to act as a template to convert normal cellular protein (PrP(C)) to its distinctive pathogenic conformation, the two forms of prion protein (PrP) must interact closely. The neuronal receptor that rapidly endocytoses PrP(C) is the low...... clusters 2 and 4, PrP(C) and PrP(Sc) fibrils bind only to receptor cluster 4. PrP(Sc) fibrils out-compete PrP(C) for internalization. When endocytosed, PrP(Sc) fibrils are routed to lysosomes, rather than recycled to the cell surface with PrP(C). Thus, although LRP1 binds both forms of PrP, it traffics...

  19. Chimeric proteins containing the cytoplasmic domains of the mannose 6-phosphate receptors codistribute with the endogenous receptors.

    Science.gov (United States)

    Mauxion, F; Schmidt, A; Le Borgne, R; Hoflack, B

    1995-02-01

    We have constructed and transiently expressed in HeLa cells a series of hybrid proteins in which the cytoplasmic domain or both the transmembrane and the cytoplasmic domains of the mannose 6-phosphate/insulin-like growth factor II receptor were fused to the ectodomain of the hemagglutinin of the influenza virus (HA), a typical plasma membrane protein. In addition, we have expressed a hybrid protein containing the luminal domain of HA fused to the transmembrane and cytoplasmic tail of the cation-dependent mannose 6-phosphate receptor. These hybrids were transported through and sorted from the secretory pathway as shown by acquisition of endo-H resistant oligosaccharides and their ability to recruit the Golgi assembly proteins AP-1 on the Golgi membrane. Like the mannose 6-phosphate receptors (MPRs), these hybrid proteins are also present in small amounts at the cell surface where they are likely to undergo endocytosis as disruption of the endocytosis signals contained in the MPR cytoplasmic domains induces their accumulation at the cell surface. Double immunofluorescence studies indicate that these chimeras codistribute with the endogenous MPRs at steady state. The results suggest that the cytoplasmic domains of the MPRs are sufficient to determine the steady-state distribution of the full-length proteins.

  20. A Molecular Mechanism for Sequential Activation of a G Protein-Coupled Receptor

    DEFF Research Database (Denmark)

    Grundmann, Manuel; Tikhonova, Irina G; Hudson, Brian D

    2016-01-01

    Ligands targeting G protein-coupled receptors (GPCRs) are currently classified as either orthosteric, allosteric, or dualsteric/bitopic. Here, we introduce a new pharmacological concept for GPCR functional modulation: sequential receptor activation. A hallmark feature of this is a stepwise ligand...... and pharmacological perturbations along with computational methods, and propose a kinetic model applicable to the analysis of sequential receptor activation. We envision this form of dynamic agonism as a common principle of nature to spatiotemporally encode cellular information....

  1. Endothelial protein C receptor-dependent antichemotactic effects of canine protein C.

    Science.gov (United States)

    Wong, Valerie M; Côté, Olivier; Bienzle, Dorothee; Hayes, M Anthony; Wood, R Darren

    2017-02-01

    OBJECTIVE To determine whether canine protein C (CnPC) had antichemotactic effects on canine neutrophils, whether endothelial protein C receptor (EPCR) was expressed on canine neutrophils, and the role of EPCR in neutrophil chemotaxis. SAMPLE Neutrophils isolated from blood samples from healthy dogs (n = 6) and sick dogs with (2) or without (3) an inflammatory leukogram. PROCEDURES Neutrophils were analyzed by reverse transcriptase PCR assay and flow cytometry for detection of EPCR mRNA and protein expression, respectively. Neutrophils were incubated with CnPC zymogen or canine activated protein C (CnAPC), with or without RCR-379 (an anti-human EPCR antibody). Neutrophils were then allowed to migrate through a filter membrane toward a chemokine. Untreated neutrophils served as positive control samples. Migration was quantified by fluorescence measurement, and chemotaxis index (Chx) values (fluorescence of test sample/fluorescence of positive control sample) were computed. RESULTS The cDNA for EPCR was amplified, and EPCR expression was detected on neutrophil surfaces. Obtained Chx values were significantly higher in cells treated with RCR-379 than in cells treated with CnPC or CnAPC alone. The Chx values for neutrophils treated with RCR-379 were not significantly different from 1, whereas those for neutrophils treated without RCR-379 were significantly less than 1. The effects of RCR-379 on neutrophil migration were independent of concentration or activation status of protein C. CONCLUSIONS AND CLINICAL RELEVANCE Canine neutrophils expressed EPCR, and inhibition of neutrophil chemotaxis by CnPC and CnAPC depended on EPCR. Interventions with EPCR signaling may have therapeutic application in dogs.

  2. Glucocorticoids curtail stimuli-induced CREB phosphorylation in TRH neurons through interaction of the glucocorticoid receptor with the catalytic subunit of protein kinase A.

    Science.gov (United States)

    Sotelo-Rivera, Israim; Cote-Vélez, Antonieta; Uribe, Rosa-María; Charli, Jean-Louis; Joseph-Bravo, Patricia

    2017-03-01

    Corticosterone prevents cold-induced stimulation of thyrotropin-releasing hormone (Trh) expression in rats, and the stimulatory effect of dibutyryl cyclic-adenosine monophosphate (dB-cAMP) on Trh transcription in hypothalamic cultures. We searched for the mechanism of this interference. Immunohistochemical analyses of phosphorylated cAMP-response element binding protein (pCREB) were performed in the paraventricular nucleus (PVN) of Wistar rats, and in cell cultures of 17-day old rat hypothalami, or neuroblastoma SH-SY5Y cells. Cultures were incubated 1h with dB-cAMP, dexamethasone and both drugs combined; their nuclear extracts were used for chromatin immunoprecipitation; cytosolic or nuclear extracts for coimmunoprecipitation analyses of catalytic subunit of protein kinase A (PKAc) and of glucocorticoid receptor (GR); their subcellular distribution was analyzed by immunocytochemistry. Cold exposure increased pCREB in TRH neurons of rats PVN, effect blunted by corticosterone previous injection. Dexamethasone interfered with forskolin increase in nuclear pCREB and its binding to Trh promoter; antibodies against histone deacetylase-3 precipitated chromatin from nuclear extracts of hypothalamic cells treated with tri-iodothyronine but not with dB-cAMP + dexamethasone, discarding chromatin compaction as responsible mechanism. Co-immunoprecipitation analyses of cytosolic or nuclear extracts showed protein:protein interactions between activated GR and PKAc. Immunocytochemical analyses of hypothalamic or SH-SY5Y cells revealed diminished nuclear translocation of PKAc and GR in cells incubated with forskolin + dexamethasone, compared to either forskolin or dexamethasone alone. Glucocorticoids and cAMP exert mutual inhibition of Trh transcription through interaction of activated glucocorticoid receptor with protein kinase A catalytic subunit, reducing their nuclear translocation, limiting cAMP-response element binding protein phosphorylation and its binding to Trh promoter.

  3. Molecular identification of a Drosophila G protein-coupled receptor specific for crustacean cardioactive peptide

    DEFF Research Database (Denmark)

    Cazzamali, Giuseppe; Hauser, Frank; Kobberup, Sune

    2003-01-01

    The Drosophila Genome Project website (www.flybase.org) contains the sequence of an annotated gene (CG6111) expected to code for a G protein-coupled receptor. We have cloned this receptor and found that its gene was not correctly predicted, because an annotated neighbouring gene (CG14547) was also...... part of the receptor gene. DNA corresponding to the corrected gene CG6111 was expressed in Chinese hamster ovary cells, where it was found to code for a receptor that could be activated by low concentrations of crustacean cardioactive peptide, which is a neuropeptide also known to occur in Drosophila...... and other insects (EC(50), 5.4 x 10(-10)M). Other known Drosophila neuropeptides, such as adipokinetic hormone, did not activate the receptor. The receptor is expressed in all developmental stages from Drosophila, but only very weakly in larvae. In adult flies, the receptor is mainly expressed in the head...

  4. Interaction between the p21ras GTPase activating protein and the insulin receptor

    NARCIS (Netherlands)

    Pronk, G.J.; Medema, R.H.; Burgering, B.M.T.; Clark, R.; McCormick, F.; Bos, J.L.

    1992-01-01

    We investigated the involvement of the p21ras-GTPase activating protein (GAP) in insulin-induced signal transduction. In cells overexpressing the insulin receptor, we did not observe association between GAP and the insulin receptor after insulin treatment nor the phosphorylation of GAP on tyrosine

  5. Fibroblast growth factor receptor 3 protein is overexpressed in oral and oropharyngeal squamous cell carcinoma

    NARCIS (Netherlands)

    Koole, Koos; van Kempen, Pauline M W; Swartz, Justin E|info:eu-repo/dai/nl/413983390; Peeters, Ton; van Diest, Paul J|info:eu-repo/dai/nl/075281775; Koole, Ron|info:eu-repo/dai/nl/123508126; van Es, Robert J. J.|info:eu-repo/dai/nl/216460646; Willems, Stefan M|info:eu-repo/dai/nl/33189582X

    Fibroblast growth factor receptor 3 (FGFR3) is a member of the fibroblast growth factor receptor tyrosine kinase family. It has been identified as a promising therapeutic target in multiple types of cancer. We have investigated FGFR3 protein expression and FGFR3 gene copy-numbers in a single

  6. Structure of the full-length glucagon class B G-protein-coupled receptor

    NARCIS (Netherlands)

    Zhang, Haonan; Qiao, Anna; Yang, Dehua; Yang, Linlin; Dai, Antao; de Graaf, C.; Reedtz-Runge, Steffen; Dharmarajan, Venkatasubramanian; Zhang, Hui; Han, Gye Won; Grant, Thomas D.; Sierra, Raymond G.; Weierstall, Uwe; Nelson, Garrett; Liu, Wei; Wu, Yanhong; Ma, Limin; Cai, Xiaoqing; Lin, Guangyao; Wu, Xiaoai; Geng, Zhi; Dong, Yuhui; Song, Gaojie; Griffin, Patrick R.; Lau, Jesper; Cherezov, Vadim; Yang, Huaiyu; Hanson, Michael A.; Stevens, Raymond C.; Zhao, Qiang; Jiang, Hualiang; Wang, Ming Wei; Wu, Beili

    2017-01-01

    The human glucagon receptor, GCGR, belongs to the class B G-protein-coupled receptor family and plays a key role in glucose homeostasis and the pathophysiology of type 2 diabetes. Here we report the 3.0 Å crystal structure of full-length GCGR containing both the extracellular domain and

  7. Analysis of the protein related receptor GPR92 in G-cells

    Directory of Open Access Journals (Sweden)

    Amelie Therese Rettenberger

    2015-09-01

    Full Text Available A continuous assessment of ingested food in the gastric lumen is essential for fine-tuning the digestive activities, including the secretion of the regulatory hormones such as gastrin. It has been proposed that G-cells may be able to sense the amount of ingested proteins and adjust the secretion of gastrin accordingly. Our previous studies have shown that G-cells express suitable receptor types, most notably the peptone-receptor GPR92 and the amino acid receptors GPRC6A and CaSR; however, their relative importance remained unclear. To determine the relative quantity of each receptor type, individual G-cells isolated from the transgenic mouse line mGas-EGFP were analyzed by means of a Liquid Chromatography Tandem-Mass Spectrometry (LC-MS/MS procedure. The results indicate that the relative amount of receptor protein for GPR92 was much higher than for the receptor types GPRC6A and CaSR. These findings support the notion that the peptone-receptor GPR92 may be particularly relevant for sensing partially digested protein products. This view was supported by the finding that a high-protein diet affected the expression level of the peptone-receptor GPR92 in the gastric antrum as well as in the circumvallate papillae.

  8. G-protein coupled progesterone receptors in the plasma membrane of fungus Rhizopus nigricans.

    Science.gov (United States)

    Bavec, A; Slajpah, M; Lenasi, H; Yorko, M; Breskvar, K

    2000-01-01

    We have demonstrated simultaneous existence of progesterone receptors and GTPase activity in the membranes prepared from the filamentous fungus Rhizopus nigricans. The results obtained with pertussis toxin treated fungal mycelium suggest that these receptors do not couple to Gi-Go-proteins and play a role in the induction of steroid hydroxylating enzyme system by steroid substrates in the fungus.

  9. The Role of Rab Proteins in Neuronal Cells and in the Trafficking of Neurotrophin Receptors

    Directory of Open Access Journals (Sweden)

    Cecilia Bucci

    2014-10-01

    Full Text Available Neurotrophins are a family of proteins that are important for neuronal development, neuronal survival and neuronal functions. Neurotrophins exert their role by binding to their receptors, the Trk family of receptor tyrosine kinases (TrkA, TrkB, and TrkC and p75NTR, a member of the tumor necrosis factor (TNF receptor superfamily. Binding of neurotrophins to receptors triggers a complex series of signal transduction events, which are able to induce neuronal differentiation but are also responsible for neuronal maintenance and neuronal functions. Rab proteins are small GTPases localized to the cytosolic surface of specific intracellular compartments and are involved in controlling vesicular transport. Rab proteins, acting as master regulators of the membrane trafficking network, play a central role in both trafficking and signaling pathways of neurotrophin receptors. Axonal transport represents the Achilles' heel of neurons, due to the long-range distance that molecules, organelles and, in particular, neurotrophin-receptor complexes have to cover. Indeed, alterations of axonal transport and, specifically, of axonal trafficking of neurotrophin receptors are responsible for several human neurodegenerative diseases, such as Huntington’s disease, Alzheimer’s disease, amyotrophic lateral sclerosis and some forms of Charcot-Marie-Tooth disease. In this review, we will discuss the link between Rab proteins and neurotrophin receptor trafficking and their influence on downstream signaling pathways.

  10. Use of Designer G Protein-Coupled Receptors to Dissect Metabolic Pathways.

    Science.gov (United States)

    Wess, Jürgen

    2016-09-01

    G protein-coupled receptors (GPCRs) regulate virtually all metabolic processes, including glucose and energy homeostasis. Recently, the use of designer GPCRs referred to as designer receptors exclusively activated by designer drug (DREADDs) has made it possible to dissect metabolically relevant GPCR signaling pathways in a temporally and spatially controlled fashion in vivo. Published by Elsevier Ltd.

  11. Differential palmitoylation directs the AMPA receptor-binding protein ABP to spines or to intracellular clusters.

    Science.gov (United States)

    DeSouza, Sunita; Fu, Jie; States, Bradley A; Ziff, Edward B

    2002-05-01

    Long-term changes in excitatory synapse strength are thought to reflect changes in synaptic abundance of AMPA receptors mediated by receptor trafficking. AMPA receptor-binding protein (ABP) and glutamate receptor-interacting protein (GRIP) are two similar PDZ (postsynaptic density 95/Discs large/zona occludens 1) proteins that interact with glutamate receptors 2 and 3 (GluR2 and GluR3) subunits. Both proteins have proposed roles during long-term potentiation and long-term depression in the delivery and anchorage of AMPA receptors at synapses. Here we report a variant of ABP-L (seven PDZ form of ABP) called pABP-L that is palmitoylated at a cysteine residue at position 11 within a novel 18 amino acid N-terminal leader sequence encoded through differential splicing. In cultured hippocampal neurons, nonpalmitoylated ABP-L localizes with internal GluR2 pools expressed from a Sindbis virus vector, whereas pABP-L is membrane targeted and associates with surface-localized GluR2 receptors at the plasma membrane in spines. Mutation of Cys-11 to alanine blocks the palmitoylation of pABP-L and targets the protein to intracellular clusters, confirming that targeting the protein to spines is dependent on palmitoylation. Non-palmitoylated GRIP is primarily intracellular, but a chimera with the pABP-L N-terminal palmitoylation sequence linked to the body of the GRIP protein is targeted to spines. We suggest that pABP-L and ABP-L provide, respectively, synaptic and intracellular sites for the anchorage of AMPA receptors during receptor trafficking to and from the synapse.

  12. Characterization of umami receptor and coupling G protein in mouse taste cells.

    Science.gov (United States)

    Narukawa, Masataka; Kitagawa-Iseki, Keiko; Oike, Hideaki; Abe, Keiko; Mori, Tomohiko; Hayashi, Yukako

    2008-08-06

    Taste receptor cells (TRCs) express multiple umami receptors. We performed physiological investigations to determine whether umami-responding cells in taste buds possess G protein-coupled receptors and to determine what type of G proteins exist if any. To clarify the components that participate in intracellular umami signal transduction in mouse, we recorded the activation of TRCs. TRCs treated with the G protein inhibitor GDP-beta-S lost umami-induced inward currents. Treatment with the Galphai inhibitor, pertussis toxin, did not increase the intracellular Ca2+ level in many TRCs. Immunohistochemical analysis revealed that a subset of TRCs responding to umami stimuli expressed alpha-gustducin. Thus, we demonstrated that umami stimuli were received by G protein-coupled receptors that function together with some of the Galphai family members.

  13. Chronic regulation of colonic epithelial secretory function by activation of G protein-coupled receptors.

    LENUS (Irish Health Repository)

    Toumi, F

    2011-02-01

    Enteric neurotransmitters that act at G protein-coupled receptors (GPCRs) are well known to acutely promote epithelial Cl(-) and fluid secretion. Here we examined if acute GPCR activation might have more long-term consequences for epithelial secretory function.

  14. High glucose enhances cAMP level and extracellular signal-regulated kinase phosphorylation in Chinese hamster ovary cell: Usage of Br-cAMP in foreign protein β-galactosidase expression.

    Science.gov (United States)

    Lin, Hsiao-Hsien; Lee, Tsung-Yih; Liu, Ting-Wei; Tseng, Ching-Ping

    2017-07-01

    Glucose is a carbon source for Chinese hamster ovary (CHO) cell growth, while low growth rate is considered to enhance the production of recombinant proteins. The present study reveals that glucose concentrations higher than 1 g/L reduce the growth rate and substantially increase in cAMP (∼300%) at a high glucose concentration (10 g/L). High glucose also enhances the phosphorylation of extracellular signal-regulated kinase (ERK) and p27 kip by Western blot analysis. To determine whether the phosphorylation of ERK is involved in the mechanism, a cyclic-AMP dependent protein kinase A (PKA) inhibitor (H-8) or MEK (MAPKK) inhibitor (PD98059) was added to block ERK phosphorylation. We show that both the high glucose-induced ERK phosphorylation and growth rate return to baseline levels. These results suggest that the cAMP/PKA and MAP signaling pathways are involved in the abovementioned mechanism. Interestingly, the direct addition of 8-bromo-cAMP (Br-cAMP), a membrane-permeable cAMP analog, can mimic the similar effects produced by high glucose. Subsequently Br-cAMP could induce β-galactosidase (β-Gal) recombinant protein expression by 1.6-fold. Furthermore, Br-cAMP can additionally enhance the β-Gal production (from 2.8- to 4.5-fold) when CHO cells were stimulated with glycerol, thymidine, dimethyl sulfoxide, pentanoic acid, or sodium butyrate. Thus, Br-cAMP may be used as an alternative agent in promoting foreign protein expression for CHO cells. Copyright © 2017. Published by Elsevier B.V.

  15. Docosahexaenoic acid, G protein-coupled receptors, and melanoma: is G protein-coupled receptor 40 a potential therapeutic target?

    Science.gov (United States)

    Nehra, Deepika; Pan, Amy H; Le, Hau D; Fallon, Erica M; Carlson, Sarah J; Kalish, Brian T; Puder, Mark

    2014-05-15

    To determine the effect of docosahexaenoic acid (DHA) on the growth of human melanoma in vitro and in vivo and to better understand the potential role of the G protein-coupled receptors (GPRs) in mediating this effect. For in vitro studies, human melanoma and control fibroblast cells were treated with DHA and TAK-875 (selective GPR40 agonist) and a cell viability assay was performed to determine cell counts. A murine subcutaneous xenograft model of human melanoma was used to test the effect of dietary treatment with an omega-3 fatty acid (FA) rich diet compared with an omega-6 FA rich diet on the growth of human melanoma in vivo. A similar animal model was used to test the effect of oral TAK-875 on the growth of established melanoma tumors in vivo. DHA has an inhibitory effect on the growth of human melanoma both in vitro and in vivo. Tumors from animals on the omega-3 FA rich diet were 69% smaller in weight (P = 0.005) and 76% smaller in volume compared with tumors from animals on the omega-6 FA rich diet. TAK-875 has an inhibitory effect on the growth of human melanoma both in vitro and in vivo. Tumors from animals treated with TAK-875 were 46% smaller in weight (P = 0.07), 62% smaller in volume (P = 0.03), and grew 77% slower (P = 0.04) compared with the placebo group. DHA and TAK-875 have a profound and selective inhibitory effect on the growth of human melanoma both in vitro and in vivo. Copyright © 2014 Elsevier Inc. All rights reserved.

  16. Peroxisome proliferator-activated receptor delta (PPARdelta )-mediated regulation of preadipocyte proliferation and gene expression is dependent on cAMP signaling

    DEFF Research Database (Denmark)

    Hansen, Jacob B.; Zhang, H; Rasmussen, T H

    2001-01-01

    The peroxisome proliferator-activated receptor gamma (PPARgamma) is a key regulator of terminal adipocyte differentiation. PPARdelta is expressed in preadipocytes, but the importance of this PPAR subtype in adipogenesis has been a matter of debate. Here we present a critical evaluation of the role...... of PPARdelta in adipocyte differentiation. We demonstrate that treatment of NIH-3T3 fibroblasts overexpressing PPARdelta with standard adipogenic inducers led to induction of PPARgamma2 expression and terminal adipocyte differentiation in a manner that was strictly dependent on simultaneous administration...... expression of PPARgamma and ALBP/aP2, but only modestly promoted terminal differentiation as determined by lipid accumulation. Finally, we provide evidence that synergistic activation of PPARdelta promotes mitotic clonal expansion in 3T3-L1 cells with or without forced expression of PPARdelta. In conclusion...

  17. Biophysical approaches to G protein-coupled receptors: Structure, function and dynamics

    Science.gov (United States)

    Chollet, André; Turcatti, Gerardo

    1999-05-01

    G protein-coupled receptors (GPCR) represent a large family of drug targets for which there is no high-resolution structural information. In order to understand the mechanisms of ligand recognition and receptor activation, there is a strong need for novel biophysical methods. In this Perspective we provide an overview of recent experimental approaches used to explore the molecular architecture and dynamics of GPCR and their interactions with ligands and G proteins using biophysical, non-crystallographic, methods.

  18. Running Boot Camp

    CERN Document Server

    Toporek, Chuck

    2008-01-01

    When Steve Jobs jumped on stage at Macworld San Francisco 2006 and announced the new Intel-based Macs, the question wasn't if, but when someone would figure out a hack to get Windows XP running on these new "Mactels." Enter Boot Camp, a new system utility that helps you partition and install Windows XP on your Intel Mac. Boot Camp does all the heavy lifting for you. You won't need to open the Terminal and hack on system files or wave a chicken bone over your iMac to get XP running. This free program makes it easy for anyone to turn their Mac into a dual-boot Windows/OS X machine. Running Bo

  19. Probing intermolecular protein-protein interactions in the calcium-sensing receptor homodimer using bioluminescence resonance energy transfer (BRET)

    DEFF Research Database (Denmark)

    Jensen, Anders A.; Hansen, Jakob L; Sheikh, Søren P

    2002-01-01

    The calcium-sensing receptor (CaR) belongs to family C of the G-protein coupled receptor superfamily. The receptor is believed to exist as a homodimer due to covalent and non-covalent interactions between the two amino terminal domains (ATDs). It is well established that agonist binding to family C......-induced intermolecular movements in the CaR homodimer using the new bioluminescence resonance energy transfer technique, BRET2, which is based on the transference of energy from Renilla luciferase (Rluc) to the green fluorescent protein mutant GFP2. We tagged CaR with Rluc and GFP2 at different intracellular locations....... Stable and highly receptor-specific BRET signals were obtained in tsA cells transfected with Rluc- and GFP2-tagged CaRs under basal conditions, indicating that CaR is constitutively dimerized. However, the signals were not enhanced by the presence of agonist. These results could indicate that at least...

  20. Nuclear localization of bradykinin B(2) receptors reflects binding to the nuclear envelope protein lamin C.

    Science.gov (United States)

    Takano, Masaoki; Kanoh, Akira; Amako, Katsumi; Otani, Mieko; Sano, Keiji; Kanazawa-Hamada, Michiko; Matsuyama, Shogo

    2014-01-15

    The mechanism of action of bradykinin (BK), a pro-inflammatory mediator, is thought to be mediated by specific cell surface membrane bradykinin B2 receptors. Some evidence suggests that there are both intracellular and nuclear bradykinin B2 receptors. This study identified proteins that interact with the C-terminus of the bradykinin B2 receptor (in particular, the nuclear membrane protein lamin C), using the yeast two-hybrid system. The motif of the C-terminal domain (CT) mutant 303-320 in bradykinin B2 receptor was identified as a lamin C protein binding motif. Immunohistochemistry revealed colocalization of FLAG- bradykinin B2 receptor with HA-lamin C in the nucleus of HEK 293T cells. In situ proximity ligation assay (PLA) showed that FLAG-bradykinin B2 receptor formed heterodimers with HA-lamin C in the nucleus. In addition, live cell fluorescence imaging showed that bradykinin B2 receptor-EGFP was located in the nucleus and co-localized with HcRed-lamin C. Interestingly, neither BK addition nor bradykinin B2 receptor CT mutation reduced the binding to lamin C or changed the distribution of bradykinin B2 receptor. Taken together, these findings demonstrate that bradykinin B2 receptor-lamin C heterodimers form in the nucleus independent of BK stimulation and CT mutation. We propose that heterodimerization of bradykinin B2 receptor with lamin C is essential to nuclear localization of bradykinin B2 receptor and plays an important role in cell signaling and function. © 2013 Elsevier B.V. All rights reserved.

  1. Structure-Function Analysis of Cf-9, a Receptor-Like Protein with Extracytoplasmic Leucine-Rich Repeats

    NARCIS (Netherlands)

    Hoorn, van der R.A.L.; Wulff, B.B.H.; Rivas, S.; Durrant, M.C.; Ploeg, van der A.; Wit, de P.J.G.M.; Jones, J.D.G.

    2005-01-01

    The tomato (Lycopersicon pimpinellifolium) resistance protein Cf-9 belongs to a large class of plant proteins with extracytoplasmic Leu-rich repeats (eLRRs). eLRR proteins play key roles in plant defense and development, mainly as receptor-like proteins or receptor-like kinases, conferring

  2. 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.

  3. Receptor oligomerization in family B1 of G-protein-coupled receptors

    DEFF Research Database (Denmark)

    Roed, Sarah Norklit; Ørgaard, Anne; Jørgensen, Rasmus

    2012-01-01

    , the glucagon receptor, and the receptors for parathyroid hormone (PTHR1 and PTHR2). The dysregulation of several family B1 receptors is involved in diseases, such as diabetes, chronic inflammation, and osteoporosis which underlines the pathophysiological importance of this GPCR subfamily. In spite of this......, investigation of family B1 receptor oligomerization and especially its pharmacological importance is still at an early stage. Even though GPCR oligomerization is a well-established phenomenon, there is a need for more investigations providing a direct link between these interactions and receptor functionality...... in family B1 GPCRs. One example of the functional effects of GPCR oligomerization is the facilitation of allosterism including cooperativity in ligand binding to GPCRs. Here, we review the currently available data on family B1 GPCR homo- and heteromerization, mainly based on BRET investigations. Furthermore...

  4. Protein interactome mining defines melatonin MT 1 receptors as integral component of presynaptic protein complexes of neurons

    OpenAIRE

    Benleulmi-Chaachoua, Abla; Chen, Lina; Sokolina, Kate; Wong, Victoria; Jurisica, Igor; Emerit, Michel-Boris; Darmon, Michèle; Espin, Almudena; Stagljar, Igor; Tafelmeyer, Petra; Zamponi, Gerald; Delagrange, Philippe; Maurice, Pascal; Jockers, Ralf

    2015-01-01

    International audience; In mammals, the hormone melatonin is mainly produced by the pineal gland with nocturnal peak levels. Its peripheral and central actions rely either on its intrinsic antioxidant properties or on binding to melatonin MT 1 and MT 2 receptors, belonging to the G protein-coupled receptor (GPCR) super-family. Melatonin has been reported to be involved in many functions of the central nervous system such as circadian rhythm regulation, neurotransmission, synaptic plasticity, ...

  5. Bioluminescence resonance energy transfer methods to study G protein-coupled receptor-receptor tyrosine kinase heteroreceptor complexes.

    Science.gov (United States)

    Borroto-Escuela, Dasiel O; Flajolet, Marc; Agnati, Luigi F; Greengard, Paul; Fuxe, Kjell

    2013-01-01

    A large body of evidence indicates that G protein-coupled receptors (GPCRs) and receptor tyrosine kinases (RTKs) can form heteroreceptor complexes. In these complexes, the signaling from each interacting protomer is modulated to produce an integrated and therefore novel response upon agonist(s) activation. In the GPCR-RTK heteroreceptor complexes, GPCRs can activate RTK in the absence of added growth factor through the use of RTK signaling molecules. This integrative phenomenon is reciprocal and can place also RTK signaling downstream of GPCR. Formation of either stable or transient complexes by these two important classes of membrane receptors is involved in regulating all aspects of receptor function, from ligand binding to signal transduction, trafficking, desensitization, and downregulation among others. Functional phenomena can be modulated with conformation-specific inhibitors that stabilize defined GPCR states to abrogate both GPCR agonist- and growth factor-stimulated cell responses or by means of small interfering heteroreceptor complex interface peptides. The bioluminescence resonance energy transfer (BRET) technology has emerged as a powerful method to study the structure of heteroreceptor complexes closely associated with the study of receptor-receptor interactions in such complexes. In this chapter, we provide an overview of different BRET(2) assays that can be used to study the structure of GPCR-RTK heteroreceptor complexes and their functions. Various experimental designs for optimization of these experiments are also described. Copyright © 2013 Elsevier Inc. All rights reserved.

  6. Some properties of human neuronal α7 nicotinic acetylcholine receptors fused to the green fluorescent protein

    Science.gov (United States)

    Palma, Eleonora; Mileo, Anna M.; Martínez-Torres, Ataúlfo; Eusebi, Fabrizio; Miledi, Ricardo

    2002-01-01

    The functional properties and cellular localization of the human neuronal α7 nicotinic acetylcholine (AcCho) receptor (α7 AcChoR) and its L248T mutated (mut) form were investigated by expressing them alone or as gene fusions with the enhanced version of the green fluorescent protein (GFP). Xenopus oocytes injected with wild-type (wt), mutα7, or the chimeric subunit cDNAs expressed receptors that gated membrane currents when exposed to AcCho. As already known, AcCho currents generated by wtα7 receptors decay much faster than those elicited by the mutα7 receptors. Unexpectedly, the fusion of GFP to the wt and mutated α7 receptors led to opposite results: the AcCho-current decay of the wt receptors became slower, whereas that of the mutated receptors was accelerated. Furthermore, repetitive applications of AcCho led to a considerable “run-down” of the AcCho currents generated by mutα7-GFP receptors, whereas those of the wtα7-GFP receptors remained stable or increased in amplitude. The AcCho-current run-down of mutα7-GFP oocytes was accompanied by a marked decrease of α-bungarotoxin binding activity. Fluorescence, caused by the chimeric receptors expressed, was seen over the whole oocyte surface but was more intense and abundant in the animal hemisphere, whereas it was much weaker in the vegetal hemisphere. We conclude that fusion of GFP to wtα7 and mutα7 receptors provides powerful tools to study the distribution and function of α7 receptors. We also conclude that fused genes do not necessarily recapitulate all of the properties of the original receptors. This fact must be borne close in mind whenever reporter genes are attached to proteins. PMID:11891308

  7. Regulator of G-protein signaling Gβ5-R7 is a crucial activator of muscarinic M3 receptor-stimulated insulin secretion.

    Science.gov (United States)

    Wang, Qiang; Pronin, Alexey N; Levay, Konstantin; Almaca, Joana; Fornoni, Alessia; Caicedo, Alejandro; Slepak, Vladlen Z

    2017-11-01

    In pancreatic β cells, muscarinic cholinergic receptor M3 (M3R) stimulates glucose-induced secretion of insulin. Regulator of G-protein signaling (RGS) proteins are critical modulators of GPCR activity, yet their role in β cells remains largely unknown. R7 subfamily RGS proteins are stabilized by the G-protein subunit Gβ5, such that the knockout of the Gnb5 gene results in degradation of all R7 subunits. We found that Gnb5 knockout in mice or in the insulin-secreting MIN6 cell line almost completely eliminates insulinotropic activity of M3R. Moreover, overexpression of Gβ5-RGS7 strongly promotes M3R-stimulated insulin secretion. Examination of this noncanonical mechanism in Gnb5-/- MIN6 cells showed that cAMP, diacylglycerol, or Ca2+ levels were not significantly affected. There was no reduction in the amplitude of free Ca2+ responses in islets from the Gnb5-/- mice, but the frequency of Ca2+ oscillations induced by cholinergic agonist was lowered by more than 30%. Ablation of Gnb5 impaired M3R-stimulated phosphorylation of ERK1/2. Stimulation of the ERK pathway in Gnb5-/- cells by epidermal growth factor restored M3R-stimulated insulin release to near normal levels. Identification of the novel role of Gβ5-R7 in insulin secretion may lead to a new therapeutic approach for improving pancreatic β-cell function.-Wang, Q., Pronin, A. N., Levay, K., Almaca, J., Fornoni, A., Caicedo, A., Slepak, V. Z. Regulator of G-protein signaling Gβ5-R7 is a crucial activator of muscarinic M3 receptor-stimulated insulin secretion. © FASEB.

  8. C1q-tumour necrosis factor-related protein 8 (CTRP8) is a novel interaction partner of relaxin receptor RXFP1 in human brain cancer cells.

    Science.gov (United States)

    Glogowska, Aleksandra; Kunanuvat, Usakorn; Stetefeld, Jörg; Patel, Trushar R; Thanasupawat, Thatchawan; Krcek, Jerry; Weber, Ekkehard; Wong, G William; Del Bigio, Marc R; Hoang-Vu, Cuong; Hombach-Klonisch, Sabine; Klonisch, Thomas

    2013-12-01

    We report a novel ligand-receptor system composed of the leucine-rich G-protein-coupled relaxin receptor, RXFP1, and the C1q-tumour necrosis factor-related protein 8 (CTRP8) in human primary brain cancer, a tumour entity devoid of the classical RXFP1 ligands, RLN1-3. In structural homology studies and computational docking experiments we delineated the N-terminal region of the globular C1q region of CTRP8 and the leucine-rich repeat units 7 and 8 of RXFP1 to mediate this new ligand-receptor interaction. CTRP8 secreted from HEK293T cells, recombinant human (rh) CTRP8, and short synthetic peptides derived from the C1q globular domain of human CTRP8 caused the activation of RXFP1 as determined by elevated intracellular cAMP levels and the induction of a marked pro-migratory phenotype in established glioblastoma (GB) cell lines and primary cells from GB patients. Employing a small competitor peptide, we were able to disrupt the CTRP8-RXFP1-induced increased GB motility. The CTRP8-RXFP1-mediated migration in GB cells involves the activation of PI3K and specific protein kinase C pathways and the increased production/secretion of the potent lysosomal protease cathepsin B (cathB), a known prognostic marker of GB. Specific inhibition of CTRP8-induced cathB activity effectively blocked the ability of primary GB to invade laminin matrices. Finally, co-immunoprecipitation studies revealed the direct interaction of human CTRP8 with RXFP1. Our results support a therapeutic approach in GB aimed at targeting multiple steps of the CTRP8-RXFP1 signalling pathway by a combined inhibitor and peptide-based strategy to block GB dissemination within the brain. Copyright © 2013 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

  9. 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...

  10. Molecular basis for activation of G protein-coupled receptor kinases

    Energy Technology Data Exchange (ETDEWEB)

    Boguth, Cassandra A.; Singh, Puja; Huang, Chih-chin; Tesmer, John J.G. (Michigan)

    2012-03-16

    G protein-coupled receptor (GPCR) kinases (GRKs) selectively recognize and are allosterically regulated by activated GPCRs, but the molecular basis for this interaction is not understood. Herein, we report crystal structures of GRK6 in which regions known to be critical for receptor phosphorylation have coalesced to stabilize the kinase domain in a closed state and to form a likely receptor docking site. The crux of this docking site is an extended N-terminal helix that bridges the large and small lobes of the kinase domain and lies adjacent to a basic surface of the protein proposed to bind anionic phospholipids. Mutation of exposed, hydrophobic residues in the N-terminal helix selectively inhibits receptor, but not peptide phosphorylation, suggesting that these residues interact directly with GPCRs. Our structural and biochemical results thus provide an explanation for how receptor recognition, phospholipid binding, and kinase activation are intimately coupled in GRKs.

  11. Molecular evolution of a chordate specific family of G protein-coupled receptors

    Directory of Open Access Journals (Sweden)

    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

  12. The cAMP Response Element Binding protein (CREB) is activated by Insulin-like Growth Factor-1 (IGF-1) and regulates myostatin gene expression in skeletal myoblast

    Energy Technology Data Exchange (ETDEWEB)

    Zuloaga, R. [Facultad de Ciencias Biológicas, Universidad Andres Bello, Santiago (Chile); Fuentes, E.N.; Molina, A. [Facultad de Ciencias Biológicas, Universidad Andres Bello, Santiago (Chile); Interdisciplinary Center for Aquaculture Research (INCAR), Víctor Lamas 1290, PO Box 160-C, Concepción (Chile); Valdés, J.A., E-mail: jvaldes@unab.cl [Facultad de Ciencias Biológicas, Universidad Andres Bello, Santiago (Chile); Interdisciplinary Center for Aquaculture Research (INCAR), Víctor Lamas 1290, PO Box 160-C, Concepción (Chile)

    2013-10-18

    Highlights: •IGF-1 induces the activation of CREB via IGF-1R/PI3K/PLC signaling pathway. •Calcium dependent signaling pathways regulate myostatin gene expression. •IGF-1 regulates myostatin gene expression via CREB transcription in skeletal myoblast. -- Abstract: Myostatin, a member of the Transforming Growth Factor beta (TGF-β) superfamily, plays an important role as a negative regulator of skeletal muscle growth and differentiation. We have previously reported that IGF-1 induces a transient myostatin mRNA expression, through the activation of the Nuclear Factor of Activated T cells (NFAT) in an IP{sub 3}/calcium-dependent manner. Here we examined the activation of CREB transcription factor as downstream targets of IGF-1 during myoblast differentiation and its role as a regulator of myostatin gene expression. In cultured skeletal myoblast, IGF-1 induced the phosphorylation and transcriptional activation of CREB via IGF-1 Receptor/Phosphatidylinositol 3-Kinase (PI3K)/Phospholipase C gamma (PLC γ), signaling pathways. Also, IGF-1 induced calcium-dependent molecules such as Calmodulin Kinase II (CaMK II), Extracellular signal-regulated Kinases (ERK), Protein Kinase C (PKC). Additionally, we examined myostatin mRNA levels and myostatin promoter activity in differentiated myoblasts stimulated with IGF-1. We found a significant increase in mRNA contents of myostatin and its reporter activity after treatment with IGF-1. The expression of myostatin in differentiated myoblast was downregulated by the transfection of siRNA–CREB and by pharmacological inhibitors of the signaling pathways involved in CREB activation. By using pharmacological and genetic approaches together these data demonstrate that IGF-1 regulates the myostatin gene expression via CREB transcription factor during muscle cell differentiation.

  13. Registration Day-Camp 2016

    CERN Multimedia

    Nursery School

    2016-01-01

    Reminder Registration for the CERN Staff Association Day-camp are open for children from 4 to 6 years old More information on the website: http://nurseryschool.web.cern.ch/. The day-camp is open to all children. An inscription per week is proposed, cost 480.-CHF/week, lunch included The camp will be open weeks 27, 28, 29 and 30, from 8:30 am to 5:30 pm. For further questions, thanks you for contacting us by email at Summer.Camp@cern.ch.

  14. G protein-coupled receptors self-assemble in dynamics simulations of model bilayers

    NARCIS (Netherlands)

    Periole, Xavier; Huber, Thomas; Marrink, Siewert-Jan; Sakmar, Thomas P.

    2007-01-01

    Many integral membrane proteins assemble to form oligomeric structures in biological membranes. In particular, seven-transmembrane helical G protein-coupled receptors (GPCRs) appear to self-assemble constitutively in membranes, but the mechanism and physiological role of this assembly are unknown.

  15. The c-erb-A protein is a high-affinity receptor for thyroid hormone

    DEFF Research Database (Denmark)

    Sap, J; Muñoz, A; Damm, K

    1987-01-01

    Hormone binding and localization of the c-erb-A protein suggest that it is a receptor for thyroid hormone, a nuclear protein that binds to DNA and activates transcription. In contrast, the product of the viral oncogene v-erb-A is defective in binding the hormone but is still located in the nucleus....

  16. The Arabidopsis kinase-associated protein phosphatase controls internalization of the somatic embryogenesis receptor kinase 1

    NARCIS (Netherlands)

    Shah, K.; Russinova, E.; Gadella, T.W.J.; Willemse, J.; Vries, de S.C.

    2002-01-01

    The AtSERK1 protein is a plasma membrane-located LRR receptor-like serine threonine kinase that is transiently expressed during plant embryogenesis. Our results show that AtSERK1 interacts with the kinase-associated protein phosphatase (KAPP) in vitro. The kinase interaction (KI) domain of KAPP does

  17. Factor VIII interacts with the endocytic receptor low-density lipoprotein receptor-related protein 1 via an extended surface comprising "hot-spot" lysine residues

    NARCIS (Netherlands)

    Van Den Biggelaar, Maartje|info:eu-repo/dai/nl/304831433; Madsen, Jesper J.; Faber, Johan H.; Zuurveld, Marleen G.; Van Der Zwaan, Carmen; Olsen, Ole H.; Stennicke, Henning R.; Mertens, Koen|info:eu-repo/dai/nl/070940258; Meijer, Alexander B.

    2015-01-01

    Background: It is unclear how the LDL receptor family binds large protein ligands. Results: HDX and lysine scanning identified factor (F)VIII regions and specific lysine residues binding low-density lipoprotein receptor-related protein 1 (LRP1). Conclusion: FVIII-LRP1 interaction involves multiple

  18. Ligand-specific regulation of the extracellular surface of a G-protein-coupled receptor

    DEFF Research Database (Denmark)

    Bokoch, Michael P; Zou, Yaozhong; Rasmussen, Søren Gøgsig Faarup

    2010-01-01

    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...... 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...

  19. Species difference in the G protein selectivity of the human and bovine A1-adenosine receptor.

    Science.gov (United States)

    Jockers, R; Linder, M E; Hohenegger, M; Nanoff, C; Bertin, B; Strosberg, A D; Marullo, S; Freissmuth, M

    1994-12-23

    The purified bovine brain A1-adenosine receptor has previously been shown to discriminate among closely related G protein alpha-subunits. To obtain analogous information for the human receptor, the cDNA coding for the human A1-adenosine receptor was inserted into a plasmid placing the synthesis of the receptor protein under the control of the MalE promoter. Following induction by maltose, active receptor accumulated in Escherichia coli membranes. Binding of the antagonist 8-[3H]cyclopentyl-1,3-dipropylxanthine to E. coli membranes (KD approximately 2 nM, Bmax approximately 0.2-0.4 pmol/mg) showed the appropriate pharmacological profile. Incubation of E. coli membranes with purified Go,i-reconstituted guanine nucleotide-sensitive high affinity binding of the agonist (-)[125I] N6-3-(iodo-4-hydroxyphenylisopropyl)adenosine to the receptor (KD approximately 1 nM). In the presence of purified beta gamma-subunit, the recombinant receptor interacted equally well with the recombinant G protein alpha-subunits Gi alpha-1, Gi alpha-2, Gi alpha-3; G(o) alpha displayed a lower affinity for the receptor while Gs alpha was inactive. Parallel experiments were carried out in bovine and human brain membranes pretreated with N-ethylmaleimide to inactivate the endogenous G(o)/Gi proteins; Gi alpha-3 was most potent in reconstituting 125I-HPIA binding to bovine membranes, while Gi alpha-1, Gi alpha-2, and G(o) alpha displayed similar affinities. However, in human membranes, Gi alpha-1, Gi alpha-2, and Gi alpha-3, were equipotent and high concentrations of G(o) alpha were required to promote 125I-HPIA binding. These observations show (i) that functional human A1-adenosine receptors were synthesized in E. coli; (ii) that the pattern of G protein coupling is identical for the recombinant human A1-receptor and its counterpart in the native membrane; (iii) and that species differences between bovine and human receptor exist not only in their pharmacological profile but also in their G

  20. Functional cyclic AMP response element in the breast cancer resistance protein (BCRP/ABCG2) promoter modulates epidermal growth factor receptor pathway- or androgen withdrawal-mediated BCRP/ABCG2 transcription in human cancer cells.

    Science.gov (United States)

    Xie, Yi; Nakanishi, Takeo; Natarajan, Karthika; Safren, Lowell; Hamburger, Anne W; Hussain, Arif; Ross, Douglas D

    2015-03-01

    Phosphorylated cyclic-AMP (cAMP) response element binding protein (p-CREB) is a downstream effector of a variety of important signaling pathways. We investigated whether the human BCRP promoter contains a functional cAMP response element (CRE). 8Br-cAMP, a cAMP analogue, increased the activity of a BCRP promoter reporter construct and BCRP mRNA in human carcinoma cells. Epidermal growth factor receptor (EGFR) pathway activation also led to an increase in p-CREB and in BCRP promoter reporter activity via two major downstream EGFR signaling pathways: the phosphotidylinositol-3-kinase (PI3K)/AKT pathway and the mitogen-activated protein kinase (MAPK) pathway. EGF treatment increased the phosphorylation of EGFR, AKT, ERK and CREB, while simultaneously enhancing BCRP mRNA and functional protein expression. EGF-stimulated CREB phosphorylation and BCRP induction were diminished by inhibition of EGFR, PI3K/AKT or RAS/MAPK signaling. CREB silencing using RNA interference reduced basal levels of BCRP mRNA and diminished the induction of BCRP by EGF. Chromatin immunoprecipitation assays confirmed that a putative CRE site on the BCRP promoter bound p-CREB by a point mutation of the CRE site abolished EGF-induced stimulation of BCRP promoter reporter activity. Furthermore, the CREB co-activator, cAMP-regulated transcriptional co-activator (CRTC2), is involved in CREB-mediated BCRP transcription: androgen depletion of LNCaP human prostate cancer cells increased both CREB phosphorylation and CRTC2 nuclear translocation, and enhanced BCRP expression. Silencing CREB or CRTC2 reduced basal BCRP expression and BCRP induction under androgen-depletion conditions. This novel CRE site plays a central role in mediating BCRP gene expression in several human cancer cell lines following activation of multiple cancer-relevant signaling pathways. Published by Elsevier B.V.

  1. The role of receptor-like proteins in Arabidopsis development

    NARCIS (Netherlands)

    Wang, G.

    2009-01-01

    An intriguing and long-standing question in developmental biology is how plant cells communicate with each other and sense signals from their surrounding environment. Through research over past decades, it became clear that plant cells use membrane-localized receptors to perceive signals from their

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

    DEFF Research Database (Denmark)

    Steen, Anne; Larsen, Olav; Thiele, Stefanie

    2014-01-01

    a single chemokine may bind to several receptors - in both cases with the same functional outcome. The ubiquitous biased signaling confers a hitherto unknown specificity to the chemokine system with a complex interaction pattern that is better described as promiscuous with context-defined roles...

  3. Membrane cholesterol access into a G-protein-coupled receptor

    Czech Academy of Sciences Publication Activity Database

    Guixa-González, R.; Albasanz, J. L.; Rodriguez-Espigares, I.; Pastor, M.; Sanz, F.; Martí-Solano, M.; Manna, M.; Martinez-Seara, Hector; Hildebrand, P. W.; Martín, M.; Selent, J.

    2017-01-01

    Roč. 8, Feb 21 (2017), č. článku 14505. ISSN 2041-1723 Institutional support: RVO:61388963 Keywords : postmortem orbitofrontal cortex * A(2A) adenosine receptor * molecular dynamics Subject RIV: CF - Physical ; Theoretical Chemistry Impact factor: 12.124, year: 2016 https://www.nature.com/articles/ncomms14505

  4. No ordinary boot camp.

    Science.gov (United States)

    Tichy, N M

    2001-04-01

    Many companies now run boot camps--comprehensive orientation programs designed to help new hires hit the ground running. They're intense and intimidating, and new employees emerge from them with strong bonds to other recruits and to the organization. But at Trilogy, organizational consultant Noel Tichy discovered one program that's a breed apart. In this article, Tichy gives us a detailed tour of Trilogy's boot camp, Trilogy University, to demonstrate why it's so different--and so effective. Like the best boot camps, it serves as an immersion in both the technical skills new recruits will need for their jobs and Trilogy's corporate culture, which emphasizes risk-taking, teamwork, humility, and a strong customer focus. But this is a new-employee orientation session that's so fundamental to the company as a whole that it's presided over by the CEO and top corporate executives for fully six months of the year. Why? In two three-month sessions, these top executives hone their own strategic thinking about the company as they decide what to teach the new recruits each session. They also find the company's next generation of new products as they judge the innovative ideas the recruits are tasked with developing--making the program Trilogy's main R&D engine. And they pull the company's rising technical stars into mentoring roles for the new recruits, helping to build the next generation of top leadership. After spending months on-site studying Trilogy University, Tichy came away highly impressed by the power of the virtuous teaching cycle the program has set in motion. Leaders of the organization are learning from recruits at the same time that the recruits are learning from the leaders. It's a model, he argues, that other companies would do well to emulate.

  5. The role of insulin receptor substrate (IRS) proteins in oncogenic transformation.

    Science.gov (United States)

    Gorgisen, G; Gulacar, I M; Ozes, O N

    2017-01-30

    Insulin Receptor Substrate (IRS) proteins are the main cytoplasmic adaptor molecules involved in transducing extracellular signals from receptors to downstream proteins. This protein family have pivotal roles on maintenance, distribution and regulation of signaling networks. Since IRS1/2 interact with and transmits signals from the receptors of insulin, Insulin Like Growth Factor 1 (IGF1), prolactin, growth hormone (GH), leptin, Vascular Endothelial Growth Factor (VEGF), TrkB, ALK and integrins this promoted scientist to think that IRS1 may have functions in cell proliferation, tumorigenesis and metastasis. Therefore, over the past decade, studies on IRS proteins and their functions in cancer has been increased and these studies provided valuable results claiming the involvement of IRS1/2 in cancer development. In this review, we discuss the function and contributions of IRS1 and IRS2 in development of  breast cancer.

  6. The significance of G protein-coupled receptor crystallography for drug discovery.

    Science.gov (United States)

    Salon, John A; Lodowski, David T; Palczewski, Krzysztof

    2011-12-01

    Crucial as molecular sensors for many vital physiological processes, seven-transmembrane domain G protein-coupled receptors (GPCRs) comprise the largest family of proteins targeted by drug discovery. Together with structures of the prototypical GPCR rhodopsin, solved structures of other liganded GPCRs promise to provide insights into the structural basis of the superfamily's biochemical functions and assist in the development of new therapeutic modalities and drugs. One of the greatest technical and theoretical challenges to elucidating and exploiting structure-function relationships in these systems is the emerging concept of GPCR conformational flexibility and its cause-effect relationship for receptor-receptor and receptor-effector interactions. Such conformational changes can be subtle and triggered by relatively small binding energy effects, leading to full or partial efficacy in the activation or inactivation of the receptor system at large. Pharmacological dogma generally dictates that these changes manifest themselves through kinetic modulation of the receptor's G protein partners. Atomic resolution information derived from increasingly available receptor structures provides an entrée to the understanding of these events and practically applying it to drug design. Supported by structure-activity relationship information arising from empirical screening, a unified structural model of GPCR activation/inactivation promises to both accelerate drug discovery in this field and improve our fundamental understanding of structure-based drug design in general. This review discusses fundamental problems that persist in drug design and GPCR structural determination.

  7. A New Molecular Mechanism To Engineer Protean Agonism at a G Protein-Coupled Receptor.

    Science.gov (United States)

    De Min, Anna; Matera, Carlo; Bock, Andreas; Holze, Janine; Kloeckner, Jessica; Muth, Mathias; Traenkle, Christian; De Amici, Marco; Kenakin, Terry; Holzgrabe, Ulrike; Dallanoce, Clelia; Kostenis, Evi; Mohr, Klaus; Schrage, Ramona

    2017-04-01

    Protean agonists are of great pharmacological interest as their behavior may change in magnitude and direction depending on the constitutive activity of a receptor. Yet, this intriguing phenomenon has been poorly described and understood, due to the lack of stable experimental systems and design strategies. In this study, we overcome both limitations: First, we demonstrate that modulation of the ionic strength in a defined experimental set-up allows for analysis of G protein-coupled receptor activation in the absence and presence of a specific amount of spontaneous receptor activity using the muscarinic M2 acetylcholine receptor as a model. Second, we employ this assay system to show that a dualsteric design principle, that is, molecular probes, carrying two pharmacophores to simultaneously adopt orthosteric and allosteric topography within a G protein-coupled receptor, may represent a novel approach to achieve protean agonism. We pinpoint three molecular requirements within dualsteric compounds that elicit protean agonism at the muscarinic M2 acetylcholine receptor. Using radioligand-binding and functional assays, we posit that dynamic ligand binding may be the mechanism underlying protean agonism of dualsteric ligands. Our findings provide both new mechanistic insights into the still enigmatic phenomenon of protean agonism and a rationale for the design of such compounds for a G protein-coupled receptor. Copyright © 2017 by The American Society for Pharmacology and Experimental Therapeutics.

  8. Delayed toxicity associated with soluble anthrax toxin receptor decoy-Ig fusion protein treatment.

    Directory of Open Access Journals (Sweden)

    Diane Thomas

    Full Text Available Soluble receptor decoy inhibitors, including receptor-immunogloubulin (Ig fusion proteins, have shown promise as candidate anthrax toxin therapeutics. These agents act by binding to the receptor-interaction site on the protective antigen (PA toxin subunit, thereby blocking toxin binding to cell surface receptors. Here we have made the surprising observation that co-administration of receptor decoy-Ig fusion proteins significantly delayed, but did not protect, rats challenged with anthrax lethal toxin. The delayed toxicity was associated with the in vivo assembly of a long-lived complex comprised of anthrax lethal toxin and the receptor decoy-Ig inhibitor. Intoxication in this system presumably results from the slow dissociation of the toxin complex from the inhibitor following their prolonged circulation. We conclude that while receptor decoy-Ig proteins represent promising candidates for the early treatment of B. anthracis infection, they may not be suitable for therapeutic use at later stages when fatal levels of toxin have already accumulated in the bloodstream.

  9. Pharmacological prion protein silencing accelerates central nervous system autoimmune disease via T cell receptor signalling.

    Science.gov (United States)

    Hu, Wei; Nessler, Stefan; Hemmer, Bernhard; Eagar, Todd N; Kane, Lawrence P; Leliveld, S Rutger; Müller-Schiffmann, Andreas; Gocke, Anne R; Lovett-Racke, Amy; Ben, Li-Hong; Hussain, Rehana Z; Breil, Andreas; Elliott, Jeffrey L; Puttaparthi, Krishna; Cravens, Petra D; Singh, Mahendra P; Petsch, Benjamin; Stitz, Lothar; Racke, Michael K; Korth, Carsten; Stüve, Olaf

    2010-02-01

    The primary biological function of the endogenous cellular prion protein has remained unclear. We investigated its biological function in the generation of cellular immune responses using cellular prion protein gene-specific small interfering ribonucleic acid in vivo and in vitro. Our results were confirmed by blocking cellular prion protein with monovalent antibodies and by using cellular prion protein-deficient and -transgenic mice. In vivo prion protein gene-small interfering ribonucleic acid treatment effects were of limited duration, restricted to secondary lymphoid organs and resulted in a 70% reduction of cellular prion protein expression in leukocytes. Disruption of cellular prion protein signalling augmented antigen-specific activation and proliferation, and enhanced T cell receptor signalling, resulting in zeta-chain-associated protein-70 phosphorylation and nuclear factor of activated T cells/activator protein 1 transcriptional activity. In vivo prion protein gene-small interfering ribonucleic acid treatment promoted T cell differentiation towards pro-inflammatory phenotypes and increased survival of antigen-specific T cells. Cellular prion protein silencing with small interfering ribonucleic acid also resulted in the worsening of actively induced and adoptively transferred experimental autoimmune encephalomyelitis. Finally, treatment of myelin basic protein(1-11) T cell receptor transgenic mice with prion protein gene-small interfering ribonucleic acid resulted in spontaneous experimental autoimmune encephalomyelitis. Thus, central nervous system autoimmune disease was modulated at all stages of disease: the generation of the T cell effector response, the elicitation of T effector function and the perpetuation of cellular immune responses. Our findings indicate that cellular prion protein regulates T cell receptor-mediated T cell activation, differentiation and survival. Defects in autoimmunity are restricted to the immune system and not the central

  10. Marlin-1, a novel RNA-binding protein associates with GABA receptors.

    Science.gov (United States)

    Couve, Andrés; Restituito, Sophie; Brandon, Julia M; Charles, Kelly J; Bawagan, Hinayana; Freeman, Katie B; Pangalos, Menelas N; Calver, Andrew R; Moss, Stephen J

    2004-04-02

    GABA(B) receptors are heterodimeric G protein-coupled receptors that mediate slow synaptic inhibition in the central nervous system. Whereas heterodimerization between GABA(B) receptor GABA(B)R1 and GABA(B)R2 subunits is essential for functional expression, how neurons coordinate the assembly of these critical receptors remains to be established. Here we have identified Marlin-1, a novel GABA(B) receptor-binding protein that associates specifically with the GABA(B)R1 subunit in yeast, tissue culture cells, and neurons. Marlin-1 is expressed in the brain and exhibits a granular distribution in cultured hippocampal neurons. Marlin-1 binds different RNA species including the 3'-untranslated regions of both the GABA(B)R1 and GABA(B)R2 mRNAs in vitro and also associates with RNA in cultured neurons. Inhibition of Marlin-1 expression via small RNA interference technology results in enhanced intracellular levels of the GABA(B)R2 receptor subunit without affecting the level of GABA(B)R1. Together our results suggest that Marlin-1 functions to regulate the cellular levels of GABA(B) R2 subunits, which may have significant effects on the production of functional GABA(B) receptor heterodimers. Therefore, our observations provide an added level of regulation for the control of GABA(B) receptor expression and for the efficacy of inhibitory synaptic transmission.

  11. Peripheral-type benzodiazepine receptor: a protein of mitochondrial outer membranes utilizing porphyrins as endogenous ligands

    Energy Technology Data Exchange (ETDEWEB)

    Snyder, S.H.; Verma, A.; Trifiletti, R.R.

    1987-10-01

    The peripheral-type benzodiazepine receptor is a site identified by its nanomolar affinity for (/sup 3/H)diazepam, similar to the affinity of diazepam for the central-type benzodiazepine receptor in the brain. The peripheral type benzodiazepine receptor occurs in many peripheral tissues but has discrete localizations as indicated by autoradiographic studies showing uniquely high densities of the receptors in the adrenal cortex and in Leydig cells of the testes. Subcellular localization studies reveal a selective association of the receptors with the outer membrane of mitochondria. Photoaffinity labeling of the mitochondrial receptor with (/sup 3/H)flunitrazepam reveals two discrete labeled protein bands of 30 and 35 kDa, respectively. The 35-kDa band appears to be identical with the voltage-dependent anion channel protein porin. Fractionation of numerous peripheral tissues reveals a single principal endogenous ligand for the receptor, consisting of porphyrins, which display nanomolar affinity. Interactions of porphyrins with the mitochondrial receptor may clarify its physiological role and account for many pharmacological actions of benzodiazepines.

  12. Noradrenaline represses PPAR (peroxisome-proliferator-activated receptor) γ2 gene expression in brown adipocytes: intracellular signalling and effects on PPARγ2 and PPARγ1 protein levels

    Science.gov (United States)

    Lindgren, Eva M.; Nielsen, Ronni; Petrovic, Natasa; Jacobsson, Anders; Mandrup, Susanne; Cannon, Barbara; Nedergaard, Jan

    2004-01-01

    PPAR (peroxisome-proliferator-activated receptor) γ is expressed in brown and white adipose tissues and is involved in the control of differentiation and proliferation. Noradrenaline stimulates brown pre-adipocyte proliferation and brown adipocyte differentiation. The aim of the present study was thus to investigate the influence of noradrenaline on PPARγ gene expression in brown adipocytes. In primary cultures of brown adipocytes, PPARγ2 mRNA levels were 20-fold higher than PPARγ1 mRNA levels. PPARγ expression occurred during both the proliferation and the differentiation phases, with the highest mRNA levels being found at the time of transition between the phases. PPARγ2 mRNA levels were downregulated by noradrenaline treatment (EC50, 0.1 μM) in both proliferative and differentiating cells, with a lagtime of 1 h and lasting up to 4 h, after which expression gradually recovered. The down-regulation was β-adrenoceptor-induced and intracellularly mediated via cAMP and protein kinase A; the signalling pathway did not involve phosphoinositide 3-kinase, Src, p38 mitogen-activated protein kinase or extracellular-signal-regulated kinases 1 and 2. Treatment of the cells with the protein synthesis inhibitor cycloheximide not only abolished the noradrenaline-induced down-regulation of PPARγ2 mRNA, but also in itself induced PPARγ2 hyperexpression. The down-regulation was probably the result of suppression of transcription. The down-regulation of PPARγ2 mRNA resulted in similar down-regulation of PPARγ2 and phosphoPPARγ2 protein levels. Remarkably, the level of PPARγ1 protein was similar to that of PPARγ2 (despite almost no PPARγ1 mRNA), and the down-regulation by noradrenaline demonstrated similar kinetics to that of PPARγ2; thus PPARγ1 was apparently translated from the PPARγ2 template. It is suggested that β-adrenergic stimulation via cAMP and protein kinase A represses PPARγ gene expression, leading to reduction of PPARγ2 mRNA levels, which is

  13. Rapid activation of inwardly rectifying potassium channels by immobile G-protein-coupled receptors.

    Science.gov (United States)

    Lober, Robert M; Pereira, Miguel A; Lambert, Nevin A

    2006-11-29

    G-protein-coupled receptors (GPCRs) mediate slow synaptic transmission and many other effects of small molecule and peptide neurotransmitters. In the standard model of GPCR signaling, receptors and G-proteins diffuse laterally within the plane of the plasma membrane and encounter each other by random collision. This model predicts that signaling will be most efficient if both GPCRs and G-proteins are free to diffuse, thus maximizing collision frequency. However, neuronal GPCRs are often recruited to and enriched at specific synaptic locations, suggesting receptor mobility is restricted in these cells. Here, we test the hypothesis that restricting GPCR mobility impairs signaling in neurons by limiting the frequency of collisions between receptors and G-proteins. Mu-opioid receptors (MORs) were immobilized on the surface of cerebellar granule neurons by avidin-mediated cross-linking, and inwardly rectifying potassium (GIRK) channels were used as rapid indicators of G-protein activation. Mobile and immobile MORs activated GIRK channels with the same onset kinetics and agonist sensitivity in these neurons. In a heterologous expression system, GFP (green fluorescent protein)-tagged G alpha(oA) subunits remained mobile after cross-linking, but their mobility was reduced in the presence of immobile MORs, suggesting that these receptors and subunits were transiently precoupled. In addition, channel activation could be reconstituted with immobile GPCRs, G-protein heterotrimers, and GIRK channels. These results show that collision frequency is not rate-limiting for G-protein activation in CNS neurons, and are consistent with the idea that signaling components are compartmentalized or preassembled.

  14. DEVELOPMENT OF PHARMACOLOGICAL TOOLS FOR THE IDENTIFICATION OF G PROTEIN-COUPLED RECEPTORS LIGANDS

    OpenAIRE

    Gilissen, Julie

    2016-01-01

    G protein-coupled receptors (GPCRs) represent the protein family most successfully targeted for treating human diseases. They couple to G proteins to mobilize second messenger pathways that lead to cellular responses and ultimately to physiological changes. However many are poorly characterized with few ligands reported or remain completely orphans. Therefore, there is a growing need for screening-compatible and sensitive assays in order to identify new ligands. The present project aims at...

  15. On the G-Protein-Coupled Receptor Heteromers and Their Allosteric Receptor-Receptor Interactions in the Central Nervous System: Focus on Their Role in Pain Modulation

    Directory of Open Access Journals (Sweden)

    Dasiel O. Borroto-Escuela

    2013-01-01

    Full Text Available The modulatory role of allosteric receptor-receptor interactions in the pain pathways of the Central Nervous System and the peripheral nociceptors has become of increasing interest. As integrators of nociceptive and antinociceptive wiring and volume transmission signals, with a major role for the opioid receptor heteromers, they likely have an important role in the pain circuits and may be involved in acupuncture. The delta opioid receptor (DOR exerts an antagonistic allosteric influence on the mu opioid receptor (MOR function in a MOR-DOR heteromer. This heteromer contributes to morphine-induced tolerance and dependence, since it becomes abundant and develops a reduced G-protein-coupling with reduced signaling mainly operating via β-arrestin2 upon chronic morphine treatment. A DOR antagonist causes a return of the Gi/o binding and coupling to the heteromer and the biological actions of morphine. The gender- and ovarian steroid-dependent recruitment of spinal cord MOR/kappa opioid receptor (KOR heterodimers enhances antinociceptive functions and if impaired could contribute to chronic pain states in women. MOR1D heterodimerizes with gastrin-releasing peptide receptor (GRPR in the spinal cord, mediating morphine induced itch. Other mechanism for the antinociceptive actions of acupuncture along meridians may be that it enhances the cross-desensitization of the TRPA1 (chemical nociceptor-TRPV1 (capsaicin receptor heteromeric channel complexes within the nociceptor terminals located along these meridians. Selective ionotropic cannabinoids may also produce cross-desensitization of the TRPA1-TRPV1 heteromeric nociceptor channels by being negative allosteric modulators of these channels leading to antinociception and antihyperalgesia.

  16. LDL receptor-GFP fusion proteins: new tools for the characterization of disease-causing mutations in the LDL receptor gene

    DEFF Research Database (Denmark)

    Holst, Henrik Uffe; Dagnæs-Hansen, Frederik; Corydon, Thomas Juhl

    2001-01-01

    The function of a series of LDL receptor GFP fusion proteins with different, flexible, unstructured spacer regions was analysed. An optimised version of the fusion protein was used to analyse the effect of a LDL receptor mutation (W556S) found in FH patients and characterized as transport defective....... In cultured liver cells this mutation was found to inhibit the transport of LDL receptor GFP fusion protein to the cell surface, thus leading to impaired internalisation of fluorescent labelled LDL. Co-locallisation studies confirmed the retention of the mutant protein in the endoplasmic reticulum....

  17. Lactoferrin binding protein B - a bi-functional bacterial receptor protein.

    Directory of Open Access Journals (Sweden)

    Nicholas K H Ostan

    2017-03-01

    Full Text Available Lactoferrin binding protein B (LbpB is a bi-lobed outer membrane-bound lipoprotein that comprises part of the lactoferrin (Lf receptor complex in Neisseria meningitidis and other Gram-negative pathogens. Recent studies have demonstrated that LbpB plays a role in protecting the bacteria from cationic antimicrobial peptides due to large regions rich in anionic residues in the C-terminal lobe. Relative to its homolog, transferrin-binding protein B (TbpB, there currently is little evidence for its role in iron acquisition and relatively little structural and biophysical information on its interaction with Lf. In this study, a combination of crosslinking and deuterium exchange coupled to mass spectrometry, information-driven computational docking, bio-layer interferometry, and site-directed mutagenesis was used to probe LbpB:hLf complexes. The formation of a 1:1 complex of iron-loaded Lf and LbpB involves an interaction between the Lf C-lobe and LbpB N-lobe, comparable to TbpB, consistent with a potential role in iron acquisition. The Lf N-lobe is also capable of binding to negatively charged regions of the LbpB C-lobe and possibly other sites such that a variety of higher order complexes are formed. Our results are consistent with LbpB serving dual roles focused primarily on iron acquisition when exposed to limited levels of iron-loaded Lf on the mucosal surface and effectively binding apo Lf when exposed to high levels at sites of inflammation.

  18. Summer Camp, July 2016

    CERN Multimedia

    Staff Association

    2016-01-01

    During the month of July, the Staff Association’s Children’s Day-Care Centre and School EVEE held a summer camp for 4- to 6-year-olds. 24 children altogether joined in on the adventures. On the summer camp, the children got to “travel” to a different continent of the world every week. Day after day, they would pass through make-believe Customs upon arrival and get their passports stamped by a “customs officer”. For the first week, we went on a trip to Africa. In the spirit of the theme, the children got to do plenty of crafts and coloring, make their own little bindles and play various games. They even had the chance to visit the Museum of Ethnography in Geneva (MEG), learn to play the balafon and make musical instruments with Sterrenlab. For the second week, we set off to discover the Americas, exploring both the South and the North. Alongside different workshops (singing, dancing, storytelling, crafts), the children could enjoy several special ac...

  19. Streptococcus pyogenes CAMP factor attenuates phagocytic activity of RAW 264.7 cells.

    Science.gov (United States)

    Kurosawa, Mie; Oda, Masataka; Domon, Hisanori; Saitoh, Issei; Hayasaki, Haruaki; Terao, Yutaka

    2016-02-01

    Streptococcus pyogenes produces molecules that inhibit the function of human immune system, thus allowing the pathogen to grow and spread in tissues. It is known that S. pyogenes CAMP factor increases erythrocytosis induced by Staphylococcus aureus β-hemolysin. However, the effects of CAMP factor for immune cells are unclear. In this study, we investigated the effects of CAMP factor to macrophages. Western blotting analysis demonstrated that all examined strains expressed CAMP factor protein. In the presence of calcium or magnesium ion, CAMP factor was significantly released in the supernatant. In addition, both culture supernatant from S. pyogenes strain SSI-9 and recombinant CAMP factor dose-dependently induced vacuolation in RAW 264.7 cells, but the culture supernatant from Δcfa isogenic mutant strain did not. CAMP factor formed oligomers in RAW 264.7 cells in a time-dependent manner. CAMP factor suppressed cell proliferation via G2 phase cell cycle arrest without inducing cell death. Furthermore, CAMP factor reduced the uptake of S. pyogenes and phagocytic activity indicator by RAW 264.7 cells. These results suggest that CAMP factor works as a macrophage dysfunction factor. Therefore, we conclude that CAMP factor allows S. pyogenes to escape the host immune system, and contribute to the spread of streptococcal infection. Copyright © 2015 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.

  20. GPR99, a new G protein-coupled receptor with homology to a new subgroup of nucleotide receptors

    Directory of Open Access Journals (Sweden)

    Chica Schaller H

    2002-07-01

    Full Text Available Abstract Background Based on sequence similarity, the superfamily of G protein-coupled receptors (GPRs can be subdivided into several subfamilies, the members of which often share similar ligands. The sequence data provided by the human genome project allows us to identify new GPRs by in silico homology screening, and to predict their ligands. Results By searching the human genomic database with known nucleotide receptors we discovered the gene for GPR99, a new orphan GPR. The mRNA of GPR99 was found in kidney and placenta. Phylogenetic analysis groups GPR99 into the P2Y subfamily of GPRs. Based on the phylogenetic tree we propose a new classification of P2Y nucleotide receptors into two subgroups predicting a nucleotide ligand for GPR99. By assaying known nucleotide ligands on heterologously expressed GPR99, we could not identify specifically activating substances, indicating that either they are not agonists of GPR99 or that GPR99 was not expressed at the cell surface. Analysis of the chromosomal localization of all genes of the P2Y subfamily revealed that all members of subgroup "a" are encoded by less than 370 kb on chromosome 3q24, and that the genes of subgroup "b" are clustered on one hand to chromosome 11q13.5 and on the other on chromosome 3q24-25.1 close to the subgroup "a" position. Therefore, the P2Y subfamily is a striking example for local gene amplification. Conclusions We identified a new orphan receptor, GPR99, with homology to the family of G protein-coupled nucleotide receptors. Phylogenetic analysis separates this family into different subgroups predicting a nucleotide ligand for GPR99.

  1. REEPs are membrane shaping adapter proteins that modulate specific g protein-coupled receptor trafficking by affecting ER cargo capacity.

    Directory of Open Access Journals (Sweden)

    Susann Björk

    Full Text Available Receptor expression enhancing proteins (REEPs were identified by their ability to enhance cell surface expression of a subset of G protein-coupled receptors (GPCRs, specifically GPCRs that have proven difficult to express in heterologous cell systems. Further analysis revealed that they belong to the Yip (Ypt-interacting protein family and that some REEP subtypes affect ER structure. Yip family comparisons have established other potential roles for REEPs, including regulation of ER-Golgi transport and processing/neuronal localization of cargo proteins. However, these other potential REEP functions and the mechanism by which they selectively enhance GPCR cell surface expression have not been clarified. By utilizing several REEP family members (REEP1, REEP2, and REEP6 and model GPCRs (α2A and α2C adrenergic receptors, we examined REEP regulation of GPCR plasma membrane expression, intracellular processing, and trafficking. Using a combination of immunolocalization and biochemical methods, we demonstrated that this REEP subset is localized primarily to ER, but not plasma membranes. Single cell analysis demonstrated that these REEPs do not specifically enhance surface expression of all GPCRs, but affect ER cargo capacity of specific GPCRs and thus their surface expression. REEP co-expression with α2 adrenergic receptors (ARs revealed that this REEP subset interacts with and alter glycosidic processing of α2C, but not α2A ARs, demonstrating selective interaction with cargo proteins. Specifically, these REEPs enhanced expression of and interacted with minimally/non-glycosylated forms of α2C ARs. Most importantly, expression of a mutant REEP1 allele (hereditary spastic paraplegia SPG31 lacking the carboxyl terminus led to loss of this interaction. Thus specific REEP isoforms have additional intracellular functions besides altering ER structure, such as enhancing ER cargo capacity, regulating ER-Golgi processing, and interacting with select cargo

  2. REEPs are membrane shaping adapter proteins that modulate specific g protein-coupled receptor trafficking by affecting ER cargo capacity.

    Science.gov (United States)

    Björk, Susann; Hurt, Carl M; Ho, Vincent K; Angelotti, Timothy

    2013-01-01

    Receptor expression enhancing proteins (REEPs) were identified by their ability to enhance cell surface expression of a subset of G protein-coupled receptors (GPCRs), specifically GPCRs that have proven difficult to express in heterologous cell systems. Further analysis revealed that they belong to the Yip (Ypt-interacting protein) family and that some REEP subtypes affect ER structure. Yip family comparisons have established other potential roles for REEPs, including regulation of ER-Golgi transport and processing/neuronal localization of cargo proteins. However, these other potential REEP functions and the mechanism by which they selectively enhance GPCR cell surface expression have not been clarified. By utilizing several REEP family members (REEP1, REEP2, and REEP6) and model GPCRs (α2A and α2C adrenergic receptors), we examined REEP regulation of GPCR plasma membrane expression, intracellular processing, and trafficking. Using a combination of immunolocalization and biochemical methods, we demonstrated that this REEP subset is localized primarily to ER, but not plasma membranes. Single cell analysis demonstrated that these REEPs do not specifically enhance surface expression of all GPCRs, but affect ER cargo capacity of specific GPCRs and thus their surface expression. REEP co-expression with α2 adrenergic receptors (ARs) revealed that this REEP subset interacts with and alter glycosidic processing of α2C, but not α2A ARs, demonstrating selective interaction with cargo proteins. Specifically, these REEPs enhanced expression of and interacted with minimally/non-glycosylated forms of α2C ARs. Most importantly, expression of a mutant REEP1 allele (hereditary spastic paraplegia SPG31) lacking the carboxyl terminus led to loss of this interaction. Thus specific REEP isoforms have additional intracellular functions besides altering ER structure, such as enhancing ER cargo capacity, regulating ER-Golgi processing, and interacting with select cargo proteins

  3. G protein-coupled receptor signaling complexity in neuronal tissue: implications for novel therapeutics.

    Science.gov (United States)

    Maudsley, Stuart; Martin, Bronwen; Luttrell, Louis M

    2007-02-01

    The manipulation of transmembrane signaling by G protein-coupled receptors (GPCRs) constitutes perhaps the single most important therapeutic target in medicine. Therapeutics acting on GPCRs have traditionally been classified as agonists, partial agonists, or antagonists based on a two state model of receptor function embodied in the ternary complex model. Over the past decade, however, many lines of investigation have shown that GPCR signaling exhibits greater diversity and 'texture' than previously appreciated. Signal diversity arises from numerous factors, among them the ability of receptors to adopt multiple 'active' states with different effector coupling profiles, the formation of receptor dimers that exhibit unique pharmacology, signaling, and trafficking, the dissociation of receptor 'activation' from desensitization and internalization, and the discovery that non-G protein effectors mediate some aspects of GPCR signaling. At the same time, clustering of GPCRs with their downstream effectors in membrane microdomains, and interactions between receptors and a plethora of multidomain scaffolding proteins and accessory/chaperone molecules confers signal preorganization, efficiency, and specificity. More importantly it is likely that alteration in the interactions of these proteins with GPCRs may occur in aging or neurodegenerative disorders, thus defining a distinct 'pharmacology' from that seen in young organisms or normal physiology. In this context, the concept of agonist selective trafficking of receptor signaling, which recognizes that a bound ligand may select between a menu of 'active' receptor conformations and induce only a subset of the possible response profile, presents the opportunity to develop drugs that change the quality as well as the quantity of efficacy and enhance these qualities for specific disorders or other paradigms. As a more comprehensive understanding of the complexity of GPCR signaling is developed, the rational design of ligands

  4. G protein-coupled receptor systems and their lipid environment in health disorders during aging

    OpenAIRE

    Alemany, Regina; Perona, Javier S.; Sánchez-Dominguez, José M.; Montero, Emilio; Cañizares, Julio; Bressani, Ricardo; Escribá, Pablo V.; Ruiz-Gutierrez, Valentina

    2007-01-01

    Cells, tissues and organs undergo phenotypic changes and deteriorate as they age. Cell growth arrest and hyporesponsiveness to extrinsic stimuli are all hallmarks of senescent cells. Most such external stimuli received by a cell are processed by two different cell membrane systems: receptor tyrosine kinases (RTKs) and G protein-coupled receptors (GPCRs). GPCRs form the largest gene family in the human genome and they are involved in most relevant physiological functions. Given the changes obs...

  5. Borna disease virus P protein affects neural transmission through interactions with gamma-aminobutyric acid receptor-associated protein.

    Science.gov (United States)

    Peng, Guiqing; Yan, Yan; Zhu, Chengliang; Wang, Shiqun; Yan, Xiaohong; Lu, Lili; Li, Wei; Hu, Jing; Wei, Wei; Mu, Yongxin; Chen, Yanni; Feng, Yong; Gong, Rui; Wu, Kailang; Zhang, Fengmin; Zhang, Xiaolian; Zhu, Ying; Wu, Jianguo

    2008-12-01

    Borna disease virus (BDV) is one of the infectious agents that causes diseases of the central nervous system in a wide range of vertebrate species and, perhaps, in humans. The phosphoprotein (P) of BDV, an essential cofactor of virus RNA-dependent RNA polymerase, is required for virus replication. In this study, we identified the gamma-aminobutyric acid receptor-associated protein (GABARAP) with functions in neurobiology as one of the viral P protein-interacting cellular factors by using an approach of phage display-based protein-protein interaction analysis. Direct binding between GABARAP and P protein was confirmed by coimmunoprecipitation, protein pull-down, and mammalian two-hybrid analyses. GABARAP originally was identified as a linker between the gamma-aminobutyric acid receptor (GABAR) and the microtubule to regulate receptor trafficking and plays important roles in the regulation of the inhibitory neural transmitter gamma-aminobutyric acid (GABA). We showed that GABARAP colocalizes with P protein in the cells infected with BDV or transfected with the P gene, which resulted in shifting the localization of GABARAP from the cytosol to the nucleus. We further demonstrated that P protein blocks the trafficking of GABAR, a principal GABA-gated ion channel that plays important roles in neural transmission, to the surface of cells infected with BDV or transfected with the P gene. We proposed that during BDV infection, P protein binds to GABARAP, shifts the distribution of GABARAP from the cytoplasm to the nucleus, and disrupts the trafficking of GABARs to the cell membranes, which may result in the inhibition of GABA-induced currents and in the enhancement of hyperactivity and anxiety.

  6. Identification of a novel protein-protein interaction motif mediating interaction of GPCR-associated sorting proteins with G protein-coupled receptors

    DEFF Research Database (Denmark)

    Bornert, Olivier; Møller, Thor Christian; Boeuf, Julien

    2013-01-01

    GPCR desensitization and down-regulation are considered key molecular events underlying the development of tolerance in vivo. Among the many regulatory proteins that are involved in these complex processes, GASP-1 have been shown to participate to the sorting of several receptors toward the degra......GPCR desensitization and down-regulation are considered key molecular events underlying the development of tolerance in vivo. Among the many regulatory proteins that are involved in these complex processes, GASP-1 have been shown to participate to the sorting of several receptors toward...... the degradation pathway. This protein belongs to the recently identified GPCR-associated sorting proteins (GASPs) family that comprises ten members for which structural and functional details are poorly documented. We present here a detailed structure-function relationship analysis of the molecular interaction...... between GASPs and a panel of GPCRs. In a first step, GST-pull down experiments revealed that all the tested GASPs display significant interactions with a wide range of GPCRs. Importantly, the different GASP members exhibiting the strongest interaction properties were also characterized by the presence...

  7. Relaxin family peptide receptors Rxfp1 and Rxfp2: mapping of the mRNA and protein distribution in the reproductive tract of the male rat

    Directory of Open Access Journals (Sweden)

    Porto Catarina S

    2007-07-01

    Full Text Available Abstract Background Relaxin is the endogenous ligand of the G-protein coupled receptor RXFP1, previously known as LGR7. In humans relaxin can also activate, but with lower affinity, the closely related receptor for the insulin-like peptide from Leydig cells, RXFP2, previously known as LGR8. The lack of relaxin impairs male fertility but the precise distribution and the function of relaxin receptors in the male reproductive tract is not known. We investigated the distribution of Rxfp1 and Rxfp2 in the reproductive tract of the male rat and the function of relaxin in the vas deferens, a tissue with high expression of both receptors. Methods The presence of mRNA for Rxfp1 and Rxfp2 was investigated in testes, cultured Sertoli cells, epididymis, vas deferens, seminal vesicle, prostate, and spermatozoa by RT-PCR and Southern blot. Protein expression in the testis, vas deferens, primary culture of Sertoli cells, and spermatozoa was assessed by immunohistochemistry and immunofluorescence. The role of relaxin in the vas deferens was evaluated by contractility studies and radioimmunoassay of cAMP production. The effect of relaxin on mRNA levels for metalloproteinase-7 was measured by Northern blot. Results Transcripts for Rxfp1 and Rxfp2 were present in almost all parts of the male reproductive tract, with high levels in testis and vas deferens. Both receptors were immunolocalized in late stage germ cells but not in mature spermatozoa, although mRNAs for both receptors were also present in mature spermatozoa. Rxfp1 but not Rxfp2 was detected in cultured Sertoli cells. Strong immunostaining for Rxfp1 and Rxfp2 was seen in muscular and epithelial layers of the vas deferens and in arteriolar walls. Relaxin did not affect contractility and cyclic AMP production of the vas deferens, but increased the levels of mRNA for metalloproteinase-7. Conclusion Rxfp1 and Rxfp2 are widely and similarly distributed throughout the male reproductive tract. Our results

  8. Cloning of human genes encoding novel G protein-coupled receptors

    Energy Technology Data Exchange (ETDEWEB)

    Marchese, A.; Docherty, J.M.; Heiber, M. [Univ. of Toronto, (Canada)] [and others

    1994-10-01

    We report the isolation and characterization of several novel human genes encoding G protein-coupled receptors. Each of the receptors contained the familiar seven transmembrane topography and most closely resembled peptide binding receptors. Gene GPR1 encoded a receptor protein that is intronless in the coding region and that shared identity (43% in the transmembrane regions) with the opioid receptors. Northern blot analysis revealed that GPR1 transcripts were expressed in the human hippocampus, and the gene was localized to chromosome 15q21.6. Gene GPR2 encoded a protein that most closely resembled an interleukin-8 receptor (51% in the transmembrane regions), and this gene, not expressed in the six brain regions examined, was localized to chromosome 17q2.1-q21.3. A third gene, GPR3, showed identity (56% in the transmembrane regions) with a previously characterized cDNA clone from rat and was localized to chromosome 1p35-p36.1. 31 refs., 5 figs., 1 tab.

  9. A urokinase receptor-associated protein with specific collagen binding properties

    DEFF Research Database (Denmark)

    Behrendt, N; Jensen, O N; Engelholm, L H

    2000-01-01

    molecular weight urokinase receptor-associated protein. The tryptic peptide mixture derived from a cross-linked complex of pro-urokinase and the latter protein was analyzed by nanoelectrospray tandem mass spectrometric sequencing. This analysis identified the novel protein as the human homologue of a murine...... membrane-bound lectin with hitherto unknown function. The human cDNA was cloned and sequenced. The protein, designated uPARAP, is a member of the macrophage mannose receptor protein family and contains a putative collagen-binding (fibronectin type II) domain in addition to 8 C-type carbohydrate recognition...... domains. It proved capable of binding strongly to a single type of collagen, collagen V. This collagen binding reaction at the exact site of plasminogen activation on the cell may lead to adhesive functions as well as a contribution to cellular degradation of collagen matrices....

  10. CCR11 is a functional receptor for the monocyte chemoattractant protein family of chemokines.

    Science.gov (United States)

    Schweickart, V L; Epp, A; Raport, C J; Gray, P W

    2000-03-31

    Chemokines mediate their diverse activities through G protein-coupled receptors. The human homolog of the bovine orphan receptor PPR1 shares significant similarity to chemokine receptors. Transfection of this receptor into murine L1.2 cells resulted in responsiveness to monocyte chemoattractant protein (MCP)-4, MCP-2, and MCP-1 in chemotaxis assays. Binding studies with radiolabeled MCP-4 demonstrated a single high affinity binding site with an IC(50) of 0.14 nM. As shown by competition binding, other members of the MCP family also recognized this receptor. MCP-2 was the next most potent ligand, with an IC(50) of 0.45 nM. Surprisingly, eotaxin (IC(50) = 6.7 nM) and MCP-3 (IC(50) = 4.1 nM) bind with greater affinity than MCP-1 (IC(50) = 10.7 nM) but only act as agonists in chemotaxis assays at 100-fold higher concentrations. Because of high affinity binding and functional chemotactic responses, we have termed this receptor CCR11. The gene for CCR11 was localized to human chromosome 3q22, which is distinct from most CC chemokine receptor genes at 3p21. Northern blot hybridization was used to identify CCR11 expression in heart, small intestine, and lung. Thus CCR11 shares functional similarity to CCR2 because it recognizes members of the MCP family, but CCR11 has a distinct expression pattern.

  11. 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...... some mutants show enhanced binding affinity to the foreign pheromone, others only display weakened interactions with the network's negative regulators. Importantly, the latter changes have a limited impact on overall pathway regulation, despite their considerable effect on sensitivity. Our results...

  12. A Computational Modeling and Simulation Approach to Investigate Mechanisms of Subcellular cAMP Compartmentation.

    Directory of Open Access Journals (Sweden)

    Pei-Chi Yang

    2016-07-01

    Full Text Available Subcellular compartmentation of the ubiquitous second messenger cAMP has been widely proposed as a mechanism to explain unique receptor-dependent functional responses. How exactly compartmentation is achieved, however, has remained a mystery for more than 40 years. In this study, we developed computational and mathematical models to represent a subcellular sarcomeric space in a cardiac myocyte with varying detail. We then used these models to predict the contributions of various mechanisms that establish subcellular cAMP microdomains. We used the models to test the hypothesis that phosphodiesterases act as functional barriers to diffusion, creating discrete cAMP signaling domains. We also used the models to predict the effect of a range of experimentally measured diffusion rates on cAMP compartmentation. Finally, we modeled the anatomical structures in a cardiac myocyte diad, to predict the effects of anatomical diffusion barriers on cAMP compartmentation. When we incorporated experimentally informed model parameters to reconstruct an in silico subcellular sarcomeric space with spatially distinct cAMP production sites linked to caveloar domains, the models predict that under realistic conditions phosphodiesterases alone were insufficient to generate significant cAMP gradients. This prediction persisted even when combined with slow cAMP diffusion. When we additionally considered the effects of anatomic barriers to diffusion that are expected in the cardiac myocyte dyadic space, cAMP compartmentation did occur, but only when diffusion was slow. Our model simulations suggest that additional mechanisms likely contribute to cAMP gradients occurring in submicroscopic domains. The difference between the physiological and pathological effects resulting from the production of cAMP may be a function of appropriate compartmentation of cAMP signaling. Therefore, understanding the contribution of factors that are responsible for coordinating the spatial and

  13. G protein-coupled receptor inactivation by an allosteric inverse-agonist antibody

    Science.gov (United States)

    Hino, Tomoya; Arakawa, Takatoshi; Iwanari, Hiroko; Yurugi-Kobayashi, Takami; Ikeda-Suno, Chiyo; Nakada-Nakura, Yoshiko; Kusano-Arai, Osamu; Weyand, Simone; Shimamura, Tatsuro; Nomura, Norimichi; Cameron, Alexander D.; Kobayashi, Takuya; Hamakubo, Takao; Iwata, So; Murata, Takeshi

    2011-01-01

    G protein-coupled receptors (GPCRs) are the largest class of cell-surface receptors, and these membrane proteins exist in equilibrium between inactive and active states.1-13 Conformational changes induced by extracellular ligands binding to GPCRs result in a cellular response through the activation of G-proteins. The A2A adenosine receptor (A2AAR) is responsible for regulating blood flow to the cardiac muscle and is important in the regulation of glutamate and dopamine release in the brain.14 In this study, we have successfully raised a mouse monoclonal antibody against human A2AAR that prevents agonist but not antagonist binding to the extracellular ligand-binding pocket. The structure of the A2AAR-antibody Fab fragment (Fab2838) complex reveals that the fragment, unexpectedly, recognises the intracellular surface of A2AAR and that its complementarity determining region, CDR-H3, penetrates into the receptor. CDR-H3 is located in a similar position to the G-protein C-terminal fragment in the active opsin structure1 and to the CDR-3 of the nanobody in the active β2 adrenergic receptor structure2 but locks the A2AAR in an inactive conformation. These results shed light on a novel strategy to modulate GPCR activity. PMID:22286059

  14. Chromatin Modulatory Proteins and Olfactory Receptor Signaling in the Refinement and Maintenance of Fruitless Expression in Olfactory Receptor Neurons.

    Directory of Open Access Journals (Sweden)

    Catherine E Hueston

    2016-04-01

    Full Text Available During development, sensory neurons must choose identities that allow them to detect specific signals and connect with appropriate target neurons. Ultimately, these sensory neurons will successfully integrate into appropriate neural circuits to generate defined motor outputs, or behavior. This integration requires a developmental coordination between the identity of the neuron and the identity of the circuit. The mechanisms that underlie this coordination are currently unknown. Here, we describe two modes of regulation that coordinate the sensory identities of Drosophila melanogaster olfactory receptor neurons (ORNs involved in sex-specific behaviors with the sex-specific behavioral circuit identity marker fruitless (fru. The first mode involves a developmental program that coordinately restricts to appropriate ORNs the expression of fru and two olfactory receptors (Or47b and Ir84a involved in sex-specific behaviors. This regulation requires the chromatin modulatory protein Alhambra (Alh. The second mode relies on the signaling from the olfactory receptors through CamK and histone acetyl transferase p300/CBP to maintain ORN-specific fru expression. Our results highlight two feed-forward regulatory mechanisms with both developmentally hardwired and olfactory receptor activity-dependent components that establish and maintain fru expression in ORNs. Such a dual mechanism of fru regulation in ORNs might be a trait of neurons driving plastic aspects of sex-specific behaviors.

  15. Characterization of rabbit ileal receptors for Clostridium difficile toxin A. Evidence for a receptor-coupled G protein

    Energy Technology Data Exchange (ETDEWEB)

    Pothoulakis, C.; LaMont, J.T.; Eglow, R.; Gao, N.; Rubins, J.B.; Theoharides, T.C.; Dickey, B.F. (Boston Univ. School of Medicine, MA (USA))

    1991-07-01

    The purpose of this study was to characterize the surface receptor for toxin A, the enterotoxin from Clostridium difficile, on rabbit intestinal brush borders (BB) and on rat basophilic leukemia (RBL) cells. Purified toxin A was radiolabeled using a modified Bolton-Hunter method to sp act 2 microCi/micrograms, with retention of full biologic activity. 3H-Toxin A bound specifically to a single class of receptors on rabbit BB and on RBL cells with dissociation constants of 5.4 x 10(-8) and 3.5 x 10(-8) M, respectively. RBL cells were highly sensitive to toxin A (cell rounding) and had 180,000 specific binding sites per cell, whereas IMR-90 fibroblasts were far less sensitive to toxin A and lacked detectable specific binding sites. Exposure of BB to trypsin or chymotrypsin significantly reduced 3H-toxin A specific binding. Preincubation of BB with Bandeirea simplicifolia (BS-1) lectin also reduced specific binding, and CHAPS-solubilized receptors could be immobilized with WGA-agarose. The addition of 100 nM toxin A accelerated the association of 35S-GTP gamma S with rabbit ileal BB, and preincubation of BB with the GTP analogues GTP gamma S or Gpp(NH)p, significantly reduced 3H-toxin A specific binding. Our data indicate that the membrane receptor for toxin A is a galactose and N-acetyl-glucosamine-containing glycoprotein which appears to be coupled to a G protein.

  16. A Novel T55A Variant of Gsα Associated with Impaired cAMP Production, Bone Fragility, and Osteolysis

    Directory of Open Access Journals (Sweden)

    Kelly Wentworth

    2016-01-01

    Full Text Available G-protein coupled receptors (GPCRs mediate a wide spectrum of biological activities. The GNAS complex locus encodes the stimulatory alpha subunit of the guanine nucleotide binding protein (Gsα and regulates production of the second messenger cyclic AMP (cAMP. Loss-of-function GNAS mutations classically lead to Albright’s Hereditary Osteodystrophy (AHO and pseudohypoparathyroidism, often with significant effects on bone formation and mineral metabolism. We present the case of a child who exhibits clinical features of osteolysis, multiple childhood fractures, and neonatal SIADH. Exome sequencing revealed a novel de novo heterozygous missense mutation of GNAS (c.163AcAMP activity associated with this mutation. We identified a 64% decrease in isoproterenol-induced cAMP production in vitro, compared to wild type, consistent with loss of Gsα activity. Despite a significant decrease in isoproterenol-induced cAMP production in vitro, this mutation did not produce a classical AHO phenotype in our patient; however, it may account for her presentation with childhood fractures and osteolysis.

  17. Genetically encoding an electrophilic amino acid for protein stapling and covalent binding to native receptors.

    Science.gov (United States)

    Chen, Xiao-Hua; Xiang, Zheng; Hu, Ying S; Lacey, Vanessa K; Cang, Hu; Wang, Lei

    2014-09-19

    Covalent bonds can be generated within and between proteins by an unnatural amino acid (Uaa) reacting with a natural residue through proximity-enabled bioreactivity. Until now, Uaas have been developed to react mainly with cysteine in proteins. Here we genetically encoded an electrophilic Uaa capable of reacting with histidine and lysine, thereby expanding the diversity of target proteins and the scope of the proximity-enabled protein cross-linking technology. In addition to efficient cross-linking of proteins inter- and intramolecularly, this Uaa permits direct stapling of a protein α-helix in a recombinant manner and covalent binding of native membrane receptors in live cells. The target diversity, recombinant stapling, and covalent targeting of endogenous proteins enabled by this versatile Uaa should prove valuable in developing novel research tools, biological diagnostics, and therapeutics by exploiting covalent protein linkages for specificity, irreversibility, and stability.

  18. Targeting of vasoactive intestinal peptide receptor 2, VPAC2, a secretin family G-protein coupled receptor, to primary cilia

    Directory of Open Access Journals (Sweden)

    Livana Soetedjo

    2013-05-01

    Primary cilia protrude from the cell surface of many cell types in the human body and function as cellular antennae via ciliary membrane localized receptors. Neurons and glial cells in the brain possess primary cilia, and the malfunction of primary cilia may contribute to neurological deficits present in many cilia-associated disorders. Several rhodopsin family G-protein coupled receptors (GPCRs are specifically localized to a subset of neuronal primary cilia. However, whether other family GPCRs target to neuronal cilia and whether glial primary cilia harbor any GPCRs are not known. We conducted a screening of GPCRs to determine their ability to target to primary cilia, and identified a secretin family member, Vasoactive Intestinal Receptor 2 (VPAC2, as a novel ciliary GPCR. Here, we show that endogenous VPAC2 targets to primary cilia in various brain regions, including the suprachiasmatic nuclei and the thalamus. Surprisingly, VPAC2 not only localizes to neuronal cilia but also to glial cilia. In addition, we show that VPAC2's C-terminus is both necessary and sufficient for its ciliary targeting and we define a novel ciliary targeting signal: the tetrapeptide RDYR motif in the C-terminus of VPAC2. Furthermore, we demonstrate that VPAC2 ciliary targeting is dependent on Tubby, the BBSome (a complex of Bardet–Biedl syndrome proteins and the BBSome targeting factor, Arl6.

  19. Expression of androgen receptor and its association with estrogen receptor and androgen receptor downstream proteins in normal/benign breast luminal epithelium.

    Science.gov (United States)

    Wang, Xi; Yarid, Nicole; McMahon, Loralee; Yang, Qi; Hicks, David G

    2014-08-01

    The androgen receptor (AR) is strongly expressed in the majority of breast carcinomas, but its role in breast hormonal carcinogenesis is not clear. We believe a better knowledge of the biology of normal/benign breast tissue will be the key to understanding this process. Using standard immunohistochemical staining on consecutive sections and dual immunohistochemical labeling, we studied the expression pattern of AR and estrogen receptor (ER) in normal/benign breast luminal epithelial cells. We found that most of the AR-positive cells are also ER positive, about 10% of the cells are AR-positive only, whereas ER-positive only cells are uncommon, a distribution pattern of hormone receptor expression similar to what was revealed in invasive breast carcinomas. Whereas the expression of AR downstream proteins, such as prostate-specific antigen and gross cystic disease fluid protein, was either negative or unrelated to the AR status. We conclude that AR and ER expression status in invasive breast carcinomas reflects that of their progenitor cells in terminal duct lobular units. Our study did not reveal the expression of AR downstream proteins in normal/benign luminal epithelial cells at the regular immunohistochemistry level.

  20. 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.

  1. Fluorescent Approaches for Understanding Interactions of Ligands with G Protein Coupled Receptors

    Science.gov (United States)

    Sridharan, Rajashri; Zuber, Jeffrey; Connelly, Sara M.; Mathew, Elizabeth; Dumont, Mark E.

    2014-01-01

    G Protein Coupled Receptors (GPCRs) are responsible for a wide variety of signaling responses in diverse cell types. Despite major advances in the determination of structures of this class of receptors, the underlying mechanisms by which binding of different types of ligands specifically elicits particular signaling responses remains unclear. The use of fluorescence spectroscopy can provide important information about the process of ligand binding and ligand dependent conformational changes in receptors, especially kinetic aspects of these processes, that can be difficult to extract from x-ray structures. We present an overview of the extensive array of fluorescent ligands that have been used in studies of GPCRs and describe spectroscopic approaches for assaying binding and probing the environment of receptor-bound ligands with particular attention to examples involving yeast pheromone receptors. In addition, we discuss the use of fluorescence spectroscopy for detecting and characterizing conformational changes in receptors induced by the binding of ligands. Such studies have provided strong evidence for diversity of receptor conformations elicited by different ligands, consistent with the idea that GPCRs are not simple on and off switches. This diversity of states constitutes an underlying mechanistic basis for biased agonism, the observation that different stimuli can produce different responses from a single receptor. It is likely that continued technical advances will allow fluorescence spectroscopy to play an important role in continued probing of structural transitions in GPCRs. PMID:24055822

  2. The origins of diversity and specificity in g protein-coupled receptor signaling.

    Science.gov (United States)

    Maudsley, Stuart; Martin, Bronwen; Luttrell, Louis M

    2005-08-01

    The modulation of transmembrane signaling by G protein-coupled receptors (GPCRs) constitutes the single most important therapeutic target in medicine. Drugs acting on GPCRs have traditionally been classified as agonists, partial agonists, or antagonists based on a two-state model of receptor function embodied in the ternary complex model. Over the past decade, however, many lines of investigation have shown that GPCR signaling exhibits greater diversity and "texture" than previously appreciated. Signal diversity arises from numerous factors, among which are the ability of receptors to adopt multiple "active" states with different effector-coupling profiles; the formation of receptor dimers that exhibit unique pharmacology, signaling, and trafficking; the dissociation of receptor "activation" from desensitization and internalization; and the discovery that non-G protein effectors mediate some aspects of GPCR signaling. At the same time, clustering of GPCRs with their downstream effectors in membrane microdomains and interactions between receptors and a plethora of multidomain scaffolding proteins and accessory/chaperone molecules confer signal preorganization, efficiency, and specificity. In this context, the concept of agonist-selective trafficking of receptor signaling, which recognizes that a bound ligand may select between a menu of active receptor conformations and induce only a subset of the possible response profile, presents the opportunity to develop drugs that change the quality as well as the quantity of efficacy. As a more comprehensive understanding of the complexity of GPCR signaling is developed, the rational design of ligands possessing increased specific efficacy and attenuated side effects may become the standard mode of drug development.

  3. β2 adrenergic receptor fluorescent protein fusions traffic to the plasma membrane and retain functionality.

    Directory of Open Access Journals (Sweden)

    Jaclyn Bubnell

    Full Text Available Green fluorescent protein (GFP has proven useful for the study of protein interactions and dynamics for the last twenty years. A variety of new fluorescent proteins have been developed that expand the use of available excitation spectra. We have undertaken an analysis of seven of the most useful fluorescent proteins (XFPs, Cerulean (and mCerulean3, Teal, GFP, Venus, mCherry and TagRFP657, as fusions to the archetypal G-protein coupled receptor, the β2 adrenergic receptor (β2AR. We have characterized these β2AR::XFP fusions in respect to membrane trafficking and G-protein activation. We noticed that in the mouse neural cell line, OP 6, that membrane bound β2AR::XFP fusions robustly localized in the filopodia identical to gap::XFP fusions. All β2AR::XFP fusions show responses indistinguishable from each other and the non-fused form after isoprenaline exposure. Our results provide a platform by which G-protein coupled receptors can be dissected for their functionality.

  4. Structure--Function Studies on Receptor Activation of Photoactive Yellow Protein

    Science.gov (United States)

    Kaledhonkar, Sandip; Dai, Shuo; Rathod, Rachana; Hoff, Wouter; Xie, Aihua; Xie Collaboration; Hoff Collaboration

    2013-03-01

    Biological signaling in cells starts with detection of stimuli from ever changing environment, results in relay of signal, and finishes with particular cellular response. Photoactive yellow protein (PYP) from a salt loving Halorhodospira halophila bacterium is a blue light photoreceptor protein for negative phototaxis and a structural prototype of PAS domain superfamily of signaling and regulatory proteins. Upon absorption of a blue photon by its negatively charged p-coumaric acid (pCA) chromophore, the receptor state (off-state) undergoes photocyclic process, leading to large amplitude protein quake that results in PYP receptor activation. To understand the structural basis of receptor activation we employ time-resolved FTIR spectroscopic techniques combined with site-specific mutation to search for a key residue involved in protein quake. We will discuss the strategies and experimental results in light of hydrogen bonding network, active site structure and protein quake in PYP. The signaling mechanism leaned from PYP may have implication to understand signal transduction in other proteins.

  5. Isothermal chemical denaturation to determine binding affinity of small molecules to G-protein coupled receptors.

    Science.gov (United States)

    Ross, Patrick; Weihofen, Wilhelm; Siu, Fai; Xie, Amy; Katakia, Hetal; Wright, S Kirk; Hunt, Ian; Brown, Richard K; Freire, Ernesto

    2015-03-15

    The determination of accurate binding affinities is critical in drug discovery and development. Several techniques are available for characterizing the binding of small molecules to soluble proteins. The situation is different for integral membrane proteins. Isothermal chemical denaturation has been shown to be a valuable biophysical method to determine, in a direct and label-free fashion, the binding of ligands to soluble proteins. In this study, the application of isothermal chemical denaturation was applied to an integral membrane protein, the A2a G-protein coupled receptor. Binding affinities for a set of 19 small molecule agonists/antagonists of the A2a receptor were determined and found to be in agreement with data from surface plasmon resonance and radioligand binding assays previously reported in the literature. Therefore, isothermal chemical denaturation expands the available toolkit of biophysical techniques to characterize and study ligand binding to integral membrane proteins, specifically G-protein coupled receptors in vitro. Copyright © 2014 Elsevier Inc. All rights reserved.

  6. Computational Analysis of the CB1 Carboxyl-terminus in the Receptor-G Protein Complex

    OpenAIRE

    Shim, Joong-Youn; Khurana, Leepakshi; Kendall, Debra A.

    2016-01-01

    Despite the important role of the carboxyl-terminus (Ct) of the activated brain cannabinoid receptor one (CB1) in the regulation of G protein signaling, a structural understanding of interactions with G proteins is lacking. This is largely due to the highly flexible nature of the CB1 Ct that dynamically adapts its conformation to the presence of G proteins. In the present study, we explored how the CB1 Ct can interact with the G protein by building on our prior modeling of the CB1-Gi complex ...

  7. The prion protein as a receptor for amyloid-beta

    NARCIS (Netherlands)

    Kessels, Helmut W.; Nguyen, Louis N.; Nabavi, Sadegh; Malinow, Roberto

    2010-01-01

    Increased levels of brain amyloid-beta, a secreted peptide cleavage product of amyloid precursor protein (APP), is believed to be critical in the aetiology of Alzheimer's disease. Increased amyloid-beta can cause synaptic depression, reduce the number of spine protrusions (that is, sites of synaptic

  8. AT(1) receptor Gαq protein-independent signalling transcriptionally activates only a few genes directly, but robustly potentiates gene regulation from the β2-adrenergic receptor

    DEFF Research Database (Denmark)

    Christensen, Gitte Lund; Knudsen, Steen; Schneider, Mikael

    2011-01-01

    The angiotensin II type 1 receptor (AT(1)R) is known to signal through heterotrimeric G proteins, and Gαq protein-independent signalling has only recently gained appreciation for profound impact on a diverse range of biological functions. β-Arrestins, among other central mediators of Gαq protein-...

  9. The activation mechanisms of G protein-coupled receptors : the case of the adenosine A2B and HCA2/3 receptors

    NARCIS (Netherlands)

    Liu, R.

    2016-01-01

    Identifying and elucidating the functions and activation of GPCRs will provide opportunities for novel drug discovery. We confirmed that a yeast system with an extended library of G proteins is very well suited for the study of GPCR activation, G protein coupling profiles, receptor-G protein binding

  10. A novel human gene encoding a G-protein-coupled receptor (GPR15) is located on chromosome 3

    Energy Technology Data Exchange (ETDEWEB)

    Heiber, M.; Marchese, A.; O`Dowd, B.F. [Univ. of Toronto, Ontario (Canada)] [and others

    1996-03-05

    We used sequence similarities among G-protein-coupled receptor genes to discover a novel receptor gene. Using primers based on conserved regions of the opioid-related receptors, we isolated a PCR product that was used to locate the full-length coding region of a novel human receptor gene, which we have named GPR15. A comparison of the amino acid sequence of the receptor gene, which we have named GPR15. A comparison of the amino acid sequence of the receptor encoded by GPR15 with other receptors revealed that it shared sequence identity with the angiotensin II AT1 and AT2 receptors, the interleukin 8b receptor, and the orphan receptors GPR1 and AGTL1. GPR15 was mapped to human chromosome 3q11.2-q13.1. 12 refs., 2 figs.

  11. The Use of FRET/FLIM to Study Proteins Interacting with Plant Receptor Kinases.

    Science.gov (United States)

    Weidtkamp-Peters, Stefanie; Stahl, Yvonne

    2017-01-01

    The investigation of protein interactions in living plant tissue has become of increasing importance in recent years. A high spatial and temporal resolution for the observation of in vivo protein interaction is needed, e.g., in order to follow changes of plant receptor kinase interactions and complex formation over time. In vivo fluorescence or Förster resonance energy transfer (FRET) measurements allow for detailed analyses of interacting proteins in their natural environment at a subcellular level. Especially FRET-FLIM (fluorescence lifetime imaging microscopy) measurements provide an extremely powerful and reliable tool meeting the demands for investigating in vivo protein interaction quantitatively and with high precision. Here, we will describe in detail how to practically perform in vivo FRET measurements of receptor kinases in plants and discuss potential pitfalls and points of consideration.

  12. 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 the partial inverse agonist carazolol at 2.4 angstrom resolution. The structure provides a high-resolution view of a human G protein-coupled receptor bound to a diffusible ligand. Ligand-binding site accessibility is enabled by the second extracellular loop, which is held out of the binding cavity by a pair...

  13. The Concise Guide to Pharmacology 2013/14: G Protein-Coupled Receptors

    Science.gov (United States)

    Alexander, Stephen PH; Benson, Helen E; Faccenda, Elena; Pawson, Adam J; Sharman, Joanna L; Spedding, Michael; Peters, John A; Harmar, Anthony J

    2013-01-01

    The Concise Guide to PHARMACOLOGY 2013/14 provides concise overviews of the key properties of over 2000 human drug targets with their pharmacology, plus links to an open access knowledgebase of drug targets and their ligands (www.guidetopharmacology.org), which provides more detailed views of target and ligand properties. The full contents can be found at http://onlinelibrary.wiley.com/doi/10.1111/bph.12444/full. G protein-coupled receptors are one of the seven major pharmacological targets into which the Guide is divided, with the others being G protein-coupled receptors, ligand-gated ion channels, ion channels, catalytic receptors, nuclear hormone receptors, transporters and enzymes. These are presented with nomenclature guidance and summary information on the best available pharmacological tools, alongside key references and suggestions for further reading. A new landscape format has easy to use tables comparing related targets. It is a condensed version of material contemporary to late 2013, which is presented in greater detail and constantly updated on the website www.guidetopharmacology.org, superseding data presented in previous Guides to Receptors and Channels. It is produced in conjunction with NC-IUPHAR and provides the official IUPHAR classification and nomenclature for human drug targets, where appropriate. It consolidates information previously curated and displayed separately in IUPHAR-DB and the Guide to Receptors and Channels, providing a permanent, citable, point-in-time record that will survive database updates. PMID:24517644

  14. Agouti protein is an antagonist of the melanocyte-stimulating-hormone receptor.

    Science.gov (United States)

    Lu, D; Willard, D; Patel, I R; Kadwell, S; Overton, L; Kost, T; Luther, M; Chen, W; Woychik, R P; Wilkison, W O

    1994-10-27

    The genetic loci agouti and extension control the relative amounts of eumelanin (brown-black) and phaeomelanin (yellow-red) pigments in mammals: extension encodes the receptor for melanocyte-stimulating hormone (MSH) and agouti encodes a novel 131-amino-acid protein containing a signal sequence. Agouti, which is produced in the hair follicle, acts on follicular melanocytes to inhibit alpha-MSH-induced eumelanin production, resulting in the subterminal band of phaeomelanin often visible in mammalian fur. Here we use partially purified agouti protein to demonstrate that agouti is a high-affinity antagonist of the MSH receptor and blocks alpha-MSH stimulation of adenylyl cyclase, the effector through which alpha-MSH induces eumelanin synthesis. Agouti was also found to be an antagonist of the melanocortin-4 receptor, a related MSH-binding receptor. Consequently, the obesity caused by ectopic expression of agouti in the lethal yellow (Ay) mouse may be due to the inhibition of melanocortin receptor(s) outside the hair follicle.

  15. The repertoire of G-protein-coupled receptors in Xenopus tropicalis

    Directory of Open Access Journals (Sweden)

    Hu Yinghe

    2009-06-01

    Full Text Available Abstract Background The G-protein-coupled receptor (GPCR superfamily represents the largest protein family in the human genome. These proteins have a variety of physiological functions that give them well recognized roles in clinical medicine. In Xenopus tropicalis, a widely used animal model for physiology research, the repertoire of GPCRs may help link the GPCR evolutionary history in vertebrates from teleost fish to mammals. Results We have identified 1452 GPCRs in the X. tropicalis genome. Phylogenetic analyses classified these receptors into the following seven families: Glutamate, Rhodopsin, Adhesion, Frizzled, Secretin, Taste 2 and Vomeronasal 1. Nearly 70% of X. tropicalis GPCRs are represented by the following three types of receptors thought to receive chemosensory information from the outside world: olfactory, vomeronasal 1 and vomeronasal 2 receptors. Conclusion X. tropicalis shares a more similar repertoire of GPCRs with mammals than it does with fish. An examination of the three major groups of receptors related to olfactory/pheromone detection shows that in X. tropicalis, these groups have undergone lineage specific expansion. A comparison of GPCRs in X. tropicalis, teleost fish and mammals reveals the GPCR evolutionary history in vertebrates.

  16. Heterologous production, characterization and isolation of selected G protein-coupled receptors for structural studies

    OpenAIRE

    Shukla, Arun Kumar

    2006-01-01

    G protein-coupled receptors (GPCRs) play regulatory roles in many different physiological processes and they represent one of the most important class of drug targets. However, due to the lack of three-dimensional structures, structure based drug design has not been possible. The major bottleneck in getting three-dimensional crystal structure of GPCRs is to obtain milligram quantities of pure, homogenous and stable protein. Therefore, during my Ph.D. thesis, I focused on expression, character...

  17. Emerging issues in receptor protein tyrosine phosphatase function: lifting fog or simply shifting?

    DEFF Research Database (Denmark)

    Petrone, A; Sap, J

    2000-01-01

    Transmembrane (receptor) tyrosine phosphatases are intimately involved in responses to cell-cell and cell-matrix contact. Several important issues regarding the targets and regulation of this protein family are now emerging. For example, these phosphatases exhibit complex interactions...... with signaling pathways involving SRC family kinases, which result from their ability to control phosphorylation of both activating and inhibitory sites in these kinases and possibly also their substrates. Similarly, integrin signaling illustrates how phosphorylation of a single protein, or the activity...

  18. Slave Labor Camps of the Third Reich.

    Science.gov (United States)

    Stone, Adolf

    1983-01-01

    Describes the ground rules used by Nazi architects in choosing the sites for slave labor camps. While some, like Auschwitz, became extermination camps, others also produced armaments. One camp, Theresienstadt, became a "model" camp to show to reporters and Red Cross representatives. (CS)

  19. Interactions of the NPXY microdomains of the low density lipoprotein receptor-related protein 1.

    Science.gov (United States)

    Guttman, Miklos; Betts, Gina N; Barnes, Helen; Ghassemian, Majid; van der Geer, Peter; Komives, Elizabeth A

    2009-11-01

    The low density lipoprotein receptor-related protein 1 (LRP1) mediates internalization of a large number of proteins and protein-lipid complexes and is widely implicated in Alzheimer's disease. The cytoplasmic domain of LRP1 (LRP1-CT) can be phosphorylated by activated protein-tyrosine kinases at two NPXY motifs in LRP1-CT; Tyr 4507 is readily phosphorylated and must be phosphorylated before phosphorylation of Tyr 4473 occurs. Pull-down experiments from brain lysate revealed numerous proteins binding to LRP1-CT, but the results were highly variable. To separate which proteins bind to each NPXY motif and their phosphorylation dependence, each NPXY motif microdomain was prepared in both phosphorylated and non-phosphorylated forms and used to probe rodent brain extracts for binding proteins. Proteins that bound specifically to the microdomains were identified by LC-MS/MS, and confirmed by Western blot. Recombinant proteins were then tested for binding to each NPXY motif. The NPXY(4507) (membrane distal) was found to interact with a large number of proteins, many of which only bound the tyrosine-phosphorylated form. This microdomain also bound a significant number of other proteins in the unphosphorylated state. Many of the interactions were later confirmed to be direct with recombinant proteins. The NPXY(4473) (membrane proximal) bound many fewer proteins and only to the phosphorylated form.

  20. Interactions of the NPXY microdomains of the LDL Receptor-Related Protein 1

    Science.gov (United States)

    Guttman, Miklos; Betts, Gina N.; Barnes, Helen; Ghassemian, Majid; van der Geer, Peter; Komives, Elizabeth A.

    2010-01-01

    The LDL receptor-related protein 1 (LRP1) mediates internalization of a large number of proteins and protein-lipid complexes and is widely implicated in Alzheimer's disease. The cytoplasmic domain of LRP1 (LRP1-CT) can be phosphorylated by activated protein-tyrosine kinases at two NPXY motifs in LRP1-CT; Tyr 4507 is readily phosphorylated and must be phosphorylated before phosphorylation of Tyr 4473 occurs. Pull-down experiments from brain lysate revealed numerous proteins binding to LRP1-CT, but the results were highly variable. To separate which proteins bind to each NPXY motif and their phosphorylation dependence, each NPXY motif microdomain was prepared in both phosphorylated and non-phosphorylated forms and used to probe rodent brain extracts for binding proteins. Proteins that bound specifically to the microdomains were identified by LC-MS/MS, and confirmed by western blot. Recombinant proteins were then tested for binding to each NPXY motif. The NPXY4507 (membrane distal) was found to interact with a large number of proteins, many of which only bound the tyrosine-phosphorylated form. This microdomain also bound a significant number of other proteins in the unphosphorylated state. Many of the interactions were later confirmed to be direct with recombinant proteins. The NPXY4473 (membrane proximal) bound many fewer proteins and only to the phosphorylated form. PMID:19771558

  1. Assessment and Challenges of Ligand Docking into Comparative Models of G-Protein Coupled Receptors

    DEFF Research Database (Denmark)

    Nguyen, E.D.; Meiler, J.; Norn, C.

    2013-01-01

    The rapidly increasing number of high-resolution X-ray structures of G-protein coupled receptors (GPCRs) creates a unique opportunity to employ comparative modeling and docking to provide valuable insight into the function and ligand binding determinants of novel receptors, to assist in virtual...... and side-chain conformational space with Rosetta can be leveraged to meet this challenge. This study performs unbiased comparative modeling and docking methodologies using 14 distinct high-resolution GPCRs and proposes knowledge-based filtering methods for improvement of sampling performance...... and identification of correct ligand-receptor interactions. On average, top ranked receptor models built on template structures over 50% sequence identity are within 2.9 Å of the experimental structure, with an average root mean square deviation (RMSD) of 2.2 Å for the transmembrane region and 5 Å for the second...

  2. Broad-spectrum L-amino acid sensing by class 3 G-protein-coupled receptors.

    Science.gov (United States)

    Conigrave, Arthur D; Hampson, David R

    2006-12-01

    The sensing of nutrients is essential to the control of growth and metabolism. Although the sensing mechanisms responsible for the detection and coordination of metabolic responses to some nutrients, most notably glucose, are well understood, the molecular basis of amino acid sensing by cells and tissues is only now emerging. In this article, we consider evidence that some members of G-protein-coupled receptor class 3 are broad-spectrum amino acid sensors that couple changes in extracellular amino acid levels to the activation of intracellular signaling pathways. In particular, we consider both the molecular basis of specific and broad-spectrum amino acid sensing by different members of class 3 and the physiological significance of broad spectrum amino acid sensing by the extracellular calcium-sensing receptor, heterodimeric taste receptors and the recently "deorphanized" receptor GPRC6A and its goldfish homolog, the 5.24 chemoreceptor.

  3. 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...

  4. From atomic structures to neuronal functions of g protein-coupled receptors.

    Science.gov (United States)

    Palczewski, Krzysztof; Orban, Tivadar

    2013-07-08

    G protein-coupled receptors (GPCRs) are essential mediators of signal transduction, neurotransmission, ion channel regulation, and other cellular events. GPCRs are activated by diverse stimuli, including light, enzymatic processing of their N-termini, and binding of proteins, peptides, or small molecules such as neurotransmitters. GPCR dysfunction caused by receptor mutations and environmental challenges contributes to many neurological diseases. Moreover, modern genetic technology has helped identify a rich array of mono- and multigenic defects in humans and animal models that connect such receptor dysfunction with disease affecting neuronal function. The visual system is especially suited to investigate GPCR structure and function because advanced imaging techniques permit structural studies of photoreceptor neurons at both macro and molecular levels that, together with biochemical and physiological assessment in animal models, provide a more complete understanding of GPCR signaling.

  5. Transferrin protein nanospheres: a nanoplatform for receptor-mediated cancer cell labeling and gene delivery

    Science.gov (United States)

    McDonald, Michael A.; Spurlin, Tighe A.; Tona, Alessandro; Elliott, John T.; Halter, Michael; Plant, Anne L.

    2010-02-01

    This paper presents preliminary results on the use of transferrin protein nanospheres (TfpNS) for targeting cancer cells in vitro. Protein nanospheres represent an easily prepared and modifiable nanoplatform for receptor-specific targeting, molecular imaging and gene delivery. Rhodamine B isothiocyanate conjugated TfpNS (RBITC-TfpNS) show significantly enhanced uptake in vitro in SK-MEL-28 human malignant melanoma cells known to overexpress transferrin receptors compared to controls. RBITCTfpNS labeling of the cancer cells is due to transferrin receptor-mediated uptake, as demonstrated by competitive inhibition with native transferrin. Initial fluorescence microscopy studies indicate GFP plasmid can be transfected into melanoma cells via GFP plasmid encapsulated by TfpNS.

  6. GPCR-OKB: the G Protein Coupled Receptor Oligomer Knowledge Base.

    NARCIS (Netherlands)

    Khelashvili, G.; Dorff, K.; Shan, J.; Camacho-Artacho, M.; Skrabanek, L.; Vroling, B.; Bouvier, M.; Devi, L.A.; George, S.R.; Javitch, J.A.; Lohse, M.J.; Milligan, G.; Neubig, R.R.; Palczewski, K.; Parmentier, M.; Pin, J.P.; Vriend, G.; Campagne, F.; Filizola, M.

    2010-01-01

    SUMMARY: Rapid expansion of available data about G Protein Coupled Receptor (GPCR) dimers/oligomers over the past few years requires an effective system to organize this information electronically. Based on an ontology derived from a community dialog involving colleagues using experimental and

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

    NARCIS (Netherlands)

    Liebscher, Ines; Ackley, Brian; Araç, Demet; Ariestanti, Donna M.; Aust, Gabriela; Bae, Byoung-Il; Bista, Bigyan R.; Bridges, James P.; Duman, Joseph G.; Engel, Felix B.; Giera, Stefanie; Goffinet, André M.; Hall, Randy A.; Hamann, Jörg; Hartmann, Nicole; Lin, Hsi-Hsien; Liu, Mingyao; Luo, Rong; Mogha, Amit; Monk, Kelly R.; Peeters, Miriam C.; Prömel, Simone; Ressl, Susanne; Schiöth, Helgi B.; Sigoillot, Séverine M.; Song, Helen; Talbot, William S.; Tall, Gregory G.; White, James P.; Wolfrum, Uwe; Xu, Lei; Piao, Xianhua

    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 alpha-helix-a structural feature of all GPCRs-the class of aGPCRs is characterized by the presence of a large N-terminal extracellular

  8. Vitamin D receptor protein is associated with interleukin-6 in human skeletal muscle

    Science.gov (United States)

    Vitamin D is associated with skeletal muscle physiology and function and may play a role in intramuscular inflammation, possibly via the vitamin D receptor (VDR). We conducted two studies to examine (1) whether serum 25-hydroxyvitamin D (25OHD) and/or intramuscular VDR protein concentrations are ass...

  9. COLOCALIZATION OF MUSCARINIC ACETYLCHOLINE-RECEPTORS AND PROTEIN KINASE-C-GAMMA IN RAT PARIETAL CORTEX

    NARCIS (Netherlands)

    VANDERZEE, EA; STROSBERG, AD; BOHUS, B; LUITEN, PGM

    The present investigation analyzes the cellular distribution of muscarinic acetylcholine receptors (mAChRs) and the gamma isoform of protein kinase C (PKC) in the rat parietal cortex employing the monoclonal antibodies M35 and 36G9, respectively. Muscarinic cholinoceptive neurons were most present

  10. Subcellular localization and oligomerization of the Arabidopsis thaliana somatic embryogenesis receptor kinase 1 protein

    NARCIS (Netherlands)

    Shah, K.; Gadella, T.W.J.; Erp, van H.; Hecht, V.; Vries, de S.C.

    2001-01-01

    The Arabidopsis thaliana somatic embryogenesis receptor kinase 1 (AtSERK1) gene is expressed in developing ovules and early embryos. AtSERK1 is also transiently expressed during somatic embryogenesis. The predicted AtSERK1 protein contains an extracellular domain with a leucine zipper motif followed

  11. Subcellular localization and oligomerization of the Aradopsis thaliana somatic embryogenesis receptor kinase 1 protein

    NARCIS (Netherlands)

    Shah, K.; Gadella, Th.W.J.; van Erp, H.; Hecht, V.; de Vries, S.C.

    2001-01-01

    The Arabidopsis thaliana somatic embryogenesis receptor kinase 1 (AtSERK1) gene is expressed in developing ovules and early embryos. AtSERK1 is also transiently expressed during somatic embryogenesis. The predicted AtSERK1 protein contains an extracellular domain with a leucine zipper motif followed

  12. LDL receptor-related protein 5 (LRP5) affects bone accrual and eye development

    NARCIS (Netherlands)

    Gong, Y.; Slee, R. B.; Fukai, N.; Rawadi, G.; Roman-Roman, S.; Reginato, A. M.; Wang, H.; Cundy, T.; Glorieux, F. H.; Lev, D.; Zacharin, M.; Oexle, K.; Marcelino, J.; Suwairi, W.; Heeger, S.; Sabatakos, G.; Apte, S.; Adkins, W. N.; Allgrove, J.; Arslan-Kirchner, M.; Batch, J. A.; Beighton, P.; Black, G. C.; Boles, R. G.; Boon, L. M.; Borrone, C.; Brunner, H. G.; Carle, G. F.; Dallapiccola, B.; de Paepe, A.; Floege, B.; Halfhide, M. L.; Hall, B.; Hennekam, R. C.; Hirose, T.; Jans, A.; Jüppner, H.; Kim, C. A.; Keppler-Noreuil, K.; Kohlschuetter, A.; Lacombe, D.; Lambert, M.; Lemyre, E.; Letteboer, T.; Peltonen, L.; Ramesar, R. S.; Romanengo, M.; Somer, H.; Steichen-Gersdorf, E.; Steinmann, B.; Sullivan, B.; Superti-Furga, A.; Swoboda, W.; van den Boogaard, M. J.; van Hul, W.; Vikkula, M.; Votruba, M.; Zabel, B.; Garcia, T.; Baron, R.; Olsen, B. R.; Warman, M. L.

    2001-01-01

    In humans, low peak bone mass is a significant risk factor for osteoporosis. We report that LRP5, encoding the low-density lipoprotein receptor-related protein 5, affects bone mass accrual during growth. Mutations in LRP5 cause the autosomal recessive disorder osteoporosis-pseudoglioma syndrome

  13. LDL receptor-related protein 5 (LRP5) affects bone accrual and eye development.

    NARCIS (Netherlands)

    Gong, Y.; Slee, R.B.; Fukai, N.; Rawadi, G.; Roman-Roman, S.; Reginato, A.M.; Wang, H.; Cundy, T.; Glorieux, F.H.; Lev, D.; Zacharin, M.; Oexle, K.; Marcelino, J.; Suwairi, W.; Heeger, S.; Sabatakos, G.; Apte, S.; Adkins, W.; Allgrove, J.; Arslan-Kirchner, M.; Batch, J.A.; Beighton, P.; Black, G.C.M.; Boles, R.G.; Boon, L.; Borrone, C.; Brunner, H.G.; Carle, G.F.; Dallapicola, B.; Paepe, A. de; Floege, B.; Halfhide, M.L.; Hall, B.D.; Hennekam, R.C.M.; Hirose, T.; Jans, A.; Juppner, H.; Kim, C.; Keppler-Noreuil, K.; Kohlschuetter, A.; Lacombe, D.; Lambert, M.; Lemyre, E.; Letteboer, T.; Peltonen, L.; Ramesar, R.S.; Romanengo, M.; Somer, H.; Steichen-Gersdorf, E.; Steinmann, B.; Sullivan, B.; Superti-Furga, A.; Swoboda, W.; Boogaard, M.J. van den; Hul, W. van; Vikkula, M.; Votruba, M.; Zabel, B.; Garcia, T.; Baron, R.; Olsen, B.R.; Warman, M.L.

    2001-01-01

    In humans, low peak bone mass is a significant risk factor for osteoporosis. We report that LRP5, encoding the low-density lipoprotein receptor-related protein 5, affects bone mass accrual during growth. Mutations in LRP5 cause the autosomal recessive disorder osteoporosis-pseudoglioma syndrome

  14. New insights into the structure of Class B G protein-coupled receptors

    NARCIS (Netherlands)

    Hollenstein, H.; de Graaf, C.; Bortolato, A.; Wang, M-W; Marshall, F.; Stevens, R.C.

    2014-01-01

    The secretin-like (class B) family of G protein-coupled receptors (GPCRs) are key players in hormonal homeostasis and are interesting drug targets for the treatment of several metabolic disorders (such as type 2 diabetes, osteoporosis, and obesity) and nervous system diseases (such as migraine,

  15. Wnt proteins regulate acetylcholine receptor clustering in muscle cells.

    Science.gov (United States)

    Zhang, Bin; Liang, Chuan; Bates, Ryan; Yin, Yiming; Xiong, Wen-Cheng; Mei, Lin

    2012-02-06

    The neuromuscular junction (NMJ) is a cholinergic synapse that rapidly conveys signals from motoneurons to muscle cells and exhibits a high degree of subcellular specialization characteristic of chemical synapses. NMJ formation requires agrin and its coreceptors LRP4 and MuSK. Increasing evidence indicates that Wnt signaling regulates NMJ formation in Drosophila, C. elegans and zebrafish. In the study we systematically studied the effect of all 19 different Wnts in mammals on acetylcholine receptor (AChR) cluster formation. We identified five Wnts (Wnt9a, Wnt9b, Wnt10b, Wnt11, and Wnt16) that are able to stimulate AChR clustering, of which Wnt9a and Wnt11 are expressed abundantly in developing muscles. Using Wnt9a and Wnt11 as example, we demonstrated that Wnt induction of AChR clusters was dose-dependent and non-additive to that of agrin, suggesting that Wnts may act via similar pathways to induce AChR clusters. We provide evidence that Wnt9a and Wnt11 bind directly to the extracellular domain of MuSK, to induce MuSK dimerization and subsequent tyrosine phosphorylation of the kinase. In addition, Wnt-induced AChR clustering requires LRP4. These results identify Wnts as new players in AChR cluster formation, which act in a manner that requires both MuSK and LRP4, revealing a novel function of LRP4.

  16. Wnt proteins regulate acetylcholine receptor clustering in muscle cells

    Directory of Open Access Journals (Sweden)

    Zhang Bin

    2012-02-01

    Full Text Available Abstract Background The neuromuscular junction (NMJ is a cholinergic synapse that rapidly conveys signals from motoneurons to muscle cells and exhibits a high degree of subcellular specialization characteristic of chemical synapses. NMJ formation requires agrin and its coreceptors LRP4 and MuSK. Increasing evidence indicates that Wnt signaling regulates NMJ formation in Drosophila, C. elegans and zebrafish. Results In the study we systematically studied the effect of all 19 different Wnts in mammals on acetylcholine receptor (AChR cluster formation. We identified five Wnts (Wnt9a, Wnt9b, Wnt10b, Wnt11, and Wnt16 that are able to stimulate AChR clustering, of which Wnt9a and Wnt11 are expressed abundantly in developing muscles. Using Wnt9a and Wnt11 as example, we demonstrated that Wnt induction of AChR clusters was dose-dependent and non-additive to that of agrin, suggesting that Wnts may act via similar pathways to induce AChR clusters. We provide evidence that Wnt9a and Wnt11 bind directly to the extracellular domain of MuSK, to induce MuSK dimerization and subsequent tyrosine phosphorylation of the kinase. In addition, Wnt-induced AChR clustering requires LRP4. Conclusions These results identify Wnts as new players in AChR cluster formation, which act in a manner that requires both MuSK and LRP4, revealing a novel function of LRP4.

  17. Adenosine A(2A) receptor dynamics studied with the novel fluorescent agonist Alexa488-APEC.

    Science.gov (United States)

    Brand, Frank; Klutz, Athena M; Jacobson, Kenneth A; Fredholm, Bertil B; Schulte, Gunnar

    2008-08-20

    G protein-coupled receptors, such as the adenosine A(2A) receptor, are dynamic proteins, which undergo agonist-dependent redistribution from the cell surface to intracellular membranous compartments, such as endosomes. In order to study the kinetics of adenosine A(2A) receptor redistribution in living cells, we synthesized a novel fluorescent agonist, Alexa488-APEC. Alexa488-APEC binds to adenosine A(2A) (K(i)=149+/-27 nM) as well as A(3) receptors (K(i)=240+/-160 nM) but not to adenosine A(1) receptors. Further, we characterized the dose-dependent increase in Alexa488-APEC-induced cAMP production as well as cAMP response element binding (CREB) protein phosphorylation, verifying the ligand's functionality at adenosine A(2A) but not A(2B) receptors. In live-cell imaging studies, Alexa488-APEC-induced adenosine A(2A) receptor internalization, which was blocked by the competitive reversible antagonist ZM 241385 and hyperosmolaric sucrose. Further, internalized adenosine A(2A) receptors co-localized with clathrin and Rab5, indicating that agonist stimulation promotes adenosine A(2A) receptor uptake through a clathrin-dependent mechanism to Rab5-positive endosomes. The basic characterization of Alexa488-APEC described here showed that it provides a useful tool for tracing adenosine A(2A) receptors in vitro.

  18. Impaired LDL Receptor-Related Protein 1 Translocation Correlates with Improved Dyslipidemia and Atherosclerosis in apoE-Deficient Mice

    DEFF Research Database (Denmark)

    Gordts, Philip L S M; Bartelt, Alexander; Nilsson, Stefan K

    2012-01-01

    Determination of the in vivo significance of LDL receptor-related protein 1 (LRP1) dysfunction on lipid metabolism and atherosclerosis development in absence of its main ligand apoE.......Determination of the in vivo significance of LDL receptor-related protein 1 (LRP1) dysfunction on lipid metabolism and atherosclerosis development in absence of its main ligand apoE....

  19. Evidence that the newly cloned low-density-lipoprotein receptor related protein (LRP) is the alpha 2-macroglobulin receptor

    DEFF Research Database (Denmark)

    Kristensen, T; Moestrup, Søren Kragh; Gliemann, Jørgen

    1990-01-01

    these polypeptides, and analysis of a 1772 bp cDNA encoding part of the 500 kDa polypeptide provide evidence that the 500 kDa and 85 kDa chains are the alpha- and beta-subunits, respectively, of a recently cloned hepatic membrane protein, termed the low density lipoprotein receptor related protein (LRP) (Herz, J......., Hamann, U., Rogne, S., Myklebost, O., Gausepohl, H. and Stanley, K.K. (1988) EMBO J. 7, 4119-4127; Herz, J., Kowal, R.C., Goldstein, J.L. and Brown, M.S. (1990) EMBO J. 9, 1769-1776). N-terminal sequence analysis of the 40 kDa polypeptide shows that it is of distinct genetic origin. It is suggested...

  20. Evidence that the newly cloned low-density-lipoprotein receptor related protein (LRP) is the alpha 2-macroglobulin receptor

    DEFF Research Database (Denmark)

    Kristensen, Torsten; Moestrup, Søren Kragh; Gliemann, Jørgen

    1990-01-01

    , and analysis of a 1772 bp cDNA encoding part of the 500 kDa polypeptide provide evidence that the 500 kDa and 85 kDa chains are the α- and β-subunits, respectively, of a recently cloned hepatic membrane protein, termed the low density lipoprotein receptor related protein (LRP) (Herz, J., Hamann, U., Rogne, S......., Myklebost, O., Gausepohl, H. and Stanley, K.K. (1988) EMBO J. 7, 4119-4127; Herz, J., Kowal, R.C., Goldstein, J.L. and Brown, M.S. (1990) EMBO J. 9, 1769-1776). N-terminal sequence analysis of the 40 kDa polypeptide shows that it is of distinct genetic origin. It is suggested that LRP is the functional...

  1. YMCA ROCKET RAMPAGE! SUMMER CAMP

    National Research Council Canada - National Science Library

    Anonymous

    2014-01-01

    ... & Controls, sponsored the Rocket Rampagel summer camp at the YMCA in Eklton MD. On day 1, campers took Rockets 101, constructing balloon rockets and straw rockets, followed by racket manufacturing, where campers made rocket "propellant" on day 2...

  2. Registration Day-Camp 2016

    CERN Document Server

    Nursery School

    2016-01-01

    Registration for the CERN SA Day-camp are open for children from 4 to 6 years old From March 14 to 25 for children already enrolled in CERN SA EVE and School From April 4 to 15 for the children of CERN members of the personnel (MP) From April 18 for other children More information on the website: http://nurseryschool.web.cern.ch/. The day-camp is open to all children. An inscription per week is proposed, cost 480.-CHF/week, lunch included The camp will be open weeks 27, 28, 29 and 30, from 8:30 am to 5:30 pm. For further questions, thanks you for contacting us by email at Summer.Camp@cern.ch.

  3. The receptor-binding site of the measles virus hemagglutinin protein itself constitutes a conserved neutralizing epitope.

    Science.gov (United States)

    Tahara, Maino; Ohno, Shinji; Sakai, Kouji; Ito, Yuri; Fukuhara, Hideo; Komase, Katsuhiro; Brindley, Melinda A; Rota, Paul A; Plemper, Richard K; Maenaka, Katsumi; Takeda, Makoto

    2013-03-01

    Here, we provide direct evidence that the receptor-binding site of measles virus (MV) hemagglutinin protein itself forms an effective conserved neutralizing epitope (CNE). Several receptor-interacting residues constitute the CNE. Thus, viral escape from neutralization has to be associated with loss of receptor-binding activity. Since interactions with both the signaling lymphocyte activation molecule (SLAM) and nectin4 are critical for MV pathogenesis, its escape, which results from loss of receptor-binding activity, should not occur in nature.

  4. Differential regulation of cardiac excitation–contraction coupling by cAMP phosphodiesterase subtypes

    Science.gov (United States)

    Mika, Delphine; Bobin, Pierre; Pomérance, Martine; Lechêne, Patrick; Westenbroek, Ruth E.; Catterall, William A.; Vandecasteele, Grégoire; Leroy, Jérôme; Fischmeister, Rodolphe

    2013-01-01

    Aims Multiple phosphodiesterases (PDEs) hydrolyze cAMP in cardiomyocytes, but the functional significance of this diversity is not well understood. Our goal here was to characterize the involvement of three different PDEs (PDE2–4) in cardiac excitation–contraction coupling (ECC). Methods and results Sarcomere shortening and Ca2+ transients were recorded simultaneously in adult rat ventricular myocytes and ECC protein phosphorylation by PKA was determined by western blot analysis. Under basal conditions, selective inhibition of PDE2 or PDE3 induced a small but significant increase in Ca2+ transients, sarcomere shortening, and troponin I phosphorylation, whereas PDE4 inhibition had no effect. PDE3 inhibition, but not PDE2 or PDE4, increased phospholamban phosphorylation. Inhibition of either PDE2, 3, or 4 increased phosphorylation of the myosin-binding protein C, but neither had an effect on L-type Ca2+ channel or ryanodine receptor phosphorylation. Dual inhibition of PDE2 and PDE3 or PDE2 and PDE4 further increased ECC compared with individual PDE inhibition, but the most potent combination was obtained when inhibiting simultaneously PDE3 and PDE4. This combination also induced a synergistic induction of ECC protein phosphorylation. Submaximal β-adrenergic receptor stimulation increased ECC, and this effect was potentiated by individual PDE inhibition with the rank order of potency PDE4 = PDE3 > PDE2. Identical results were obtained on ECC protein phosphorylation. Conclusion Our results demonstrate that PDE2, PDE3, and PDE4 differentially regulate ECC in adult cardiomyocytes. PDE2 and PDE3 play a more prominent role than PDE4 in regulating basal cardiac contraction and Ca2+ transients. However, PDE4 becomes determinant when cAMP levels are elevated, for instance, upon β-adrenergic stimulation or PDE3 inhibition. PMID:23933582

  5. Differential regulation of cardiac excitation-contraction coupling by cAMP phosphodiesterase subtypes.

    Science.gov (United States)

    Mika, Delphine; Bobin, Pierre; Pomérance, Martine; Lechêne, Patrick; Westenbroek, Ruth E; Catterall, William A; Vandecasteele, Grégoire; Leroy, Jérôme; Fischmeister, Rodolphe

    2013-11-01

    Multiple phosphodiesterases (PDEs) hydrolyze cAMP in cardiomyocytes, but the functional significance of this diversity is not well understood. Our goal here was to characterize the involvement of three different PDEs (PDE2-4) in cardiac excitation-contraction coupling (ECC). Sarcomere shortening and Ca(2+) transients were recorded simultaneously in adult rat ventricular myocytes and ECC protein phosphorylation by PKA was determined by western blot analysis. Under basal conditions, selective inhibition of PDE2 or PDE3 induced a small but significant increase in Ca(2+) transients, sarcomere shortening, and troponin I phosphorylation, whereas PDE4 inhibition had no effect. PDE3 inhibition, but not PDE2 or PDE4, increased phospholamban phosphorylation. Inhibition of either PDE2, 3, or 4 increased phosphorylation of the myosin-binding protein C, but neither had an effect on L-type Ca(2+) channel or ryanodine receptor phosphorylation. Dual inhibition of PDE2 and PDE3 or PDE2 and PDE4 further increased ECC compared with individual PDE inhibition, but the most potent combination was obtained when inhibiting simultaneously PDE3 and PDE4. This combination also induced a synergistic induction of ECC protein phosphorylation. Submaximal β-adrenergic receptor stimulation increased ECC, and this effect was potentiated by individual PDE inhibition with the rank order of potency PDE4 = PDE3 > PDE2. Identical results were obtained on ECC protein phosphorylation. Our results demonstrate that PDE2, PDE3, and PDE4 differentially regulate ECC in adult cardiomyocytes. PDE2 and PDE3 play a more prominent role than PDE4 in regulating basal cardiac contraction and Ca(2+) transients. However, PDE4 becomes determinant when cAMP levels are elevated, for instance, upon β-adrenergic stimulation or PDE3 inhibition.

  6. The Hemoglobin Receptor Protein of Porphyromonas gingivalis Inhibits Receptor Activator NF-κB Ligand-Induced Osteoclastogenesis from Bone Marrow Macrophages

    OpenAIRE

    Fujimura, Yuji; Hotokezaka, Hitoshi; Ohara, Naoya; Naito, Mariko; Sakai, Eiko; Yoshimura, Mamiko; Narita, Yuka; Kitaura, Hideki; Yoshida, Noriaki; Nakayama, Koji

    2006-01-01

    Extracellular proteinaceous factors of Porphyromonas gingivalis, a periodontal pathogen, that influence receptor activator of nuclear factor-κB (NF-κB) ligand (RANKL)-induced osteoclastogenesis from bone marrow macrophages were investigated. The culture supernatant of P. gingivalis had the ability to inhibit RANKL-induced in vitro osteoclastogenesis. A major protein of the culture supernatant, hemoglobin receptor protein (HbR), suppressed RANKL-induced osteoclastogenesis in a dose-dependent f...

  7. Chlorella intake attenuates reduced salivary SIgA secretion in kendo training camp participants

    Directory of Open Access Journals (Sweden)

    Otsuki Takeshi

    2012-12-01

    Full Text Available Abstract Background The green alga Chlorella contains high levels of proteins, vitamins, and minerals. We previously reported that a chlorella-derived multicomponent supplement increased the secretion rate of salivary secretory immunoglobulin A (SIgA in humans. Here, we investigated whether intake of this chlorella-derived supplement attenuated the reduced salivary SIgA secretion rate during a kendo training camp. Methods Ten female kendo athletes participated in inter-university 6-day spring and 4-day summer camps. They were randomized into two groups; one took placebo tablets during the spring camp and chlorella tablets during the summer camp, while the other took chlorella tablets during the spring camp and placebo tablets during the summer camp. Subjects took these tablets starting 4 weeks before the camp until post-camp saliva sampling. Salivary SIgA concentrations were measured by ELISA. Results All subjects participated in nearly all training programs, and body-mass changes and subjective physical well-being scores during the camps were comparable between the groups. However, salivary SIgA secretion rate changes were different between these groups. Salivary SIgA secretion rates decreased during the camp in the placebo group (before vs. second, middle, and final day of camp, and after the camp: 146 ± 89 vs. 87 ± 56, 70 ± 45, 94 ± 58, and 116 ± 71 μg/min, whereas no such decreases were observed in the chlorella group (121 ± 53 vs. 113 ± 68, 98 ± 69,115 ± 80, and 128 ± 59 μg/min. Conclusion Our results suggest that a use of a chlorella-derived dietary supplement attenuates reduced salivary SIgA secretion during a training camp for a competitive sport.

  8. Chlorella intake attenuates reduced salivary SIgA secretion in kendo training camp participants

    Science.gov (United States)

    2012-01-01

    Background The green alga Chlorella contains high levels of proteins, vitamins, and minerals. We previously reported that a chlorella-derived multicomponent supplement increased the secretion rate of salivary secretory immunoglobulin A (SIgA) in humans. Here, we investigated whether intake of this chlorella-derived supplement attenuated the reduced salivary SIgA secretion rate during a kendo training camp. Methods Ten female kendo athletes participated in inter-university 6-day spring and 4-day summer camps. They were randomized into two groups; one took placebo tablets during the spring camp and chlorella tablets during the summer camp, while the other took chlorella tablets during the spring camp and placebo tablets during the summer camp. Subjects took these tablets starting 4 weeks before the camp until post-camp saliva sampling. Salivary SIgA concentrations were measured by ELISA. Results All subjects participated in nearly all training programs, and body-mass changes and subjective physical well-being scores during the camps were comparable between the groups. However, salivary SIgA secretion rate changes were different between these groups. Salivary SIgA secretion rates decreased during the camp in the placebo group (before vs. second, middle, and final day of camp, and after the camp: 146 ± 89 vs. 87 ± 56, 70 ± 45, 94 ± 58, and 116 ± 71 μg/min), whereas no such decreases were observed in the chlorella group (121 ± 53 vs. 113 ± 68, 98 ± 69,115 ± 80, and 128 ± 59 μg/min). Conclusion Our results suggest that a use of a chlorella-derived dietary supplement attenuates reduced salivary SIgA secretion during a training camp for a competitive sport. PMID:23227811

  9. 5D imaging approaches reveal the formation of distinct intracellular cAMP spatial gradients

    Science.gov (United States)

    Rich, Thomas C.; Annamdevula, Naga; Trinh, Kenny; Britain, Andrea L.; Mayes, Samuel A.; Griswold, John R.; Deal, Joshua; Hoffman, Chase; West, Savannah; Leavesley, Silas J.

    2017-02-01

    Cyclic AMP (cAMP) is a ubiquitous second messenger known to differentially regulate many cellular functions. Several lines of evidence suggest that the distribution of cAMP within cells is not uniform. However, to date, no studies have measured the kinetics of 3D cAMP distributions within cells. This is largely due to the low signal-tonoise ratio of FRET-based probes. We previously reported that hyperspectral imaging improves the signal-to-noise ratio of FRET measurements. Here we utilized hyperspectral imaging approaches to measure FRET signals in five dimensions (5D) - three spatial (x, y, z), wavelength (λ), and time (t) - allowing us to visualize cAMP gradients in pulmonary endothelial cells. cAMP levels were measured using a FRET-based sensor (H188) comprised of a cAMP binding domain sandwiched between FRET donor and acceptor - Turquoise and Venus fluorescent proteins. We observed cAMP gradients in response to 0.1 or 1 μM isoproterenol, 0.1 or 1 μM PGE1, or 50 μM forskolin. Forskolin- and isoproterenol-induced cAMP gradients formed from the apical (high cAMP) to basolateral (low cAMP) face of cells. In contrast, PGE1-induced cAMP gradients originated from both the basolateral and apical faces of cells. Data suggest that 2D (x,y) studies of cAMP compartmentalization may lead to erroneous conclusions about the existence of cAMP gradients, and that 3D (x,y,z) studies are required to assess mechanisms of signaling specificity. Results demonstrate that 5D imaging technologies are powerful tools for measuring biochemical processes in discrete subcellular domains.

  10. Basic Concepts in G-Protein-Coupled Receptor Homo- and Heterodimerization

    Directory of Open Access Journals (Sweden)

    Rafael Franco

    2007-01-01

    Full Text Available Until recently, heptahelical G-protein-coupled receptors (GPCRs were considered to be expressed as monomers on the cell surface of neuronal and non-neuronal cells. It is now becoming evident that this view must be overtly changed since these receptors can form homodimers, heterodimers, and higher-order oligomers on the plasma membrane. Here we discuss some of the basics and some new concepts of receptor homo- and heteromerization. Dimers-oligomers modify pharmacology, trafficking, and signaling of receptors. First of all, GPCR dimers must be considered as the main molecules that are targeted by neurotransmitters or by drugs. Thus, binding data must be fitted to dimer-based models. In these models, it is considered that the conformational changes transmitted within the dimer molecule lead to cooperativity. Cooperativity must be taken into account in the binding of agonists-antagonists-drugs and also in the binding of the so-called allosteric modulators. Cooperativity results from the intramolecular cross-talk in the homodimer. As an intramolecular cross-talk in the heterodimer, the binding of one neurotransmitter to one receptor often affects the binding of the second neurotransmitter to the partner receptor. Coactivation of the two receptors in a heterodimer can change completely the signaling pathway triggered by the neurotransmitter as well as the trafficking of the receptors. Heterodimer-specific drugs or dual drugs able to activate the two receptors in the heterodimer simultaneously emerge as novel and promising drugs for a variety of central nervous system (CNS therapeutic applications.

  11. Improved methodical approach for quantitative BRET analysis of G Protein Coupled Receptor dimerization.

    Directory of Open Access Journals (Sweden)

    Bence Szalai

    Full Text Available G Protein Coupled Receptors (GPCR can form dimers or higher ordered oligomers, the process of which can remarkably influence the physiological and pharmacological function of these receptors. Quantitative Bioluminescence Resonance Energy Transfer (qBRET measurements are the gold standards to prove the direct physical interaction between the protomers of presumed GPCR dimers. For the correct interpretation of these experiments, the expression of the energy donor Renilla luciferase labeled receptor has to be maintained constant, which is hard to achieve in expression systems. To analyze the effects of non-constant donor expression on qBRET curves, we performed Monte Carlo simulations. Our results show that the decrease of donor expression can lead to saturation qBRET curves even if the interaction between donor and acceptor labeled receptors is non-specific leading to false interpretation of the dimerization state. We suggest here a new approach to the analysis of qBRET data, when the BRET ratio is plotted as a function of the acceptor labeled receptor expression at various donor receptor expression levels. With this method, we were able to distinguish between dimerization and non-specific interaction when the results of classical qBRET experiments were ambiguous. The simulation results were confirmed experimentally using rapamycin inducible heterodimerization system. We used this new method to investigate the dimerization of various GPCRs, and our data have confirmed the homodimerization of V2 vasopressin and CaSR calcium sensing receptors, whereas our data argue against the heterodimerization of these receptors with other studied GPCRs, including type I and II angiotensin, β2 adrenergic and CB1 cannabinoid receptors.

  12. Base Camp Architecture

    Directory of Open Access Journals (Sweden)

    Warebi Gabriel Brisibe

    2016-03-01

    Full Text Available Longitudinal or time line studies of change in the architecture of a particular culture are common, but an area still open to further research is change across space or place. In particular, there is need for studies on architectural change of cultures stemming from the same ethnic source split between their homeland and other Diasporas. This change may range from minor deviations to drastic shifts away from an architectural norm and the accumulation of these shifts within a time frame constitutes variations. This article focuses on identifying variations in the architecture of the Ijo fishing group that migrates along the coastline of West Africa. It examines the causes of cross-cultural variation between base camp dwellings of Ijo migrant fishermen in the Bakassi Peninsula in Cameroon and Bayelsa State in Nigeria. The study draws on the idea of the inevitability of cultural and social change over time as proposed in the theories of cultural dynamism and evolution. It tests aspects of cultural transmission theory using the principal coordinates analysis to ascertain the possible causes of variation. From the findings, this research argues that migration has enhanced the forces of cultural dynamism, which have resulted in significant variations in the architecture of this fishing group.

  13. Putative Receptor Binding Domain of Bat-Derived Coronavirus HKU9 Spike Protein: Evolution of Betacoronavirus Receptor Binding Motifs.

    Science.gov (United States)

    Huang, Canping; Qi, Jianxun; Lu, Guangwen; Wang, Qihui; Yuan, Yuan; Wu, Ying; Zhang, Yanfang; Yan, Jinghua; Gao, George F

    2016-11-01

    The suggested bat origin for Middle East respiratory syndrome coronavirus (MERS-CoV) has revitalized the studies of other bat-derived coronaviruses with respect to interspecies transmission potential. Bat coronavirus (BatCoV) HKU9 is an important betacoronavirus (betaCoV) that is phylogenetically affiliated with the same genus as MERS-CoV. The bat surveillance data indicated that BatCoV HKU9 has been widely spreading and circulating in bats. This highlights the necessity of characterizing the virus for its potential to cross species barriers. The receptor binding domain (RBD) of the coronavirus spike (S) protein recognizes host receptors to mediate virus entry and is therefore a key factor determining the viral tropism and transmission capacity. In this study, the putative S RBD of BatCoV HKU9 (HKU9-RBD), which is homologous to other betaCoV RBDs that have been structurally and functionally defined, was characterized via a series of biophysical and crystallographic methods. By using surface plasmon resonance, we demonstrated that HKU9-RBD binds to neither SARS-CoV receptor ACE2 nor MERS-CoV receptor CD26. We further determined the atomic structure of HKU9-RBD, which as expected is composed of a core and an external subdomain. The core subdomain fold resembles those of other betaCoV RBDs, whereas the external subdomain is structurally unique with a single helix, explaining the inability of HKU9-RBD to react with either ACE2 or CD26. Via comparison of the available RBD structures, we further proposed a homologous intersubdomain binding mode in betaCoV RBDs that anchors the external subdomain to the core subdomain. The revealed RBD features would shed light on the evolution route of betaCoV.

  14. Characterization of glucagon-like peptide-1 receptor beta-arrestin 2 interaction: a high-affinity receptor phenotype

    DEFF Research Database (Denmark)

    Jorgensen, Rasmus; Martini, Lene; Schwartz, Thue W

    2005-01-01

    that (beta)arr2 interaction locks the receptor in a high-affinity conformation, which can be explored by some, but not all, ligands. The fusion constructs adopted a signaling phenotype governed by the tethered (beta)arr2 with an attenuated G protein-mediated cAMP signal and a higher maximal internalization...... for the fusion constructs was observed. We conclude that the glucagon-like peptide 1 fusion construct mimics the natural interaction of the receptor with (beta)arr2 with respect to binding peptide ligands, G protein-mediated signaling and internalization, and that this distinct molecular phenotype is reminiscent......To dissect the interaction between beta-arrestin ((beta)arr) and family B G protein-coupled receptors, we constructed fusion proteins between the glucagon-like peptide 1 receptor and (beta)arr2. The fusion constructs had an increase in apparent affinity selectively for glucagon, suggesting...

  15. Structure and Function of Cross-class Complexes of G Protein-coupled Secretin and Angiotensin 1a Receptors.

    Science.gov (United States)

    Harikumar, Kaleeckal G; Augustine, Mary Lou; Lee, Leo T O; Chow, Billy K C; Miller, Laurence J

    2016-08-12

    Complexes of secretin (SecR) and angiotensin 1a (Atr1a) receptors have been proposed to be functionally important in osmoregulation, providing an explanation for overlapping and interdependent functions of hormones that bind and activate different classes of GPCRs. However, the nature of these cross-class complexes has not been well characterized and their signaling properties have not been systematically explored. We now use competitive inhibition of receptor bioluminescence resonance energy transfer and bimolecular fluorescence complementation to establish the dominant functionally important state as a symmetrical homodimeric form of SecR decorated by monomeric Atr1a, interacting through lipid-exposed faces of Atr1a TM1 and TM4. Conditions increasing prevalence of this complex exhibited negative allosteric modulatory impact on secretin-stimulated cAMP responses at SecR. In contrast, activating Atr1a with full agonist in such a complex exhibited a positive allosteric modulatory impact on the same signaling event. This modulation was functionally biased, with secretin-stimulated calcium responses unaffected, whereas angiotensin-stimulated calcium responses through the complex were reduced or absent. Further supporting this interpretation, Atr1a with mutations of lipid-exposed faces of TM1 and TM4 that did not affect its ability to bind or signal, could be expressed in the same cell as SecR, yet not exhibit either the negative or positive allosteric impact on cAMP observed with the inactive or activated states of wild type Atr1a on function, and not interfere with angiotensin-stimulated calcium responses like complexes with Atr1a. This may provide a more selective means of exploring the physiologic functional impact of this cross-class receptor complex without interfering with the function of either component receptor. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  16. G protein-coupled receptor kinases: more than just kinases and not only for GPCRs

    Science.gov (United States)

    Gurevich, Eugenia V.; Tesmer, John J. G.; Mushegian, Arcady; Gurevich, Vsevolod V.

    2011-01-01

    G protein-coupled receptor (GPCR) kinases (GRKs) are best known for their role in homologous desensitization of GPCRs. GRKs phosphorylate activated receptors and promote high affinity binding of arrestins, which precludes G protein coupling. GRKs have a multidomain structure, with the kinase domain inserted into a loop of a regulator of G protein signaling homology domain. Unlike many other kinases, GRKs do not need to be phosphorylated in their activation loop to achieve an activated state. Instead, they are directly activated by docking with active GPCRs. In this manner they are able to selectively phosphorylate Ser/Thr residues on only the activated form of the receptor, unlike related kinases such as protein kinase A. GRKs also phosphorylate a variety of non-GPCR substrates and regulate several signaling pathways via direct interactions with other proteins in a phosphorylation-independent manner. Multiple GRK subtypes are present in virtually every animal cell, with the highest expression levels found in neurons, with their extensive and complex signal regulation. Insufficient or excessive GRK activity was implicated in a variety of human disorders, ranging from heart failure to depression to Parkinson’s disease. As key regulators of GPCR-dependent and -independent signaling pathways, GRKs are emerging drug targets and promising molecular tools for therapy. Targeted modulation of expression and/or of activity of several GRK isoforms for therapeutic purposes was recently validated in cardiac disorders and Parkinson’s disease. PMID:21903131

  17. 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

  18. Dimerization of Receptor Protein-Tyrosine Phosphatase alpha in living cells

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    Gadella Theodorus WJ

    2001-06-01

    Full Text Available Abstract Background Dimerization is an important regulatory mechanism of single membrane-spanning receptors. For instance, activation of receptor protein-tyrosine kinases (RPTKs involves dimerization. Structural, functional and biochemical studies suggested that the enzymatic counterparts of RPTKs, the receptor protein-tyrosine phosphatases (RPTPs, are inhibited by dimerization, but whether RPTPs actually dimerize in living cells remained to be determined. Results In order to assess RPTP dimerization, we have assayed Fluorescence Resonance Energy Transfer (FRET between chimeric proteins of cyan- and yellow-emitting derivatives of green fluorescent protein, fused to RPTPα, using three different techniques: dual wavelength excitation, spectral imaging and fluorescence lifetime imaging. All three techniques suggested that FRET occurred between RPTPα -CFP and -YFP fusion proteins, and thus that RPTPα dimerized in living cells. RPTPα dimerization was constitutive, extensive and specific. RPTPα dimerization was consistent with cross-linking experiments, using a non-cell-permeable chemical cross-linker. Using a panel of deletion mutants, we found that the transmembrane domain was required and sufficient for dimerization. Conclusions We demonstrate here that RPTPα dimerized constitutively in living cells, which may be mediated by the transmembrane domain, providing strong support for the model that dimerization is involved in regulation of RPTPs.

  19. Integrating Pharmacophore into Membrane Molecular Dynamics Simulations to Improve Homology Modeling of G Protein-coupled Receptors with Ligand Selectivity: A2A Adenosine Receptor as an Example.

    Science.gov (United States)

    Zeng, Lingxiao; Guan, Mengxin; Jin, Hongwei; Liu, Zhenming; Zhang, Liangren

    2015-12-01

    Homology modeling has been applied to fill in the gap in experimental G protein-coupled receptors structure determination. However, achievement of G protein-coupled receptors homology models with ligand selectivity remains challenging due to structural diversity of G protein-coupled receptors. In this work, we propose a novel strategy by integrating pharmacophore and membrane molecular dynamics (MD) simulations to improve homology modeling of G protein-coupled receptors with ligand selectivity. To validate this integrated strategy, the A2A adenosine receptor (A2A AR), whose structures in both active and inactive states have been established, has been chosen as an example. We performed blind predictions of the active-state A2A AR structure based on the inactive-state structure and compared the performance of different refinement strategies. The blind prediction model combined with the integrated strategy identified ligand-receptor interactions and conformational changes of key structural elements related to the activation of A2 A AR, including (i) the movements of intracellular ends of TM3 and TM5/TM6; (ii) the opening of ionic lock; (iii) the movements of binding site residues. The integrated strategy of pharmacophore with molecular dynamics simulations can aid in the optimization in the identification of side chain conformations in receptor models. This strategy can be further investigated in homology modeling and expand its applicability to other G protein-coupled receptor modeling, which should aid in the discovery of more effective and selective G protein-coupled receptor ligands. © 2015 John Wiley & Sons A/S.

  20. Structural basis for chemokine recognition and activation of a viral G protein-coupled receptor

    Energy Technology Data Exchange (ETDEWEB)

    Burg, John S.; Ingram, Jessica R.; Venkatakrishnan, A.J.; Jude, Kevin M.; Dukkipati, Abhiram; Feinberg, Evan N.; Angelini, Alessandro; Waghray, Deepa; Dror, Ron O.; Ploegh, Hidde L.; Garcia, K. Christopher (Stanford); (Stanford-MED); (Whitehead); (MIT)

    2015-03-05

    Chemokines are small proteins that function as immune modulators through activation of chemokine G protein-coupled receptors (GPCRs). Several viruses also encode chemokines and chemokine receptors to subvert the host immune response. How protein ligands activate GPCRs remains unknown. We report the crystal structure at 2.9 angstrom resolution of the human cytomegalovirus GPCR US28 in complex with the chemokine domain of human CX3CL1 (fractalkine). The globular body of CX3CL1 is perched on top of the US28 extracellular vestibule, whereas its amino terminus projects into the central core of US28. The transmembrane helices of US28 adopt an active-state-like conformation. Atomic-level simulations suggest that the agonist-independent activity of US28 may be due to an amino acid network evolved in the viral GPCR to destabilize the receptor’s inactive state.

  1. Sorting by the cytoplasmic domain of the amyloid precursor protein binding receptor SorLA

    DEFF Research Database (Denmark)

    Nielsen, Morten S; Gustafsen, Camilla; Madsen, Peder

    2007-01-01

    -formation with the amyloid precursor protein it downregulates generation of Alzheimer's disease-associated Abeta-peptide. The receptor is mainly located in vesicles, suggesting a function in protein sorting and transport. Here we examined SorLA's trafficking using full-length and chimeric receptors and find that its...... cytoplasmic tail mediates efficient Golgi body-endosome transport, as well as AP-2 complex-dependent endocytosis. Functional sorting sites were mapped to an acidic cluster-dileucine-like motif and to a GGA binding site in the C terminus. Experiments in permanently or transiently AP-1 mu1-chain-deficient cells...... established that the AP-1 adaptor complex is essential to SorLA's transport between Golgi membranes and endosomes. Our results further implicate the GGA proteins in SorLA trafficking and provide evidence that SNX1 and Vps35, as parts of the retromer complex or possibly in a separate context, are engaged...

  2. Molecular recognition of parathyroid hormone by its G protein-coupled receptor

    Energy Technology Data Exchange (ETDEWEB)

    Pioszak, Augen A.; Xu, H. Eric (Van Andel)

    2008-08-07

    Parathyroid hormone (PTH) is central to calcium homeostasis and bone maintenance in vertebrates, and as such it has been used for treating osteoporosis. It acts primarily by binding to its receptor, PTH1R, a member of the class B G protein-coupled receptor (GPCR) family that also includes receptors for glucagon, calcitonin, and other therapeutically important peptide hormones. Despite considerable interest and much research, determining the structure of the receptor-hormone complex has been hindered by difficulties in purifying the receptor and obtaining diffraction-quality crystals. Here, we present a method for expression and purification of the extracellular domain (ECD) of human PTH1R engineered as a maltose-binding protein (MBP) fusion that readily crystallizes. The 1.95-{angstrom} structure of PTH bound to the MBP-PTH1R-ECD fusion reveals that PTH docks as an amphipathic helix into a central hydrophobic groove formed by a three-layer {alpha}-{beta}-{beta}{alpha} fold of the PTH1R ECD, resembling a hot dog in a bun. Conservation in the ECD scaffold and the helical structure of peptide hormones emphasizes this hot dog model as a general mechanism of hormone recognition common to class B GPCRs. Our findings reveal critical insights into PTH actions and provide a rational template for drug design that targets this hormone signaling pathway.

  3. Functional and Structural Overview of G-Protein-Coupled Receptors Comprehensively Obtained from Genome Sequences

    Directory of Open Access Journals (Sweden)

    Makiko Suwa

    2011-04-01

    Full Text Available An understanding of the functional mechanisms of G-protein-coupled receptors (GPCRs is very important for GPCR-related drug design. We have developed an integrated GPCR database (SEVENS http://sevens.cbrc.jp/ that includes 64,090 reliable GPCR genes comprehensively identified from 56 eukaryote genome sequences, and overviewed the sequences and structure spaces of the GPCRs. In vertebrates, the number of receptors for biological amines, peptides, etc. is conserved in most species, whereas the number of chemosensory receptors for odorant, pheromone, etc. significantly differs among species. The latter receptors tend to be single exon type or a few exon type and show a high ratio in the numbers of GPCRs, whereas some families, such as Class B and Class C receptors, have long lengths due to the presence of many exons. Statistical analyses of amino acid residues reveal that most of the conserved residues in Class A GPCRs are found in the cytoplasmic half regions of transmembrane (TM helices, while residues characteristic to each subfamily found on the extracellular half regions. The 69 of Protein Data Bank (PDB entries of complete or fragmentary structures could be mapped on the TM/loop regions of Class A GPCRs covering 14 subfamilies.

  4. 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).

  5. Increases in cAMP, MAPK Activity and CREB Phosphorylation during REM Sleep: Implications for REM Sleep and Memory Consolidation

    OpenAIRE

    Luo, Jie; Phan, Trongha X.; Yang, Yimei; Garelick, Michael G.; Storm, Daniel R.

    2013-01-01

    The cyclic adenosine monophosphate (cAMP), mitogen-activated protein kinase (MAPK) and cAMP response element-binding protein (CREB) transcriptional pathway is required for consolidation of hippocampus-dependent memory. In mice, this pathway undergoes a circadian oscillation required for memory persistence that reaches a peak during the daytime. Since mice exhibit polyphasic sleep patterns during the day, this suggested the interesting possibility that cAMP, MAPK activity and CREB phosphorylat...

  6. Computational analysis of the CB1 carboxyl-terminus in the receptor-G protein complex.

    Science.gov (United States)

    Shim, Joong-Youn; Khurana, Leepakshi; Kendall, Debra A

    2016-04-01

    Despite the important role of the carboxyl-terminus (Ct) of the activated brain cannabinoid receptor one (CB1) in the regulation of G protein signaling, a structural understanding of interactions with G proteins is lacking. This is largely due to the highly flexible nature of the CB1 Ct that dynamically adapts its conformation to the presence of G proteins. In the present study, we explored how the CB1 Ct can interact with the G protein by building on our prior modeling of the CB1-Gi complex (Shim, Ahn, and Kendall, The Journal of Biological Chemistry 2013;288:32449-32465) to incorporate a complete CB1 Ct (Glu416(Ct)-Leu472(Ct)). Based on the structural constraints from NMR studies, we employed ROSETTA to predict tertiary folds, ZDOCK to predict docking orientation, and molecular dynamics (MD) simulations to obtain two distinct plausible models of CB1 Ct in the CB1-Gi complex. The resulting models were consistent with the NMR-determined helical structure (H9) in the middle region of the CB1 Ct. The CB1 Ct directly interacted with both Gα and Gβ and stabilized the receptor at the Gi interface. The results of site-directed mutagenesis studies of Glu416(Ct), Asp423(Ct), Asp428(Ct), and Arg444(Ct) of CB1 Ct suggested that the CB1 Ct can influence receptor-G protein coupling by stabilizing the receptor at the Gi interface. This research provided, for the first time, models of the CB1 Ct in contact with the G protein. © 2016 Wiley Periodicals, Inc.

  7. Botulinum neurotoxin D uses synaptic vesicle protein SV2 and gangliosides as receptors.

    Directory of Open Access Journals (Sweden)

    Lisheng Peng

    2011-03-01

    Full Text Available Botulinum neurotoxins (BoNTs include seven bacterial toxins (BoNT/A-G that target presynaptic terminals and act as proteases cleaving proteins required for synaptic vesicle exocytosis. Here we identified synaptic vesicle protein SV2 as the protein receptor for BoNT/D. BoNT/D enters cultured hippocampal neurons via synaptic vesicle recycling and can bind SV2 in brain detergent extracts. BoNT/D failed to bind and enter neurons lacking SV2, which can be rescued by expressing one of the three SV2 isoforms (SV2A/B/C. Localization of SV2 on plasma membranes mediated BoNT/D binding in both neurons and HEK293 cells. Furthermore, chimeric receptors containing the binding sites for BoNT/A and E, two other BoNTs that use SV2 as receptors, failed to mediate the entry of BoNT/D suggesting that BoNT/D binds SV2 via a mechanism distinct from BoNT/A and E. Finally, we demonstrated that gangliosides are essential for the binding and entry of BoNT/D into neurons and for its toxicity in vivo, supporting a double-receptor model for this toxin.

  8. Repulsive guidance molecule (RGMa), a DRAGON homologue, is a bone morphogenetic protein co-receptor.

    Science.gov (United States)

    Babitt, Jodie L; Zhang, Ying; Samad, Tarek A; Xia, Yin; Tang, Jie; Campagna, Jason A; Schneyer, Alan L; Woolf, Clifford J; Lin, Herbert Y

    2005-08-19

    Bone morphogenetic proteins (BMPs) are members of the transforming growth factor beta (TGF-beta) superfamily of ligands, which regulate many mammalian physiologic and pathophysiologic processes. BMPs exert their effects through type I and type II serine/threonine kinase receptors and the Smad intracellular signaling pathway. Recently, the glycosylphosphatidylinositol (GPI)-anchored protein DRAGON was identified as a co-receptor for BMP signaling. Here, we investigate whether a homologue of DRAGON, repulsive guidance molecule (RGMa), is similarly involved in the BMP signaling pathway. We show that RGMa enhances BMP, but not TGF-beta, signals in a ligand-dependent manner in cell culture. The soluble extracellular domain of RGMa fused to human Fc (RGMa.Fc) forms a complex with BMP type I receptors and binds directly and selectively to radiolabeled BMP-2 and BMP-4. RGMa mediates BMP signaling through the classical BMP signaling pathway involving Smad1, 5, and 8, and it up-regulates endogenous inhibitor of differentiation (Id1) protein, an important downstream target of BMP signals. Finally, we demonstrate that BMP signaling occurs in neurons that express RGMa in vivo. These data are consistent with a role for RGMa as a BMP co-receptor.

  9. Dissection of Functional Residues in Receptor Activity-Modifying Proteins Through Phylogenetic and Statistical Analyses

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    Alfonso Benítez-Páez

    2008-01-01

    Full Text Available Type I and type-II functional divergences have been stated to highlight specific residues carrying out differential functions in evolutionary-divergent protein clusters from a single common ancestor. Briefly, type I analysis is based on residue constraints reflecting a gain of function just in one cluster of an entire family of proteins; while the type-II approach is based on residue constraints showing a different chemical nature in every cluster of a protein family. This last evidence is understood as differential functionality among clusters. The Receptor Activity-Modifying Proteins constitute a family characterized by its paralogous distribution in vertebrates. They are known as G-Protein Coupled Receptor modulators. Although several studies have determined their involvement in ligand binding, specificity, and enhancement of signal transduction, the responsible residues supporting those functions are unclear. Using different bioinformatic approaches, we predicted residues involved in different RAMP functional tasks. Many residues localized in an extracellular coil of RAMP proteins were predicted to be under functional divergence suggesting a gain of function in their respective proteins. Interestingly, the transmembrane region also showed important results for residues playing relevant roles where most of them showed a biased distribution on the structure. A relevant role was conferred by the enrichment of type-II residues observed in their sequences. We show a collection of residues explaining possible gain of function and differential functionality in RAMP proteins. These residues are still experimentally unexplored with regards to functionality. Finally, an evolutionary history could be discerned. Mainly, the RAMP2 cluster has evolved in a higher manner than other RAMP clusters. However, a deacceleration in the aminoacid substitution rate of RAMP2 was observed in mammals. Such effect could be caused by the co-evolution of ligands and

  10. The amphioxus (Branchiostoma floridae genome contains a highly diversified set of G protein-coupled receptors

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    Schiöth Helgi B

    2008-01-01

    Full Text Available Abstract Background G protein-coupled receptors (GPCRs are one of the largest families of genes in mammals. Branchiostoma floridae (amphioxus is one of the species most closely related species to vertebrates. Results Mining and phylogenetic analysis of the amphioxus genome showed the presence of at least 664 distinct GPCRs distributed among all the main families of GPCRs; Glutamate (18, Rhodopsin (570, Adhesion (37, Frizzled (6 and Secretin (16. Surprisingly, the Adhesion GPCR repertoire in amphioxus includes receptors with many new domains not previously observed in this family. We found many Rhodopsin GPCRs from all main groups including many amine and peptide binding receptors and several previously uncharacterized expansions were also identified. This genome has however no genes coding for bitter taste receptors (TAS2, the sweet and umami (TAS1, pheromone (VR1 or VR2 or mammalian olfactory receptors. Conclusion The amphioxus genome is remarkably rich in various GPCR subtypes while the main GPCR groups known to sense exogenous substances (such as Taste 2, mammalian olfactory, nematode chemosensory, gustatory, vomeronasal and odorant receptors in other bilateral species are absent.

  11. Co-purification of A1 adenosine receptors and guanine nucleotide-binding proteins from bovine brain.

    Science.gov (United States)

    Munshi, R; Linden, J

    1989-09-05

    A1 adenosine receptors and guanine nucleotide-binding proteins (G proteins) solubilized with 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate have been co-purified from bovine cerebral cortex. A portion of solubilized receptors which displays high affinity GTP-sensitive agonist binding (40-50%) adheres tightly to agonist affinity columns composed of N6-aminobenzyladenosine-agarose. A1 adenosine receptors and G proteins are rapidly and selectively coeluted from agonist columns by the addition of 8-p-sulfophenyltheophylline, but only in combination with Mg2+-GTP or N-ethylmaleimide, agents which lower the affinity of receptors for agonists. Purified receptors and G protein alpha-subunits can be detected with the potent A1-selective antagonist radioligand, [125I]3-(4-amino-3-iodo)phenethyl-1-propyl-8-cyclopentylxanthine (125I-BW-A844U) and [35S]guanosine 5'-3-O-(thio)triphosphate [( 35S]GTP gamma S), respectively. Pretreatment of solubilized receptors with 0.1 mM N-ethylmaleimide or 0.1 mM R-phenylisopropyladenosine abolishes adsorption of receptors and G proteins to affinity columns. Following removal of 8-p-sulfophenyltheophylline and GTP, purified receptors bind agonists (2 sites) and antagonists (1 site) with affinities similar to crude soluble receptors and typical of A1 receptors. Some receptors may be denatured as a result of purification since only 23% of the radioligand binding sites which adhere to the affinity column can be detected in the eluate. The Bmax of purified receptors, 820 +/- 100 pmol/mg protein (n = 3) is 1800-fold higher than crude soluble receptors. The specific activity of [35S]GTP gamma S binding sites in affinity column eluates is 4640 pmol/mg protein. Assuming a 1:1 stoichiometry, this specific activity indicates that receptor-G protein complexes are greater than 50% pure following affinity chromatography. The photoaffinity labeled purified receptor was identified by polyacrylamide gel electrophoresis as a single band with a

  12. The orphan G-protein-coupled receptor-encoding gene V28 is closely related to genes for chemokine receptors and is expressed in lymphoid and neural tissues.

    Science.gov (United States)

    Raport, C J; Schweickart, V L; Eddy, R L; Shows, T B; Gray, P W

    1995-10-03

    A polymerase chain reaction (PCR) strategy with degenerate primers was used to identify novel G-protein-coupled receptor-encoding genes from human genomic DNA. One of the isolated clones, termed V28, showed high sequence similarity to the genes encoding human chemokine receptors for monocyte chemoattractant protein 1 (MCP-1) and macrophage inflammatory protein 1 alpha (MIP-1 alpha)/RANTES, and to the rat orphan receptor-encoding gene RBS11. When RNA was analyzed by Northern blot, V28 was found to be most highly expressed in neural and lymphoid tissues. Myeloid cell lines, particularly THP.1 cells, showed especially high expression of V28. We have mapped V28 to human chromosome 3p21-3pter, near the MIP-1 alpha/RANTES receptor-encoding gene.

  13. Structural determinants in the second intracellular loop of the human cannabinoid CB1 receptor mediate selective coupling to Gs and Gi

    Science.gov (United States)

    Chen, XP; Yang, W; Fan, Y; Luo, JS; Hong, K; Wang, Z; Yan, JF; Chen, X; Lu, JX; Benovic, JL; Zhou, NM

    2010-01-01

    BACKGROUND AND PURPOSE The cannabinoid CB1 receptor is primarily thought to be functionally coupled to the Gi form of G proteins, through which it negatively regulates cAMP accumulation. Here, we investigated the dual coupling properties of CB1 receptors and characterized the structural determinants that mediate selective coupling to Gs and Gi. EXPERIMENTAL APPROACH A cAMP-response element reporter gene system was employed to quantitatively analyze cAMP change. CB1/CB2 receptor chimeras and site-directed mutagenesis combined with functional assays and computer modelling were used to determine the structural determinants mediating selective coupling to Gs and Gi. KEY RESULTS CB1 receptors could couple to both Gs-mediated cAMP accumulation and Gi-induced activation of ERK1/2 and Ca2+ mobilization, whereas CB2 receptors selectively coupled to Gi and inhibited cAMP production. Using CB1/CB2 chimeric receptors, the second intracellular loop (ICL2) of the CB1 receptor was identified as primarily responsible for mediating Gs and Gi coupling specificity. Furthermore, mutation of Leu-222 in ICL2 to either Ala or Pro switched G protein coupling from Gs to Gi, while to Ile or Val led to balanced coupling of the mutant receptor with Gs and Gi. CONCLUSIONS AND IMPLICATIONS The ICL2 of CB1 receptors and in particular Leu-222, which resides within a highly conserved DRY(X)5PL motif, played a critical role in Gs and Gi protein coupling and specificity. Our studies provide new insight into the mechanisms governing the coupling of CB1 receptors to G proteins and cannabinoid-induced tolerance. PMID:20735408

  14. Long-Term Memory for Place Learning Is Facilitated by Expression of cAMP Response Element-Binding Protein in the Dorsal Hippocampus

    Science.gov (United States)

    Brightwell, Jennifer J.; Smith, Clayton A.; Neve, Rachael L.; Colombo, Paul J.

    2007-01-01

    Extensive research has shown that the hippocampus is necessary for consolidation of long-term spatial memory in rodents. We reported previously that rats using a place strategy to solve a cross maze task showed sustained phosphorylation of hippocampus cyclic AMP response element-binding protein (CREB), a transcription factor implicated in…

  15. Functional Cloning of Src-like Adapter Protein-2 (SLAP-2), a Novel Inhibitor of Antigen Receptor Signaling

    OpenAIRE

    Holland, Sacha J.; Liao, X.Charlene; Mendenhall, Marcy K.; Zhou, Xiulan; Pardo, Jorge; Chu, Peter; Spencer, Collin; Fu, Alan; Sheng, Ning; Yu, Peiwen; Pali, Erlina; Nagin, Anup; Shen, Mary; Yu, Simon; Chan, Eva

    2001-01-01

    In an effort to identify novel therapeutic targets for autoimmunity and transplant rejection, we developed and performed a large-scale retroviral-based functional screen to select for proteins that inhibit antigen receptor-mediated activation of lymphocytes. In addition to known regulators of antigen receptor signaling, we identified a novel adaptor protein, SLAP-2 which shares 36% sequence similarity with the known Src-like adaptor protein, SLAP. Similar to SLAP, SLAP-2 is predominantly expr...

  16. A library of 7TM receptor C-terminal tails - Interactions with the proposed post-endocytic sorting proteins ERM-binding phosphoprotein 50 (EBP50), N-ethylmaleimide-sensitive factor (NSF), sorting nexin 1 (SNX1), and G protein-coupled receptor-associated sorting protein (GASP)

    DEFF Research Database (Denmark)

    Heydorn, A.; Sondergaard, B.P.; Ersbøll, Bjarne Kjær

    2004-01-01

    sequestration through interactions, mainly with the C-terminal intracellular tails of the receptors. A library of tails from 59 representative members of the super family of seven-transmembrane receptors was probed as glutathione S-transferase fusion proteins for interactions with four different adaptor...... only a single receptor tail, i.e. the beta(2)-adrenergic receptor, whereas N-ethylmaleimide-sensitive factor bound 11 of the tail-fusion proteins. Of the two proteins proposed to target receptors for lysosomal degradation, sorting nexin 1 (SNX1) bound 10 and the C-terminal domain of G protein......-coupled receptor-associated sorting protein bound 23 of the 59 tail proteins. Surface plasmon resonance analysis of the binding kinetics of selected hits from the glutathione S-transferase pull-down experiments, i.e. the tails of the virally encoded receptor US28 and the delta-opioid receptor, confirmed...

  17. Camp for Youth With Type 1 Diabetes.

    Science.gov (United States)

    Fegan-Bohm, Kelly; Weissberg-Benchell, Jill; DeSalvo, Daniel; Gunn, Sheila; Hilliard, Marisa

    2016-08-01

    Camps for youth with type 1 diabetes (T1D) have grown in size and scope since they first emerged in the 1920s. Anecdotal evidence suggests that attending camp with other youth with T1D is beneficial, largely attributed to sharing fun, active experiences and removing the isolation of living with diabetes. However, few studies have evaluated the psychosocial and medical impacts of T1D camp attendance during and after camp sessions. In addition, T1D camps have been a setting for numerous studies on a variety of T1D-related research questions not related to camp itself, such as testing novel diabetes management technologies in an active, non-laboratory setting. This paper reviews the evidence of psychosocial and medical outcomes associated with T1D camp attendance across the globe, provides an overview of other research conducted at camp, and offers recommendations for future research conducted at T1D camp.

  18. Challenges predicting ligand-receptor interactions of promiscuous proteins: the nuclear receptor PXR.

    Directory of Open Access Journals (Sweden)

    Sean Ekins

    2009-12-01

    Full Text Available Transcriptional regulation of some genes involved in xenobiotic detoxification and apoptosis is performed via the human pregnane X receptor (PXR which in turn is activated by structurally diverse agonists including steroid hormones. Activation of PXR has the potential to initiate adverse effects, altering drug pharmacokinetics or perturbing physiological processes. Reliable computational prediction of PXR agonists would be valuable for pharmaceutical and toxicological research. There has been limited success with structure-based modeling approaches to predict human PXR activators. Slightly better success has been achieved with ligand-based modeling methods including quantitative structure-activity relationship (QSAR analysis, pharmacophore modeling and machine learning. In this study, we present a comprehensive analysis focused on prediction of 115 steroids for ligand binding activity towards human PXR. Six crystal structures were used as templates for docking and ligand-based modeling approaches (two-, three-, four- and five-dimensional analyses. The best success at external prediction was achieved with 5D-QSAR. Bayesian models with FCFP_6 descriptors were validated after leaving a large percentage of the dataset out and using an external test set. Docking of ligands to the PXR structure co-crystallized with hyperforin had the best statistics for this method. Sulfated steroids (which are activators were consistently predicted as non-activators while, poorly predicted steroids were docked in a reverse mode compared to 5alpha-androstan-3beta-ol. Modeling of human PXR represents a complex challenge by virtue of the large, flexible ligand-binding cavity. This study emphasizes this aspect, illustrating modest success using the largest quantitative data set to date and multiple modeling approaches.

  19. 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.

  20. Crystal structure of the β2 adrenergic receptor-Gs protein complex

    Energy Technology Data Exchange (ETDEWEB)

    Rasmussen, Søren G.F.; DeVree, Brian T; Zou, Yaozhong; Kruse, Andrew C; Chung, Ka Young; Kobilka, Tong Sun; Thian, Foon Sun; Chae, Pil Seok; Pardon, Els; Calinski, Diane; Mathiesen, Jesper M; Shah, Syed T.A.; Lyons, Joseph A; Caffrey, Martin; Gellman, Samuel H; Steyaert, Jan; Skiniotis, Georgios; Weis, William I; Sunahara, Roger K; Kobilka, Brian K [Brussels; (Trinity); (Michigan); (Stanford-MED); (Michigan-Med); (UW)

    2011-12-07

    G protein-coupled receptors (GPCRs) are responsible for the majority of cellular responses to hormones and neurotransmitters as well as the senses of sight, olfaction and taste. The paradigm of GPCR signalling is the activation of a heterotrimeric GTP binding protein (G protein) by an agonist-occupied receptor. The β2 adrenergic receptor2AR) activation of Gs, the stimulatory G protein for adenylyl cyclase, has long been a model system for GPCR signalling. Here we present the crystal structure of the active state ternary complex composed of agonist-occupied monomeric β2AR and nucleotide-free Gs heterotrimer. The principal interactions between the β2AR and Gs involve the amino- and carboxy-terminal α-helices of Gs, with conformational changes propagating to the nucleotide-binding pocket. The largest conformational changes in the β2AR include a 14Å outward movement at the cytoplasmic end of transmembrane segment 6 (TM6) and an α-helical extension of the cytoplasmic end of TM5. The most surprising observation is a major displacement of the α-helical domain of Gαs relative to the Ras-like GTPase domain. This crystal structure represents the first high-resolution view of transmembrane signalling by a GPCR.

  1. Acetylation of pregnane X receptor protein determines selective function independent of ligand activation

    Energy Technology Data Exchange (ETDEWEB)

    Biswas, Arunima; Pasquel, Danielle [Albert Einstein Cancer Center, Albert Einstein College of Medicine, Bronx, NY 10461 (United States); Tyagi, Rakesh Kumar [Special Centre for Molecular Medicine, Jawaharlal Nehru University, New Delhi 110067 (India); Mani, Sridhar, E-mail: sridhar.mani@einstein.yu.edu [Albert Einstein Cancer Center, Albert Einstein College of Medicine, Bronx, NY 10461 (United States)

    2011-03-18

    Research highlights: {yields} Pregnane X receptor (PXR), a major regulatory protein, is modified by acetylation. {yields} PXR undergoes dynamic deacetylation upon ligand-mediated activation. {yields} SIRT1 partially mediates PXR deacetylation. {yields} PXR deacetylation per se induces lipogenesis mimicking ligand-mediated activation. -- Abstract: Pregnane X receptor (PXR), like other members of its class of nuclear receptors, undergoes post-translational modification [PTM] (e.g., phosphorylation). However, it is unknown if acetylation (a major and common form of protein PTM) is observed on PXR and, if it is, whether it is of functional consequence. PXR has recently emerged as an important regulatory protein with multiple ligand-dependent functions. In the present work we show that PXR is indeed acetylated in vivo. SIRT1 (Sirtuin 1), a NAD-dependent class III histone deacetylase and a member of the sirtuin family of proteins, partially mediates deacetylation of PXR. Most importantly, the acetylation status of PXR regulates its selective function independent of ligand activation.

  2. Effects of estrogen receptor modulators on cytoskeletal proteins in the central nervous system

    Science.gov (United States)

    Segura-Uribe, Julia J.; Pinto-Almazán, Rodolfo; Coyoy-Salgado, Angélica; Fuentes-Venado, Claudia E.; Guerra-Araiza, Christian

    2017-01-01

    Estrogen receptor modulators are compounds of interest because of their estrogenic agonistic/antagonistic effects and tissue specificity. These compounds have many clinical applications, particularly for breast cancer treatment and osteoporosis in postmenopausal women, as well as for the treatment of climacteric symptoms. Similar to estrogens, neuroprotective effects of estrogen receptor modulators have been described in different models. However, the mechanisms of action of these compounds in the central nervous system have not been fully described. We conducted a systematic search to investigate the effects of estrogen receptor modulators in the central nervous system, focusing on the modulation of cytoskeletal proteins. We found that raloxifene, tamoxifen, and tibolone modulate some cytoskeletal proteins such as tau, microtuble-associated protein 1 (MAP1), MAP2, neurofilament 38 (NF38) by different mechanisms of action and at different levels: neuronal microfilaments, intermediate filaments, and microtubule-associated proteins. Finally, we emphasize the importance of the study of these compounds in the treatment of neurodegenerative diseases since they present the benefits of estrogens without their side effects. PMID:28966632

  3. Engineering and optimization of an allosteric biosensor protein for peroxisome proliferator-activated receptor γ ligands

    Science.gov (United States)

    Li, Jingjing; Gierach, Izabela; Gillies, Alison; Warden, Charles D.; Wood, David W.

    2011-01-01

    The peroxisome proliferator-activated receptor gamma (PPARγ or PPARG) belongs to the nuclear receptor superfamily, and is a potential drug target for a variety of diseases. In this work, we constructed a series of bacterial biosensors for the identification of functional PPARγ ligands. These sensors entail modified Escherichia coli cells carrying a four-domain fusion protein, comprised of the PPARγ ligand binding domain (LBD), an engineered mini-intein domain, the E. coli maltose binding protein (MBD), and a thymidylate synthase (TS) reporter enzyme. E. coli cells expressing this protein exhibit hormone ligand-dependent growth phenotypes. Unlike our published estrogen (ER) and thyroid receptor (TR) biosensors, the canonical PPARγ biosensor cells displayed pronounced growth in the absence of ligand. They were able to distinguish agonists and antagonists, however, even in the absence of agonist. To improve ligand sensitivity of this sensor, we attempted to engineer and optimize linker peptides flanking the PPARγ LBD insertion point. Truncation of the original linkers led to decreased basal growth and significantly enhanced ligand sensitivity of the PPARγ sensor, while substitution of the native linkers with optimized G4S (Gly-Gly-Gly-Gly-Ser) linkers further increased the sensitivity. Our studies demonstrate that the properties of linkers, especially the C-terminal linker, greatly influence the efficiency and fidelity of the allosteric signal induced by ligand binding. Our work also suggests an approach to increase allosteric behavior in this multidomain sensor protein, without modification of the functional LBD. PMID:21893405

  4. Effects of estrogen receptor modulators on cytoskeletal proteins in the central nervous system

    Directory of Open Access Journals (Sweden)

    Julia J Segura-Uribe

    2017-01-01

    Full Text Available Estrogen receptor modulators are compounds of interest because of their estrogenic agonistic/antagonistic effects and tissue specificity. These compounds have many clinical applications, particularly for breast cancer treatment and osteoporosis in postmenopausal women, as well as for the treatment of climacteric symptoms. Similar to estrogens, neuroprotective effects of estrogen receptor modulators have been described in different models. However, the mechanisms of action of these compounds in the central nervous system have not been fully described. We conducted a systematic search to investigate the effects of estrogen receptor modulators in the central nervous system, focusing on the modulation of cytoskeletal proteins. We found that raloxifene, tamoxifen, and tibolone modulate some cytoskeletal proteins such as tau, microtuble-associated protein 1 (MAP1, MAP2, neurofilament 38 (NF38 by different mechanisms of action and at different levels: neuronal microfilaments, intermediate filaments, and microtubule-associated proteins. Finally, we emphasize the importance of the study of these compounds in the treatment of neurodegenerative diseases since they present the benefits of estrogens without their side effects.

  5. Present perspectives on the automated classification of the G-protein coupled receptors (GPCRs) at the protein sequence level

    DEFF Research Database (Denmark)

    Davies, Matthew N; Gloriam, David E; Secker, Andrew

    2011-01-01

    The G-protein coupled receptors--or GPCRs--comprise simultaneously one of the largest and one of the most multi-functional protein families known to modern-day molecular bioscience. From a drug discovery and pharmaceutical industry perspective, the GPCRs constitute one of the most commercially...... and economically important groups of proteins known. The GPCRs undertake numerous vital metabolic functions and interact with a hugely diverse range of small and large ligands. Many different methodologies have been developed to efficiently and accurately classify the GPCRs. These range from motif-based techniques...... to machine learning as well as a variety of alignment-free techniques based on the physiochemical properties of sequences. We review here the available methodologies for the classification of GPCRs. Part of this work focuses on how we have tried to build the intrinsically hierarchical nature of sequence...

  6. 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.

  7. Interaction between G Protein-Coupled Receptor 143 and Tyrosinase: Implications for Understanding Ocular Albinism Type 1.

    Science.gov (United States)

    De Filippo, Elisabetta; Schiedel, Anke C; Manga, Prashiela

    2017-02-01

    Developmental eye defects in X-linked ocular albinism type 1 are caused by G-protein coupled receptor 143 (GPR143) mutations. Mutations result in dysfunctional melanosome biogenesis and macromelanosome formation in pigment cells, including melanocytes and retinal pigment epithelium. GPR143, primarily expressed in pigment cells, localizes exclusively to endolysosomal and melanosomal membranes unlike most G protein-coupled receptors, which localize to the plasma membrane. There is some debate regarding GPR143 function and elucidating the role of this receptor may be instrumental for understanding neurogenesis during eye development and for devising therapies for ocular albinism type I. Many G protein-coupled receptors require association with other proteins to function. These G protein-coupled receptor-interacting proteins also facilitate fine-tuning of receptor activity and tissue specificity. We therefore investigated potential GPR143 interaction partners, with a focus on the melanogenic enzyme tyrosinase. GPR143 coimmunoprecipitated with tyrosinase, while confocal microscopy demonstrated colocalization of the proteins. Furthermore, tyrosinase localized to the plasma membrane when coexpressed with a GPR143 trafficking mutant. The physical interaction between the proteins was confirmed using fluorescence resonance energy transfer. This interaction may be required in order for GPR143 to function as a monitor of melanosome maturation. Identifying tyrosinase as a potential GPR143 binding protein opens new avenues for investigating the mechanisms that regulate pigmentation and neurogenesis. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

  8. Production of G protein-coupled receptors in an insect-based cell-free system.

    Science.gov (United States)

    Sonnabend, Andrei; Spahn, Viola; Stech, Marlitt; Zemella, Anne; Stein, Christoph; Kubick, Stefan

    2017-10-01

    The biochemical analysis of human cell membrane proteins remains a challenging task due to the difficulties in producing sufficient quantities of functional protein. G protein-coupled receptors (GPCRs) represent a main class of membrane proteins and drug targets, which are responsible for a huge number of signaling processes regulating various physiological functions in living cells. To circumvent the current bottlenecks in GPCR studies, we propose the synthesis of GPCRs in eukaryotic cell-free systems based on extracts generated from insect (Sf21) cells. Insect cell lysates harbor the fully active translational and translocational machinery allowing posttranslational modifications, such as glycosylation and phosphorylation of de novo synthesized proteins. Here, we demonstrate the production of several GPCRs in a eukaryotic cell-free system, performed within a short time and in a cost-effective manner. We were able to synthesize a variety of GPCRs ranging from 40 to 133 kDa in an insect-based cell-free system. Moreover, we have chosen the μ opioid receptor (MOR) as a model protein to analyze the ligand binding affinities of cell-free synthesized MOR in comparison to MOR expressed in a human cell line by "one-point" radioligand binding experiments. Biotechnol. Bioeng. 2017;114: 2328-2338. © 2017 The Authors. Biotechnology and Bioengineering Published by Wiley Periodicals, Inc. © 2017 The Authors. Biotechnology and Bioengineering Published by Wiley Periodicals, Inc.

  9. Residential summer camp intervention improves camp food environment.

    Science.gov (United States)

    Di Noia, Jennifer; Orr, Lynne; Byrd-Bredbenner, Carol

    2014-07-01

    To evaluate the effects on fruit and vegetable (FV) intake of a camp-based intervention to improve the food environment. The intervention was evaluated in a variant of the recurrent institutional cycle design in a sample of 311 youth aged 7 to 13 years. FV intake and targeted environmental variables were assessed among youth who received the intervention relative to those who attended the camp before the program was implemented. Improvements occurred in the frequency and variety of FVs served, counselor informational and instrumental support for FV consumption, and in older youth who received nutrition education lessons, perceived peer attitudes towards eating FVs and FV intake. Improving the camp food environment can improve FV intake among youth in this setting.

  10. G-Protein Sensitivity of Ligand Binding to Human Dopamine D2 and D3 Receptors Expressed in Escherichia coli : Clues for a Constrained D3 Receptor Structure

    NARCIS (Netherlands)

    Vanhauwe, Jurgen F.M.; Josson, Katty; Luyten, Walter H.M.L.; Driessen, Arnold J.; Leysen, Josée E.

    2000-01-01

    Human dopamine D2 and D3 receptors were expressed in Chinese hamster ovary (CHO) and Escherichia coli cells to compare their ligand binding properties in the presence or absence of G-proteins and to analyze their ability to interact with Gi/o-proteins. Binding affinities of agonists (dopamine,

  11. Identification of the hemoglobin scavenger receptor/CD163 as a natural soluble protein in plasma

    DEFF Research Database (Denmark)

    Møller, Holger Jon; Peterslund, Niels Anker; Graversen, Jonas Heilskov

    2002-01-01

    The hemoglobin scavenger receptor (HbSR/CD163) is an interleukin-6- and glucocorticoid-regulated macrophage/monocyte receptor for uptake of haptoglobin-hemoglobin complexes. Moreover, there are strong indications that HbSR serves an anti-inflammatory function. Immunoprecipitation and immunoblotting...... enabled identification of a soluble plasma form of HbSR (sHbSR) having an electrophoretic mobility equal to that of recombinant HbSR consisting of the extracellular domain (scavenger receptor cysteine-rich 1-9). A sandwich enzyme-linked immunosorbent assay was established and used to measure the s...... a level of sHbSR above the range of healthy persons. Patients with myelomonocytic leukemias and pneumonia/sepsis exhibited the highest levels (up to 67.3 mg/L). In conclusion, sHbSR is an abundant plasma protein potentially valuable in monitoring patients with infections and myelomonocytic leukemia....

  12. A new crystal structure fragment-based pharmacophore method for G protein-coupled receptors

    DEFF Research Database (Denmark)

    Fidom, Kimberley; Isberg, Vignir; Hauser, Alexander Sebastian

    2015-01-01

    We have developed a new method for the building of pharmacophores for G protein-coupled receptors, a major drug target family. The method is a combination of the ligand- and target-based pharmacophore methods and founded on the extraction of structural fragments, interacting ligand moiety...... for new targets. A validating retrospective virtual screening of histamine H1 and H3 receptor pharmacophores yielded area-under-the-curves of 0.88 and 0.82, respectively. The fragment-based method has the unique advantage that it can be applied to targets for which no (homologous) crystal structures...... and receptor residue pairs, from crystal structure complexes. We describe the procedure to collect a library with more than 250 fragments covering 29 residue positions within the generic transmembrane binding pocket. We describe how the library fragments are recombined and inferred to build pharmacophores...

  13. G protein-coupled receptors as new therapeutic targets for type 2 diabetes.

    Science.gov (United States)

    Reimann, Frank; Gribble, Fiona M

    2016-02-01

    G protein-coupled receptors (GPCRs) in the gut-brain-pancreatic axis are key players in the postprandial control of metabolism and food intake. A number of intestinally located receptors have been implicated in the chemo-detection of ingested nutrients, and in the pancreatic islets and nervous system GPCRs play essential roles in the detection of many hormones and neurotransmitters. Because of the diversity, cell-specific expression and 'druggability' of the GPCR superfamily, these receptors are popular targets for therapeutic development. This review will outline current and potential future approaches to develop GPCR agonists for the treatment of type 2 diabetes. This review summarises a presentation given at the 'Novel approaches to treating type 2 diabetes' symposium at the 2015 annual meeting of the EASD. It is accompanied by a commentary by the Session Chair, Michael Nauck (DOI: 10.1007/s00125-015-3823-1 ).

  14. Crosslinking photosensitized by a ruthenium chelate as a tool for labeling and topographical studies of G-protein-coupled receptors.

    Science.gov (United States)

    Duroux-Richard, Isabelle; Vassault, Philippe; Subra, Guy; Guichou, Jean-François; Richard, Eric; Mouillac, Bernard; Barberis, Claude; Marie, Jacky; Bonnafous, Jean-Claude

    2005-01-01

    The purpose was to apply oxidative crosslinking reactions to the study of recognition and signaling mechanisms associated to G-protein-coupled receptors. Using a ruthenium chelate, Ru(bipy)(3)(2+), as photosensitizer and visible light irradiation, in the presence of ammonium persulfate, we performed fast and efficient covalent labeling of the B(2) bradykinin receptor by agonist or antagonist ligands possessing a radio-iodinated phenol moiety. The chemical and topographical specificities of these crosslinking experiments were investigated. The strategy could also be applied to the covalent labeling of the B(1) bradykinin receptor, the AT(1) angiotensin II receptor, the V(1a) vasopressin receptor and the oxytocin receptor. Interestingly, we demonstrated the possibility to covalently label the AT(1) and B(2) receptors with functionalized ligands. The potential applications of metal-chelate chemistry to receptor structural and signaling studies through intramolecular or intermolecular crosslinking are presented.

  15. Lactate Receptor Sites Link Neurotransmission, Neurovascular Coupling, and Brain Energy Metabolism

    DEFF Research Database (Denmark)

    Lauritzen, Knut H; Morland, Cecilie; Puchades, Maja

    2013-01-01

    by physiological concentrations of lactate and by the specific GPR81 agonist 3,5-dihydroxybenzoate to reduce cAMP. Cerebral GPR81 is concentrated on the synaptic membranes of excitatory synapses, with a postsynaptic predominance. GPR81 is also enriched at the blood-brain-barrier: the GPR81 densities at endothelial......The G-protein-coupled lactate receptor, GPR81 (HCA1), is known to promote lipid storage in adipocytes by downregulating cAMP levels. Here, we show that GPR81 is also present in the mammalian brain, including regions of the cerebral neocortex and hippocampus, where it can be activated...

  16. Management of diabetes at summer camps.

    Science.gov (United States)

    Ciambra, Roberta; Locatelli, Chiara; Suprani, Tosca; Pocecco, Mauro

    2005-01-01

    We report our experience in the organization of diabetic children summer-camps since 1973. Guidelines for organization have been recently reported by the SIEDP (Società Italiana di Endocrinologia e Diabetologia Pediatrica). Our attention is focused on diabetes management at camp, organization and planning, medical staff composition and staff training, treatment of diabetes-related emergencies, written camp management plan, diabetes education and psychological issues at camp, prevention of possible risks, assessment of effectiveness of education in summer camps and research at camp.

  17. Haplotypes of the endothelial protein C receptor gene and Behçet's disease.

    Science.gov (United States)

    Navarro, Silvia; Bonet, Elena; Medina, Pilar; Martos, Laura; Ricart, José M; Vayá, Amparo; Todolí, José; Fontcuberta, Jordi; Estellés, Amparo; España, Francisco

    2012-04-01

    Behçet's disease is a vasculitis of unknown cause in which thrombosis occurs in about 25% of patients. Two haplotypes of the endothelial protein C receptor gene, H1 and H3, are associated with the risk of thrombosis. Thus, the objective of this study was to evaluate the influence of these haplotypes on the thrombosis risk in Behçet's disease. We evaluated the H1 and H3 haplotypes in 87 patients with Behçet's disease, 19 with and 68 without a history of thrombosis, and in 260 healthy individuals. We also measured protein C, activated protein C, and soluble endothelial protein C receptor levels in all individuals. The presence of the H1 haplotype seemed to protect Behçet's patients against thrombosis (odds ratio 0.21; 95% CI 0.1-0.8; p=0.023), whereas the frequency of the H3 haplotype was lower in patients than in control individuals (0.19; 0.1-0.5; p=0.006). Furthermore, the H1 haplotype was associated with increased levels of activated protein C, whereas the H3 haplotype was associated with the highest soluble endothelial protein C levels. Moreover, activated protein C levels were lower in patients with than in patients without posterior uveitis (p<0.001). These findings indicate that the H1 haplotype protects Behçet's patients from thrombosis, likely via increased levels of activated protein C, whereas individuals carrying the H3 haplotype seem to be protected from the clinical manifestations associated with Behçet's disease, probably via increased soluble endothelial protein C levels. Copyright © 2011 Elsevier Ltd. All rights reserved.

  18. G protein-coupled receptors in invertebrates: a state of the art.

    Science.gov (United States)

    Meeusen, Tom; Mertens, Inge; De Loof, Arnold; Schoofs, Liliane

    2003-01-01

    G protein-coupled receptors (GPCRs) constitute one of the largest and most ancient superfamilies of membrane-spanning proteins. We focus on neuropeptide GPCRs, in particular on those of invertebrates. In general, such receptors mediate the responses of signaling molecules that constitute the highest hierarchical position in the regulation of physiological processes. Until recently, only a few of these receptors were identified in invertebrates. However, the availability of a plethora of genomic information has boosted the discovery of novel members in several invertebrate species, such as Drosophila, in which 18 neuropeptide GPCRs have been characterized. The finalization of genomic projects in other invertebrates will lead to a similar expansion of GPCR understanding. Many new insights regarding neuropeptide regulation have followed from the discovery of their cognate receptors. Furthermore, information on GPCR signaling is still fragmentary and the elucidation of these pathways in model insects such as Drosophila will lead to further insights in other species, including mammals. In this review we present the current status of what is known about invertebrate GPCRs, discuss some novel perceptions that follow from the identified members, and, finally, present some future prospects.

  19. Fluorescent knock-in mice to decipher the physiopathological role of G protein-coupled receptors.

    Directory of Open Access Journals (Sweden)

    Dominique eMassotte

    2015-01-01

    Full Text Available G protein-coupled receptors (GPCRs modulate most physiological functions but are also critically involved in numerous pathological states. Approximately a third of marketed drugs target GPCRs, which places this family of receptors in the main arena of pharmacological pre-clinical and clinical research. The complexity of GPCR function demands comprehensive appraisal in native environment to collect in-depth knowledge of receptor physiopathological roles and assess the potential of therapeutic molecules. Identifying neurons expressing endogenous GPCRs is therefore essential to locate them within functional circuits whereas GPCR visualization with subcellular resolution is required to get insight into agonist-induced trafficking. Both remain frequently poorly investigated because direct visualization of endogenous receptors is often hampered by the lack of appropriate tools. Also, monitoring intracellular trafficking requires real-time visualization to gather in-depth knowledge. In this context, knock-in mice expressing a fluorescent protein or a fluorescent version of a GPCR under the control of the endogenous promoter not only help to decipher neuroanatomical circuits but also enable real-time monitoring with subcellular resolution thus providing invaluable information on their trafficking in response to a physiological or a pharmacological challenge. This review will present the animal models and discuss their contribution to the understanding of the physiopathological role of GPCRs. We will also address the drawbacks associated with this methodological approach and browse future directions.

  20. Role and therapeutic potential of G-protein coupled receptors in breast cancer progression and metastases.

    Science.gov (United States)

    Singh, Anukriti; Nunes, Jessica J; Ateeq, Bushra

    2015-09-15

    G-protein-coupled receptors (GPCRs) comprise a large family of cell-surface receptors, which have recently emerged as key players in tumorigenesis, angiogenesis and metastasis. In this review, we discussed our current understanding of the many roles played by GPCRs in general, and particularly Angiotensin II type I receptor (AGTR1), a member of the seven-transmembrane-spanning G-protein coupled receptor superfamily, and its significance in breast cancer progression and metastasis. We have also discussed different strategies for targeting AGTR1, and its ligand Angiotension II (Ang II), which might unravel unique opportunities for breast cancer prevention and treatment. For example, AGTR1 blockers (ARBs) which are already in clinical use for treating hypertension, merit further investigation as a therapeutic strategy for AGTR1-positive cancer patients and may have the potential to prevent Ang II-AGTR1 signalling mediated cancer pathogenesis and metastases. Copyright © 2015 The Authors. Published by Elsevier B.V. All rights reserved.

  1. Genome wide survey of G protein-coupled receptors in Tetraodon nigroviridis

    Directory of Open Access Journals (Sweden)

    Sowdhamini Ramanathan

    2005-07-01

    Full Text Available Abstract Background The G-protein-coupled receptors (GPCRs constitute one of the largest and most ancient superfamilies of membrane proteins. They play a central role in physiological processes affecting almost all aspects of the life cycle of an organism. Availability of the complete sets of putative members of a family from diverse species provides the basis for cross genome comparative studies. Results We have defined the repertoire of GPCR superfamily of Tetraodon complement with the availability of complete sequence of the freshwater puffer fish Tetraodon nigroviridis. Almost all 466 Tetraodon GPCRs (Tnig-GPCRs identified had a clear human homologue. 189 putative human and Tetraodon GPCR orthologous pairs could be identified. Tetraodon GPCRs are classified into five GRAFS families, by phylogenetic analysis, concurrent with human GPCR classification. Conclusion Direct comparison of GPCRs in Tetraodon and human genomes displays a high level of orthology and supports large-scale gene duplications in Tetraodon. Examples of lineage specific gene expansions were also observed in opsin and odorant receptors. The human and Tetraodon GPCR sequences are analogous in terms of GPCR subfamilies but display disproportionate numbers of rece