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Sample records for camp receptor protein

  1. Characterization of the binding of cAMP and cGMP to the CRP*598 mutant of the E. coli cAMP receptor protein.

    OpenAIRE

    Ren, Y. L.; Garges, S; Adhya, S; Krakow, J. S.

    1990-01-01

    Wild type cAMP receptor protein (CRP) activates in vitro lac transcription only in the presence of cAMP. In contrast the mutant CRP*598 (Arg-142 to His, Ala-144 to Thr) can activate lac transcription in the absence of cyclic nucleotide or at concentrations of cAMP below that required by CRP. To further characterize the properties of CRP*598, the binding of cAMP and cGMP to CRP and CRP*598 has been determined. The intrinsic binding constant (K) values obtained for cAMP binding are: CRP, 1.9 x ...

  2. 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....

  3. cAMP receptor protein (CRP) downregulates Klebsiella pneumoniae nif promoters in Escherichia coli

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

    In enteric bacteria, in response to the PTS system, the cAMP receptor protein (CRP) mediates the glucose effect, via regulating s70-dependent catabolic genes at transcriptional level. In this study, it is observed that the nitrogen fixation capacity of Klebsiella pneumoniae varies strongly when cells are grown on different carbohydrates, and this carbon effect occurs at the level of nif gene expression. Here we show that CRP can repress s54-dependent nif promoters (nifB, nifE, nifF, nifH, nifJ, nifLA and nifU), in a cAMP dependent fashion, in closed related E. coli background. Sequence analysis of these nif promoters indicates that there is no direct correlation between the fold of CRP-cAMP-mediated inhibition and the upstream cis elements at the promoters. In addition, the crp gene of K. pneumoniae has been isolated and sequenced, which is structural and functional highly homologous to that of E. coli. This suggests that CRP-cAMP-mediated inhibition on the nif promoters could be the reason for carbon effect on nitrogen fixation and thus has its physiological significance. A novel regulatory linkage between carbon metabolism and nitrogen fixation is proposed.

  4. The gene encoding cAMP receptor protein is required for competence development in Haemophilus influenzae Rd.

    OpenAIRE

    Chandler, M S

    1992-01-01

    The Haemophilus influenzae Rd strain JG87 contains a single mini-Tn10kan insertion that causes a deficiency in the development of competence for genetic transformation. The DNA fragment containing this insertion mutation, as well as the wild-type locus, was cloned, mapped, and sequenced. The sequence contained an open reading frame for a protein of 224 amino acids with a predicted Mr of 25,152. The deduced protein sequence showed strong similarity to the Escherichia coli cAMP receptor protein...

  5. G-Protein-Coupled Receptor MrgD Is a Receptor for Angiotensin-(1-7) Involving Adenylyl Cyclase, cAMP, and Phosphokinase A.

    Science.gov (United States)

    Tetzner, Anja; Gebolys, Kinga; Meinert, Christian; Klein, Sabine; Uhlich, Anja; Trebicka, Jonel; Villacañas, Óscar; Walther, Thomas

    2016-07-01

    Angiotensin (Ang)-(1-7) has cardiovascular protective effects and is the opponent of the often detrimental Ang II within the renin-angiotensin system. Although it is well accepted that the G-protein-coupled receptor Mas is a receptor for the heptapeptide, the lack in knowing initial signaling molecules stimulated by Ang-(1-7) prevented definitive characterization of ligand/receptor pharmacology as well as identification of further hypothesized receptors for the heptapeptide. The study aimed to identify a second messenger stimulated by Ang-(1-7) allowing confirmation as well as discovery of the heptapeptide's receptors. Ang-(1-7) elevates cAMP concentration in primary cells, such as endothelial or mesangial cells. Using cAMP as readout in receptor-transfected human embryonic kidney (HEK293) cells, we provided pharmacological proof that Mas is a functional receptor for Ang-(1-7). Moreover, we identified the G-protein-coupled receptor MrgD as a second receptor for Ang-(1-7). Consequently, the heptapeptide failed to increase cAMP concentration in primary mesangial cells with genetic deficiency in both Mas and MrgD Mice deficient in MrgD showed an impaired hemodynamic response after Ang-(1-7) administration. Furthermore, we excluded the Ang II type 2 receptor as a receptor for the heptapeptide but discovered that the Ang II type 2 blocker PD123319 can also block Mas and MrgD receptors. Our results lead to an expansion and partial revision of the renin-angiotensin system, by identifying a second receptor for Ang-(1-7), by excluding Ang II type 2 as a receptor for the heptapeptide, and by enforcing the revisit of such publications which concluded Ang II type 2 function by only using PD123319. PMID:27217404

  6. Mycobacterium tuberculosis cAMP Receptor Protein (Rv3676) Differs from the Escherichia coli Paradigm in Its cAMP Binding and DNA Binding Properties and Transcription Activation Properties

    OpenAIRE

    Stapleton, M.; Haq, I.; Hunt, D M; Arnvig, K. B.; Artymiuk, P. J.; Buxton, R S; Green, J

    2010-01-01

    The pathogen Mycobacterium tuberculosis produces a burst of cAMP upon infection of macrophages. Bacterial cyclic AMP receptor proteins (CRP) are transcription factors that respond to cAMP by binding at target promoters when cAMP concentrations increase. Rv3676 (CRP Mt ) is a CRP family protein that regulates expression of genes (rpfA and whiB1) that are potentially involved in M. tuberculosis persistence and/or emergence from the dormant state. Here, the CRP Mt homodimer is shown to bind two ...

  7. Mechanism of the down-regulation of cAMP receptor protein by glucose in Escherichia coli: role of autoregulation of the crp gene.

    OpenAIRE

    ISHIZUKA, H; Hanamura, A; Inada, T; Aiba, H.

    1994-01-01

    Glucose causes catabolite repression by lowering the intracellular levels of both cAMP and cAMP receptor protein (CRP) in Escherichia coli. The molecular mechanism underlying the down-regulation of CRP by glucose has been investigated. We show that glucose lowers the level of crp mRNA without affecting its stability. Replacement of the crp promoter with the bla promoter almost completely abolishes the glucose-mediated regulation of crp expression. Only a slight reduction in the crp expression...

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

    OpenAIRE

    Su Zhengchang; Xu Minli

    2009-01-01

    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 st...

  9. Profound Asymmetry in the Structure of the cAMP-free cAMP Receptor Protein (CRP) from Mycobacterium tuberculosisS⃞

    OpenAIRE

    Gallagher, D. Travis; Smith, Natasha; Kim, Sook-Kyung; Robinson, Howard; Reddy, Prasad T.

    2009-01-01

    The cyclic AMP receptor protein (CRP, also called catabolite gene activator protein or CAP) plays a key role in metabolic regulation in bacteria and has become a widely studied model allosteric transcription factor. On binding its effector cAMP in the N-terminal domain, CRP undergoes a structural transition to a conformation capable of specific DNA binding in the C-terminal domain and transcription initiation. The crystal structures of Escherichia coli CRP (EcCRP) in ...

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

    Directory of Open Access Journals (Sweden)

    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.

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

    Science.gov (United States)

    Xu, Minli; Su, Zhengchang

    2009-01-01

    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 loss of CRPs in these species

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

    Directory of Open Access Journals (Sweden)

    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

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

    Directory of Open Access Journals (Sweden)

    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.

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

    International Nuclear Information System (INIS)

    Highlights: ► Expression of GPR174 in CHO cells induces morphological changes and proliferation delay. ► These are due to increase in intracellular cAMP concentration. ► Lysophosphatidylserine was identified to stimulate GPR174 leading to activate ACase. ► The potencies of fatty acid moiety on LysoPS were oleoyl ⩾ stearoyl > palmitoyl. ► 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 Gαs protein. These results suggested that GPR174 was a putative LysoPS receptor conjugating with Gαs, and its expression induced morphological changes in CHO cells by constitutively activating adenylyl cycles accompanied with cell conjunctions and delay of proliferation.

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

    DEFF Research Database (Denmark)

    Meibom, K L; Kallipolitis, B H; Ebright, R H; Valentin-Hansen, P

    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 dimer......, the subunit proximal to CytR, functionally interacts with CytR in CRP-CytR-promoter and (CRP)(2)-CytR-promoter complexes. Our results provide information about the architecture of CRP-CytR-promoter and (CRP)(2)-CytR-promoter complexes and rule out the proposal that masking of activating region 2 of...... CRP is responsible for the transcriptional inactivity of the complexes....

  16. Crystallization and preliminary X-ray analysis of the ligand-binding domain of cAMP receptor protein

    International Nuclear Information System (INIS)

    N-terminal fragments of the cyclic AMP receptor protein from E. coli created by two different proteases, subtilisin and chymotrypsin, have been crystallized and diffracted to 2.0 and 2.8 Å resolution, respectively. The cyclic AMP receptor protein (CRP) from Escherichia coli regulates the expression of a large number of genes. In this work, CRP has been overexpressed, purified and digested by subtilisin and chymotrypsin. The fragments S-CRP (digested by subtilisin) and CH-CRP (digested by chymotrypsin) have been purified and crystallized. Crystals of S-CRP diffracted to 2.0 Å resolution and belonged to space group P21, with unit-cell parameters a = 59.7, b = 75.1, c = 128.3 Å, β = 91.5°. Crystals of CH-CRP diffracted to 2.8 Å resolution and belonged to space group P222, with unit-cell parameters a = 45.8, b = 60.9, c = 205.6 Å

  17. Cyclic AMP (cAMP) Receptor Protein-cAMP Complex Regulates Heparosan Production in Escherichia coli Strain Nissle 1917.

    Science.gov (United States)

    Yan, Huihui; Bao, Feifei; Zhao, Liping; Yu, Yanying; Tang, Jiaqin; Zhou, Xianxuan

    2015-11-01

    Heparosan serves as the starting carbon backbone for the chemoenzymatic synthesis of heparin, a widely used clinical anticoagulant drug. The availability of heparosan is a significant concern for the cost-effective synthesis of bioengineered heparin. The carbon source is known as the pivotal factor affecting heparosan production. However, the mechanism by which carbon sources control the biosynthesis of heparosan is unclear. In this study, we found that the biosynthesis of heparosan was influenced by different carbon sources. Glucose inhibits the biosynthesis of heparosan, while the addition of either fructose or mannose increases the yield of heparosan. Further study demonstrated that the cyclic AMP (cAMP)-cAMP receptor protein (CRP) complex binds to the upstream region of the region 3 promoter and stimulates the transcription of the gene cluster for heparosan biosynthesis. Site-directed mutagenesis of the CRP binding site abolished its capability of binding CRP and eliminated the stimulative effect on transcription. (1)H nuclear magnetic resonance (NMR) analysis was further performed to determine the Escherichia coli strain Nissle 1917 (EcN) heparosan structure and quantify extracellular heparosan production. Our results add to the understanding of the regulation of heparosan biosynthesis and may contribute to the study of other exopolysaccharide-producing strains. PMID:26319872

  18. Persistent cAMP Signaling by Internalized LH Receptors in Ovarian Follicles.

    Science.gov (United States)

    Lyga, Sandra; Volpe, Silvia; Werthmann, Ruth C; Götz, Konrad; Sungkaworn, Titiwat; Lohse, Martin J; Calebiro, Davide

    2016-04-01

    A crucial event in female reproduction occurs at midcycle, when a LH peak induces the final maturation of ovarian follicles. LH signals via a G protein-coupled receptor selectively expressed in the outermost follicular cell layers. However, how LH signals are relayed inside these cells and finally to the oocyte is incompletely understood. Here, we monitored LH signaling in intact ovarian follicles of transgenic mice expressing a fluorescent cAMP sensor. We found that LH stimulation induces 2 phases of cAMP signaling in all cell layers surrounding the oocyte. Interfering with LH receptor internalization abolished the second, persistent cAMP phase and partially inhibited oocyte meiosis resumption. These data suggest that persistent cAMP signals from internalized LH receptors contribute to transmitting LH effects inside follicle cells and ultimately to the oocyte. Thus, this study indicates that the recently proposed paradigm of cAMP signaling by internalized G protein-coupled receptors is implicated in receptor function and is physiologically relevant. PMID:26828746

  19. Epac and PKA: a tale of two intracellular cAMP receptors

    Institute of Scientific and Technical Information of China (English)

    Xiaodong Cheng; Zhenyu Ji; Tamara Tsalkova; Fang Mei

    2008-01-01

    cAMP-mediated signaling pathways regulate a multitude of important biological processes under both physiological and pathological conditions,including diabetes,heart failure and cancer.In eukaryotic cells,the effects of cAMP are mediated by two ubiquitously expressed intracellular cAMP receptors,the classic protein kinase A (PKA)/cAMP-dependent protein kinase and the recently discovered exchange protein directly activated by cAMP(Epac)/cAMP-regulated guanine nucleotide exchange factors.Like PKA,Epac contains an evolutionally conserved cAMP binding domain that acts as a molecular switch for sensing intracellular second messenger cAMP levels to control diverse biological functions.The existence of two families of cAMP effectors provides a mechanism for a more precise and integrated control of the cAMP signaling pathways in a spatial and temporal manner.Depending upon the specific cellular environments as well as their relative abundance,distrbution and localization,Epac and PKA may act independently,converge synergistically or oppose each other in regulating a specific cellular function.

  20. 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.

    Science.gov (United States)

    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. PMID:27189964

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

    Science.gov (United States)

    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. PMID:24626197

  2. The modulation of cell surface cAMP receptors from Dictyostelium disscoideum by ammonium sulfate

    NARCIS (Netherlands)

    Haastert, Peter J.M. van

    1985-01-01

    Dictyostelium discoideum cells contain a heterogeneous population of cell surface cAMP receptors with components possessing different affinities (Kd between 15 and 450 nM) and different off-rates of the cAMP-receptor complex (t½ between 0.7 and 150 s). The association of cAMP to the receptor and the

  3. Surface expression of GABAA receptors is transcriptionally controlled by the interplay of cAMP-response element-binding protein and its binding partner inducible cAMP early repressor.

    Science.gov (United States)

    Hu, Yinghui; Lund, Ingrid V; Gravielle, Maria C; Farb, David H; Brooks-Kayal, Amy R; Russek, Shelley J

    2008-04-01

    The regulated expression of type A gamma-aminobutyric acid (GABA) receptor (GABA(A)R) subunit genes plays a critical role in neuronal maturation and synaptogenesis. It is also associated with a variety of neurological diseases. Changes in GABA(A) receptor alpha1 subunit gene (GABRA1) expression have been reported in animal models of epilepsy, alcohol abuse, withdrawal, and stress. Understanding the genetic mechanism behind such changes in alpha subunit expression will lead to a better understanding of the role that signal transduction plays in control over GABA(A)R function and brings with it the promise of providing new therapeutic tools for the prevention or cure of a variety of neurological disorders. Here we show that activation of protein kinase C increases alpha1 subunit levels via phosphorylation of CREB (pCREB) that is bound to the GABRA1 promoter (GABRA1p). In contrast, activation of protein kinase A decreases levels of alpha1 even in the presence of pCREB. Decrease of alpha1 is dependent upon the inducible cAMP early repressor (ICER) as directly demonstrated by ICER-induced down-regulation of endogenous alpha1-containing GABA(A)Rs at the cell surface of cortical neurons. Taken together with the fact that there are less alpha1gamma2-containing GABA(A)Rs in neurons after protein kinase A stimulation and that activation of endogenous dopamine receptors down-regulates alpha1 subunit mRNA levels subsequent to induction of ICER, our studies identify a transcriptional mechanism for regulating the cell surface expression of alpha1-containing GABA(A)Rs that is dependent upon the formation of CREB heterodimers. PMID:18180303

  4. Basal and adenosine receptor-stimulated levels of cAMP are reduced in lymphocytes from alcoholic patients

    International Nuclear Information System (INIS)

    Alcoholism causes serious neurologic disease that may be due, in part, to the ability of ethanol to interact with neural cell membranes and change neuronal function. Adenosine receptors are membrane-bound proteins that appear to mediate some of the effects of ethanol in the brain. Human lymphocytes also have adenosine receptors, and their activation causes increases in cAMP levels. To test the hypothesis that basal and adenosine receptor-stimulated cAMP levels in lymphocytes might be abnormal in alcoholism, the authors studied lymphocytes from 10 alcoholic subjects, 10 age- and sex-matched normal individuals, and 10 patients with nonalcoholic liver disease. Basal and adenosine receptor-stimulated cAMP levels were reduced 75% in lymphocytes from alcoholic subjects. Also, there was a 76% reduction in ethanol stimulation of cAMP accumulation in lymphocytes from alcoholics. Similar results were demonstrable in isolated T cells. Unlike other laboratory tests examined, these measurements appeared to distinguish alcoholics from normal subjects and from patients with nonalcoholic liver disease. Reduced basal and adenosine receptor-stimulated levels of cAMP in lymphocytes from alcoholics may reflect a change in cell membranes due either to chronic alcohol abuse or to a genetic predisposition unique to alcoholic subjects

  5. Forskolin-free cAMP assay for Gi-coupled receptors.

    Science.gov (United States)

    Gilissen, Julie; Geubelle, Pierre; Dupuis, Nadine; Laschet, Céline; Pirotte, Bernard; Hanson, Julien

    2015-12-01

    G protein-coupled receptors (GPCRs) represent the most successful receptor family for treating human diseases. Many are poorly characterized with few ligands reported or remain completely orphans. Therefore, there is a growing need for screening-compatible and sensitive assays. Measurement of intracellular cyclic AMP (cAMP) levels is a validated strategy for measuring GPCRs activation. However, agonist ligands for Gi-coupled receptors are difficult to track because inducers such as forskolin (FSK) must be used and are sources of variations and errors. We developed a method based on the GloSensor system, a kinetic assay that consists in a luciferase fused with cAMP binding domain. As a proof of concept, we selected the succinate receptor 1 (SUCNR1 or GPR91) which could be an attractive drug target. It has never been validated as such because very few ligands have been described. Following analyses of SUCNR1 signaling pathways, we show that the GloSensor system allows real time, FSK-free detection of an agonist effect. This FSK-free agonist signal was confirmed on other Gi-coupled receptors such as CXCR4. In a test screening on SUCNR1, we compared the results obtained with a FSK vs FSK-free protocol and were able to identify agonists with both methods but with fewer false positives when measuring the basal levels. In this report, we validate a cAMP-inducer free method for the detection of Gi-coupled receptors agonists compatible with high-throughput screening. This method will facilitate the study and screening of Gi-coupled receptors for active ligands. PMID:26386312

  6. Membrane estrogen receptor-α levels in MCF-7 breast cancer cells predict cAMP and proliferation responses

    International Nuclear Information System (INIS)

    17β-estradiol (E2) can rapidly induce cAMP production, but the conditions under which these cAMP levels are best measured and the signaling pathways responsible for the consequent proliferative effects on breast cancer cells are not fully understood. To help resolve these issues, we compared cAMP mechanistic responses in MCF-7 cell lines selected for low (mERlow) and high (mERhigh) expression of the membrane form of estrogen receptor (mER)-α, and thus addressed the receptor subform involved in cAMP signaling. MCF-7 cells were immunopanned and subsequently separated by fluorescence activated cell sorting into mERhigh (mER-α-enriched) and mERlow (mER-α-depleted) populations. Unique (compared with previously reported) incubation conditions at 4°C were found to be optimal for demonstrating E2-induced cAMP production. Time-dependent and dose-dependent effects of E2 on cAMP production were determined for both cell subpopulations. The effects of forskolin, 8-CPT cAMP, protein kinase A inhibitor (H-89), and adenylyl cyclase inhibitor (SQ 22,536) on E2-induced cell proliferation were assessed using the crystal violet assay. We demonstrated a rapid and transient cAMP increase after 1 pmol/l E2 stimulation in mERhigh cells; at 4°C these responses were much more reliable and robust than at 37°C (the condition most often used). The loss of cAMP at 37°C was not due to export. 3-Isobutyl-1-methylxanthine (IBMX; 1 mmol/l) only partially preserved cAMP, suggesting that multiple phosphodiesterases modulate its level. The accumulated cAMP was consistently much higher in mERhigh cells than in mERlow cells, implicating mER-α levels in the process. ICI172,780 blocked the E2-induced response and 17α-estradiol did not elicit the response, also suggesting activity through an estrogen receptor. E2 dose-dependent cAMP production, although biphasic in both cell types, was responsive to 50-fold higher E2 concentrations in mERhigh cells. Proliferation of mERlow cells was stimulated

  7. Identification of secreted proteins regulated by cAMP in glioblastoma cells using glycopeptide capture and label-free quantification.

    Science.gov (United States)

    Hill, Jennifer J; Moreno, Maria J; Lam, Jean C Y; Haqqani, Arsalan S; Kelly, John F

    2009-02-01

    Exposure of glioblastoma U87MG cells to a cAMP analog leads to a decrease in proliferation, invasion, and angiogenic potential. Here, we apply a label-free MS-based approach to identify formerly N-linked glycopeptides that change in abundance upon cAMP treatment. Over 150 unique glycopeptides in three biological repetitions were quantified, leading to the identification of 14 upregulated proteins and 21 downregulated proteins due to cAMP treatment. Of these, eight have been validated, either through comparison with microarray data or by Western blot. We estimate our ability to identify differentially expressed peptides at greater than 85% in a single biological repetition, while the analysis of multiple biological repetitions lowers the false positive rate to approximately 2%. Many of the proteins identified in this study are involved in cell signaling and some, such as Tenascin C, Cathepsin L, Neuroblastoma suppressor of tumorigenicity, and AXL/UFO tyrosine-protein kinase receptor, have been previously shown to be involved in glioblastoma progression. We also identify several semitryptic peptides that increase in abundance upon cAMP treatment, suggesting that cAMP regulates protease activity in these cells. Overall, these results demonstrate the benefits of using a highly specific enrichment method for quantitative proteomic experiments. PMID:19137551

  8. Glucagon-like peptide-1 receptor is present in pancreatic acinar cells and regulates amylase secretion through cAMP.

    Science.gov (United States)

    Hou, Yanan; Ernst, Stephen A; Heidenreich, Kaeli; Williams, John A

    2016-01-01

    Glucagon-like peptide-1 (GLP-1) is a glucoincretin hormone that can act through its receptor (GLP-1R) on pancreatic β-cells and increase insulin secretion and production. GLP-1R agonists are used clinically to treat type 2 diabetes. GLP-1 may also regulate the exocrine pancreas at multiple levels, including inhibition through the central nervous system, stimulation indirectly through insulin, and stimulation directly on acinar cells. However, it has been unclear whether GLP-1R is present in pancreatic acini and what physiological functions these receptors regulate. In the current study we utilized GLP-1R knockout (KO) mice to study the role of GLP-1R in acinar cells. RNA expression of GLP-1R was detected in acutely isolated pancreatic acini. Acinar cell morphology and expression of digestive enzymes were not affected by loss of GLP-1R. GLP-1 induced amylase secretion in wild-type (WT) acini. In GLP-1R KO mice, this effect was abolished, whereas vasoactive intestinal peptide-induced amylase release in KO acini showed a pattern similar to that in WT acini. GLP-1 stimulated cAMP production and increased protein kinase A-mediated protein phosphorylation in WT acini, and these effects were absent in KO acini. These data show that GLP-1R is present in pancreatic acinar cells and that GLP-1 can regulate secretion through its receptor and cAMP signaling pathway. PMID:26542397

  9. Histamine receptors on adult rat cardiomyocytes: antagonism of alpha1-receptor stimulation of cAMP degradation

    International Nuclear Information System (INIS)

    Incubation of intact cardiomyocytes with the histamine antagonist [3H]mepyramine results in rapid reversible binding to a single class of high affinity sites (K/sub D/ = 1.2nM; 50,000 sites/myocyte). In membranes from purified myocytes histamine competition of [3H]mepyramine binding (K/sub D/ = 300nM) is not altered by GTP (10μM). Competition of [3H]mepyramine binding by H-receptor subtype-selective antagonists suggests the presence of a single class of H1-receptors. Incubation of intact myocytes with histamine (luM, H1 receptor activation) plus norepinephrine (NE 1uM, alpha1 + beta1 receptor activation) for 3 min leads to significantly more cAMP accumulation (36.5 pmol/106 myocytes) than NE alone (30 pmol/106 myocytes). Histamine alone does not alter basal cAMP = 10.4 pmol/106 myocytes, or beta1 stimulation (isoproternol, 1uM) = 39.6 pmol/106 myocytes. Cyclic AMP accumulation with NE plus prazosin 10nM, (alpha1 + beta1 + alpha1 blockade) is indistinguishable from NE + histamine, (alpha1 + beta1 + H1) stimulation. Histamine competition for [3H]prazosin binding suggests that histamine does not block alpha1 receptors on the myocyte. These data suggest that H1 receptor activation leads to antagonism of the alpha1 receptor mediated activation of cAMP phosphodiesterase the authors have recently described

  10. Regulation of nicotinic acetylcholine receptor phosphorylation in rat myotubes by forskolin and cAMP

    Energy Technology Data Exchange (ETDEWEB)

    Miles, K.; Anthony, D.T.; Rubin, L.L.; Greengard, P.; Huganir, R.L.

    1987-09-01

    The nicotinic acetylcholine receptor (Ac-ChoR) from rat myotubes prelabeled in culture with (/sup 32/P)orthophosphate was isolated by acetylcholine affinity chromatography followed by immunoaffinity chromatography. Under basal conditions, the nicotinic AcChoR was shown to be phosphorylated in situ on the ..beta.. and delta subunits. Regulation of AcChoR phosphorylation by cAMP-dependent protein kinase was explored by the addition of forskolin or cAMP analogues to prelabeled cell cultures. Forskolin, an activator of adenylate cyclase, stimulated the phosphorylation of the delta subunit 20-fold over basal phosphorylation and induced phosphorylation of the ..cap alpha.. subunit. The effect of forskolin was dose dependent with a half-maximal response at 8 ..mu..M in the presence of 35 ..mu..M Ro 20-1724, a phosphodiesterase inhibitor. Stimulation of delta subunit phosphorylation was almost maximal within 5 min, whereas stimulation of ..cap alpha.. subunit phosphorylation was not maximal until 45 min after forskolin treatment. Stimulation of AcChoR phosphorylation by 8-benzylthioadenosine 3',5'-cyclic monophosphate was identical to that obtained by forskolin. Two-dimensional thermolytic phosphopeptide maps of the delta subunit revealed a single major phosphopeptide. These results correlate closely with the observed effects of forskolin on AcChoR desensitization in muscle and suggest that cAMP-dependent phosphorylation of the delta subunit increases the rate of AcChoR desensitization in rat myotubes.

  11. Regulation of nicotinic acetylcholine receptor phosphorylation in rat myotubes by forskolin and cAMP

    International Nuclear Information System (INIS)

    The nicotinic acetylcholine receptor (Ac-ChoR) from rat myotubes prelabeled in culture with [32P]orthophosphate was isolated by acetylcholine affinity chromatography followed by immunoaffinity chromatography. Under basal conditions, the nicotinic AcChoR was shown to be phosphorylated in situ on the β and δ subunits. Regulation of AcChoR phosphorylation by cAMP-dependent protein kinase was explored by the addition of forskolin or cAMP analogues to prelabeled cell cultures. Forskolin, an activator of adenylate cyclase, stimulated the phosphorylation of the δ subunit 20-fold over basal phosphorylation and induced phosphorylation of the α subunit. The effect of forskolin was dose dependent with a half-maximal response at 8 μM in the presence of 35 μM Ro 20-1724, a phosphodiesterase inhibitor. Stimulation of δ subunit phosphorylation was almost maximal within 5 min, whereas stimulation of α subunit phosphorylation was not maximal until 45 min after forskolin treatment. Stimulation of AcChoR phosphorylation by 8-benzylthioadenosine 3',5'-cyclic monophosphate was identical to that obtained by forskolin. Two-dimensional thermolytic phosphopeptide maps of the δ subunit revealed a single major phosphopeptide. These results correlate closely with the observed effects of forskolin on AcChoR desensitization in muscle and suggest that cAMP-dependent phosphorylation of the δ subunit increases the rate of AcChoR desensitization in rat myotubes

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

    Science.gov (United States)

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

    2016-06-10

    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 that the steroid hormone 20-hydroxyecdysone (20E) activates the PKA pathway to enhance 20E-induced gene transcription in the lepidopteran insect Helicoverpa armigera The expression of the catalytic subunit 1 of PKA (PKAC1) increased during metamorphosis, and PKAC1 knockdown blocked pupation and repressed 20E-responsive gene expression. 20E regulated PKAC1 phosphorylation at threonine 200 and nuclear translocation through an ecdysone-responsive G-protein-coupled receptor 2. PKAC1 induced cAMP response element-binding protein (CREB) phosphorylation at serine 143, which bound to the cAMP response element on DNA to enhance 20E-responsive gene transcription. Through ecdysone-responsive G-protein-coupled receptor 2, 20E increased cAMP levels, which induced CREB PKA phosphorylation and 20E-responsive gene expression. This study demonstrates that the PKA/CREB pathway tightly and critically regulates 20E-induced gene transcription as well as its relationship with the 20E-induced PKC pathway. PMID:27129227

  13. Control of βAR- and N-methyl-D-aspartate (NMDA) Receptor-Dependent cAMP Dynamics in Hippocampal Neurons.

    Science.gov (United States)

    Chay, Andrew; Zamparo, Ilaria; Koschinski, Andreas; Zaccolo, Manuela; Blackwell, Kim T

    2016-02-01

    Norepinephrine, a neuromodulator that activates β-adrenergic receptors (βARs), facilitates learning and memory as well as the induction of synaptic plasticity in the hippocampus. Several forms of long-term potentiation (LTP) at the Schaffer collateral CA1 synapse require stimulation of both βARs and N-methyl-D-aspartate receptors (NMDARs). To understand the mechanisms mediating the interactions between βAR and NMDAR signaling pathways, we combined FRET imaging of cAMP in hippocampal neuron cultures with spatial mechanistic modeling of signaling pathways in the CA1 pyramidal neuron. Previous work implied that cAMP is synergistically produced in the presence of the βAR agonist isoproterenol and intracellular calcium. In contrast, we show that when application of isoproterenol precedes application of NMDA by several minutes, as is typical of βAR-facilitated LTP experiments, the average amplitude of the cAMP response to NMDA is attenuated compared with the response to NMDA alone. Models simulations suggest that, although the negative feedback loop formed by cAMP, cAMP-dependent protein kinase (PKA), and type 4 phosphodiesterase may be involved in attenuating the cAMP response to NMDA, it is insufficient to explain the range of experimental observations. Instead, attenuation of the cAMP response requires mechanisms upstream of adenylyl cyclase. Our model demonstrates that Gs-to-Gi switching due to PKA phosphorylation of βARs as well as Gi inhibition of type 1 adenylyl cyclase may underlie the experimental observations. This suggests that signaling by β-adrenergic receptors depends on temporal pattern of stimulation, and that switching may represent a novel mechanism for recruiting kinases involved in synaptic plasticity and memory. PMID:26901880

  14. Exposure to cAMP and β-adrenergic stimulation recruits CaV3 T-type channels in rat chromaffin cells through Epac cAMP-receptor proteins

    Science.gov (United States)

    Novara, M; Baldelli, P; Cavallari, D; Carabelli, V; Giancippoli, A; Carbone, E

    2004-01-01

    T-type channels are expressed weakly or not at all in adult rat chromaffin cells (RCCs) and there is contrasting evidence as to whether they play a functional role in catecholamine secretion. Here we show that 3–5 days after application of pCPT-cAMP, most RCCs grown in serum-free medium expressed a high density of low-voltage-activated T-type channels without altering the expression and characteristics of high-voltage-activated channels. The density of cAMP-recruited T-type channels increased with time and displayed the typical biophysical and pharmacological properties of low-voltage-activated Ca2+ channels: (1) steep voltage-dependent activation from −50 mV in 10 mm Ca2+, (2) slow deactivation but fast and complete inactivation, (3) full inactivation following short conditioning prepulses to −30 mV, (4) effective block of Ca2+ influx with 50 μm Ni2+, (5) comparable permeability to Ca2+ and Ba2+, and (6) insensitivity to common Ca2+ channel antagonists. The action of exogenous pCPT-cAMP (200 μm) was prevented by the protein synthesis inhibitor anisomycin and mimicked in most cells by exposure to forskolin and 1-methyl-3-isobutylxanthine (IBMX) or isoprenaline. The protein kinase A (PKA) inhibitor H89 (0.3 μm) and the competitive antagonist of cAMP binding to PKA, Rp-cAMPS, had weak or no effect on the action of pCPT-cAMP. In line with this, the selective Epac agonist 8CPT-2Me-cAMP nicely mimicked the action of pCPT-cAMP and isoprenaline, suggesting the existence of a dominant Epac-dependent recruitment of T-type channels in RCCs that may originate from the activation of β-adrenoceptors. Stimulation of β-adrenoceptors occurs autocrinally in RCCs and thus, the neosynthesis of low-voltage-activated channels may represent a new form of ‘chromaffin cell plasticity’, which contributes, by lowering the threshold of action potential firing, to increasing cell excitability and secretory activity during sustained sympathetic stimulation and/or increased

  15. Exposure to cAMP and beta-adrenergic stimulation recruits Ca(V)3 T-type channels in rat chromaffin cells through Epac cAMP-receptor proteins.

    Science.gov (United States)

    Novara, M; Baldelli, P; Cavallari, D; Carabelli, V; Giancippoli, A; Carbone, E

    2004-07-15

    T-type channels are expressed weakly or not at all in adult rat chromaffin cells (RCCs) and there is contrasting evidence as to whether they play a functional role in catecholamine secretion. Here we show that 3-5 days after application of pCPT-cAMP, most RCCs grown in serum-free medium expressed a high density of low-voltage-activated T-type channels without altering the expression and characteristics of high-voltage-activated channels. The density of cAMP-recruited T-type channels increased with time and displayed the typical biophysical and pharmacological properties of low-voltage-activated Ca(2+) channels: (1) steep voltage-dependent activation from -50 mV in 10 mm Ca(2+), (2) slow deactivation but fast and complete inactivation, (3) full inactivation following short conditioning prepulses to -30 mV, (4) effective block of Ca(2+) influx with 50 microM Ni(2+), (5) comparable permeability to Ca(2+) and Ba(2+), and (6) insensitivity to common Ca(2+) channel antagonists. The action of exogenous pCPT-cAMP (200 microM) was prevented by the protein synthesis inhibitor anisomycin and mimicked in most cells by exposure to forskolin and 1-methyl-3-isobutylxanthine (IBMX) or isoprenaline. The protein kinase A (PKA) inhibitor H89 (0.3 microM) and the competitive antagonist of cAMP binding to PKA, Rp-cAMPS, had weak or no effect on the action of pCPT-cAMP. In line with this, the selective Epac agonist 8CPT-2Me-cAMP nicely mimicked the action of pCPT-cAMP and isoprenaline, suggesting the existence of a dominant Epac-dependent recruitment of T-type channels in RCCs that may originate from the activation of beta-adrenoceptors. Stimulation of beta-adrenoceptors occurs autocrinally in RCCs and thus, the neosynthesis of low-voltage-activated channels may represent a new form of 'chromaffin cell plasticity', which contributes, by lowering the threshold of action potential firing, to increasing cell excitability and secretory activity during sustained sympathetic stimulation and

  16. G Protein-coupled receptors

    OpenAIRE

    Ross, Elliott M.

    2014-01-01

    G protein-coupled receptors and heterotrimeric G proteins can diffuse laterally in the plasma membrane such that one receptor can catalyze the activation (GDP/GTP exchange) of multiple G proteins. In some cases, these processes are fast enough to support molecular signal amplification, where a single receptor maintains the activation of multiple G proteins at steady-state. Amplification in cells is probably highly regulated. It depends upon the identities of the G receptor and G protein - som...

  17. 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…

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

    OpenAIRE

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

    2015-01-01

    The purpose of this study was to evaluate the extent of constitutive activity among orphan class-A G protein coupled receptors within the cAMP signaling pathway. Constitutive signaling was revealed by changes in gene expression under control of the cAMP response element. Gene expression was measured in Chinese hamster ovary cells transiently co-transfected with plasmids containing a luciferase reporter and orphan receptor. Criteria adopted for defining constitutive activation were: 1) 200% el...

  19. Surface Expression of GABAA Receptors Is Transcriptionally Controlled by the Interplay of cAMP-response Element-binding Protein and Its Binding Partner Inducible cAMP Early Repressor*

    OpenAIRE

    Hu, Yinghui; Lund, Ingrid V.; Gravielle, Maria C.; Farb, David H.; Brooks-Kayal, Amy R.; Russek, Shelley J.

    2008-01-01

    The regulated expression of type A γ-aminobutyric acid (GABA) receptor (GABAAR) subunit genes plays a critical role in neuronal maturation and synaptogenesis. It is also associated with a variety of neurological diseases. Changes in GABAA receptor α1 subunit gene (GABRA1) expression have been reported in animal models of epilepsy, alcohol abuse, withdrawal, and stress. Understanding the genetic mechanism behind such changes in α subunit expression will lead to a be...

  20. Structural basis for activation of G-protein-coupled receptors

    DEFF Research Database (Denmark)

    Gether, Ulrik; Asmar, Fazila; Meinild, Anne Kristine;

    2002-01-01

    Our understanding of how G-protein-coupled receptors (GPCRs) operate at the molecular level has been considerably improved over the last few years. The application of advanced biophysical techniques as well as the availability of high-resolution structural information has allowed insight both int......-expression with the cAMP sensitive Cl- channel CFTR (cystic fibrosis transmembrane conductance regulator) and electrophysiological measurements....

  1. Identification of a specific assembly of the G protein Golf as a critical and regulated module of dopamine and adenosine-activated cAMP pathways in the striatum

    Directory of Open Access Journals (Sweden)

    Denis eHervé

    2011-08-01

    Full Text Available In the principal neurons of striatum (medium spiny neurons, MSNs, cAMP pathway is primarily activated through the stimulation of dopamine D1 and adenosine A2A receptors, these receptors being mainly expressed in striatonigral and striatopallidal MSNs, respectively. Since cAMP signaling pathway could be altered in various physiological and pathological situations, including drug addiction and Parkinson’s disease, it is of crucial importance to identify the molecular components involved in the activation of this pathway. In MSNs, cAMP pathway activation is not dependent on the classical Gs GTP-binding protein but requires a specific G protein subunit heterotrimer containing Galpha-olf/beta2/gamma7 in particular association with adenylate cyclase type 5. This assembly forms an authentic functional signaling unit since loss of one of its members leads to defects of cAMP pathway activation in response to D1 or A2A receptor stimulation, inducing dramatic impairments of behavioral responses dependent on these receptors. Interestingly, D1 receptor-dependent cAMP signaling is modulated by the neuronal levels of Galpha-olf, indicating that Galpha-olf represents the rate-limiting step in this signaling cascade and could constitute a critical element for regulation of D1 receptor responses. In both Parkinsonian patients and several animal models of Parkinson’s disease, the lesion of dopamine neurons produces a prolonged elevation of Galpha-olf levels. This observation gives an explanation for the cAMP pathway hypersensitivity to D1 stimulation, occurring despite an unaltered D1 receptor density. In conclusion, alterations in the highly specialized assembly of Galpha-olf/beta2/gamma7 subunits can happen in pathological conditions, such as Parkinson’s disease, and it could have important functional consequences in relation to changes in D1 receptor signaling in the striatum.

  2. 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; Douthwaite, S R; Valentin-Hansen, P

    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...

  3. 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. PMID:23212924

  4. Receptor activity modifying proteins (RAMPs) interact with the VPAC1 receptor: evidence for differential RAMP modulation of multiple signalling pathways

    International Nuclear Information System (INIS)

    Full text: Receptor activity modifying proteins (RAMP) constitute a family of three accessory proteins that affect the expression and/or phenotype of the calcitonin receptor (CTR) or CTR-like receptor (CRLR). In this study we screened a range of class II G protein-coupled receptors (PTH1, PTH2, GHRH, VPAC1, VPAC2 receptors) for possible RAMP interactions by measurement of receptor-induced translocation of c-myc tagged RAMP1 or HA tagged RAMP3. Of these, only the VPAC1 receptor caused significant translocation of c-myc-RAMP1 or HA-RAMP3 to the cell surface. Co-transfection of VPAC1 and RAMPs did not alter 125I-VIP binding and specificity. VPAC1 receptor function was subsequently analyzed through parallel determinations of cAMP accumulation and phosphoinositide (PI) hydrolysis in the presence and absence of each of the three RAMPs. In contrast to CTR-RAMP interaction, where there was an increase in cAMP Pharmacologisand a decrease in PI hydrolysis, VPAC1-RAMP interaction was characterized by a specific increase in agonist-mediated PI hydrolysis when co-transfected with RAMP2. This change was due to an enhancement of Emax with no change in EC50 value for VIP. No significant change in cAMP accumulation was observed. This is the first demonstration of an interaction of RAMPs with a G protein-coupled receptor outside the CTR family and may suggest a more generalized role for RAMPs in modulating G protein-coupled receptor signaling. Copyright (2001) Australasian Society of Clinical and Experimental Pharmacologists and Toxicologists

  5. Development of a high throughput screen for allosteric modulators of melanocortin-4 receptor signaling using a real time cAMP assay.

    Science.gov (United States)

    Pantel, Jacques; Williams, Savannah Y; Mi, Dehui; Sebag, Julien; Corbin, Jackie D; Weaver, C David; Cone, Roger D

    2011-06-11

    The melanocortin MC(4) receptor is a potential target for the development of drugs for both obesity and cachexia. Melanocortin MC(4) receptor ligands known thus far are orthosteric agonists or antagonists, however the agonists, in particular, have generally exhibited unwanted side effects. For some receptors, allosteric modulators are expected to reduce side-effect profiles. To identify allosteric modulators of the melanocortin MC(4) receptor, we created HEK293 cell lines coexpressing the human melanocortin MC(4) receptor and a modified luciferase-based cAMP sensor. Monitoring luminescence as a readout of real-time intracellular cAMP concentration, we demonstrate that this cell line is able to report melanocortin agonist responses, as well as inverse agonist response to the physiological AgRP peptide. Based on the MC4R-GLO cell line, we developed an assay that was shown to meet HTS standards (Z'=0.50). A pilot screen run on the Microsource Spectrum compound library (n=2000) successfully identified 62 positive modulators. This screen identified predicted families of compounds: β(2)AR agonists - the β(2)AR being endogenously expressed in HEK293 cells, an adenylyl cyclase activator and finally a distribution of phosphodiesterase (PDE) inhibitors well characterized or recently identified. In this last category, we identified a structural family of coumarin-derived compounds (imperatorin, osthol and prenyletin), along with deracoxib, a drug in veterinary use for its COX2 inhibitory properties. This latter finding unveiled a new off-target mechanism of action for deracoxib as a PDE inhibitor. Overall, these data are the first report of a HTS for allosteric modulators for a Gs protein coupled receptor. PMID:21296065

  6. Expression of Tas1 Taste Receptors in Mammalian Spermatozoa: Functional Role of Tas1r1 in Regulating Basal Ca2+ and cAMP Concentrations in Spermatozoa

    Science.gov (United States)

    Meyer, Dorke; Voigt, Anja; Widmayer, Patricia; Borth, Heike; Huebner, Sandra; Breit, Andreas; Marschall, Susan; de Angelis, Martin Hrabé; Boehm, Ulrich; Meyerhof, Wolfgang; Gudermann, Thomas; Boekhoff, Ingrid

    2012-01-01

    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/or by tonic receptor activation by

  7. Autonomous and nonautonomous regulation of axis formation by antagonistic signaling via 7-span cAMP receptors and GSK3 in Dictyostelium.

    Science.gov (United States)

    Ginsburg, G T; Kimmel, A R

    1997-08-15

    Early during Dictyostelium development a fundamental cell-fate decision establishes the anteroposterior (prestalk/prespore) axis. Signaling via the 7-transmembrane cAMP receptor CAR4 is essential for creating and maintaining a normal pattern; car4-null alleles have decreased levels of prestalk-specific mRNAs but enhanced expression of prespore genes. car4- cells produce all of the signals required for prestalk differentiation but lack an extracellular factor necessary for prespore differentiation of wild-type cells. This secreted factor decreases the sensitivity of prespore cells to inhibition by the prestalk morphogen DIF-1. At the cell autonomous level, CAR4 is linked to intracellular circuits that activate prestalk but inhibit prespore differentiation. The autonomous action of CAR4 is antagonistic to the positive intracellular signals mediated by another cAMP receptor, CAR1 and/or CAR3. Additional data indicate that these CAR-mediated pathways converge at the serine/threonine protein kinase GSK3, suggesting that the anterior (prestalk)/posterior (prespore) axis of Dictyostelium is regulated by an ancient mechanism that is shared by the Wnt/Fz circuits for dorsoventral patterning during early Xenopus development and establishing Drosophila segment polarity. PMID:9284050

  8. Twitching motility and cAMP levels: signal transduction through a single methyl-accepting chemotaxis protein.

    Science.gov (United States)

    Jansari, Vibhuti H; Potharla, Vishwakanth Y; Riddell, Geoff T; Bardy, Sonia L

    2016-06-01

    The Pseudomonas aeruginosa Chp chemosensory system regulates twitching motility, intracellular adenosine 3('') 5(')-cyclic monophosphate (cAMP) levels and is postulated to be involved in directional twitching towards phosphatidylethanolamine (PE). Because PilJ is the only methyl-accepting chemotaxis protein (MCP) identified in the Chp system, we determined the role of PilJ in mediating signal transduction for the distinct outputs of this system. Mutants that lack the periplasmic domain of PilJ (pilJΔ74-273) showed lower levels of cAMP but retained directional twitching towards PE. While initial studies revealed reduced twitching motility by PilJΔ74-273, this was due to decreased cAMP levels. Our data illustrate the importance of the periplasmic domain of PilJ in regulating cAMP. This is the first time a defined domain within PilJ has been identified as having a distinct role in signal transduction. PMID:27190147

  9. Protein-Protein Interactions at the Adrenergic Receptors

    OpenAIRE

    Cotecchia, Susanna; Stanasila, Laura; Diviani, Dario

    2012-01-01

    The adrenergic receptors are among the best characterized G protein-coupled receptors (GPCRs) and knowledge on this receptor family has provided several important paradigms about GPCR function and regulation. One of the most recent paradigms initially supported by studies on adrenergic receptors is that both βarrestins and G protein-coupled receptors themselves can act as scaffolds binding a variety of proteins and this can result in growing complexity of the receptor-mediated cellular effect...

  10. Negative regulation of adenylate cyclase gene (cya) expression by cyclic AMP-cyclic AMP receptor protein in Escherichia coli: studies with cya-lac protein and operon fusion plasmids.

    OpenAIRE

    Kawamukai, M; Kishimoto, J; Utsumi, R; Himeno, M; Komano, T; Aiba, H

    1985-01-01

    We constructed cya-lac protein and operon fusion plasmids in vitro. The effect of cyclic AMP (cAMP) on cya expression was examined by measuring the synthesis of beta-galactosidase in Escherichia coli cells containing fused plasmids. In the cya-lacZ fused protein system, cya expression was strongly repressed by exogenous cAMP. Functional cAMP receptor protein (CRP) was necessary for this effect. On the other hand, in a tet-lacZ fused protein as a control system, tet expression was not affected...

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

    International Nuclear Information System (INIS)

    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 [125I]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 Ser449 to Ser467 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.

  12. Isolation of an inactive bovine heart cAMP-dependent protein kinase holoenzyme containing bound cAMP

    International Nuclear Information System (INIS)

    Bovine heart Type II cAMP-dependent protein kinase (PK) was purified to homogeneity as determined by SDS-PAGE. The purification steps were DEAE-cellulose, ammonium sulfate precipitation, phenyl-Sepharose, alumina C-γ, and HPLC-DEAE. The last step resolved two distinct peaks of cAMP dependent kinase activity (activity ratios = 0.05 - 0.10) eluting at approximately 250 (Peak 1) and 275 (Peak 2) mM NaCl. When subjected to HPLC-gel permeation they had the same Stoke's radii. Linear sucrose gradients gave S/sub 20,w/ values of 7.73 S and 7.25 S for Peaks 1 and 2, respectively. It was determined by integrating the areas of scanned SDS-PAGE bands that regulatory and catalytic subunits were present in equimolar amounts in both Peaks 1 and 2. The ratios of equilibrium [3H] cAMP binding to kinase activity for the two peaks were the same. cAMP was present in trace amounts in Peak 1 but in stoichiometric amounts in Peak 2 (between 25 and 75 % saturation of cAMP binding sites). Although SDS-PAGE analysis ruled out conversions by proteolysis or autophosphorylation-dephosphorylation, Peak 1 could be partially converted to Peak 2 by the addition of subsaturating amounts of cAMP, 3H] cAMP binding and in kinase activation (as indicated by a larger Hill coefficient) of Peak 2 relative to Peak 1 also supported the presence of cAMP in Peak 2. In conclusion, two forms of inactive PK have been isolated, one of which is a ternary complex of PK holoenzyme and cAMP. This complex could represent a cellular form of the enzyme which is primed for activation

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

    Directory of Open Access Journals (Sweden)

    M. A. Prado

    2001-01-01

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

  14. Structural Determinants in the Calcitonin Receptor-Like Receptor (Crlr) Important for Cgrp and Adrenomedullin (Am) Receptor Function of Crlr/Receptor-Activity-Modifying Protein (Ramp) 1 and Crlr/Ramp2 Heterodimers

    OpenAIRE

    W. Born; K. Leuthauser; R. Gujer; R. Muff; J.A. Fischer

    2001-01-01

    Cell surface protein cross-linking, coimmmunoprecipitation, and confocal microscopy identified CRLR/RAMP1-, CRLR/RAMP2-, and calcitonin receptor isotype 2 (CTR2)/RAMP1 heterodimers as CGRP-, AM-, and CGRP/amylin receptors, linked to cAMP production. Along these lines, effects of structural alterations in the N-terminal extracellular domain of the human CRLR on cell surface expression as well as the association with RAMP and CGRP or AM have been investigated.

  15. Cloning, chromosomal assignment, and regulation of the rat thyrotropin receptor: expression of the gene is regulated by thyrotropin, agents that increase cAMP levels, and thyroid autoantibodies.

    Science.gov (United States)

    Akamizu, T; Ikuyama, S; Saji, M; Kosugi, S; Kozak, C; McBride, O W; Kohn, L D

    1990-01-01

    A rat thyrotropin (thyroid-stimulating hormone, TSH) receptor cDNA was isolated that encoded a protein of 764 amino acids, Mr 86,528. Transfection of the cDNA caused COS-7 cells to develop a TSH-sensitive adenylate cyclase response and the ability to bind 125I-labeled TSH; both activities were similar to those of rat FRTL-5 thyroid cells and not duplicated by lutropin. The gene represented by the cDNA was assigned to mouse chromosome 12 and human chromosome 14. Northern analyses identified two species of mRNA, 5.6 and 3.3 kilobases, in FRTL-5 thyroid cells; the transcripts appeared to differ only in the extent of their 3' noncoding sequences. There were minimal amounts of the two mRNAs in rat ovary, and neither was detected in RNA preparations from rat testis, liver, lung, brain, spleen, and FRT thyroid cells, which do not have a functional TSH receptor. TSH decreased both mRNA species 3- to 4-fold within 8 hr in FRTL-5 thyroid cells; down-regulation was dependent on TSH concentration and duplicated by forskolin, cholera toxin, or 8-bromo-cAMP but not by a phorbol ester. Down-regulation was also duplicated by thyroid-stimulating autoantibodies, which increased cAMP levels, but not by thyrotropin binding-inhibiting auto-antibodies, which actually increased TSH receptor mRNA levels. Images PMID:1696008

  16. N-methyl-D-aspartate receptor activation increases cAMP levels and voltage-gated Ca2+ channel activity in area CA1 of hippocampus.

    OpenAIRE

    Chetkovich, D.M.; Gray., R; Johnston, D.; Sweatt, J D

    1991-01-01

    Tetanic stimulation of the Schaffer collateral inputs into area CA1 of the hippocampus causes N-methyl-D-aspartate (NMDA) receptor activation, an effect that contributes to the induction of long-term potentiation (LTP) in this region. The present studies demonstrate that LTP-inducing tetanic stimulation in rat hippocampal area CA1 elicited increased levels of cAMP. The elevation of cAMP was blocked by the NMDA receptor antagonist DL-2-amino-5-phosphonovaleric acid (APV). Bath application of N...

  17. Effects of low dose radiation on cAMP ,cGMP and Fos protein expressions in mouse hypothalamus

    International Nuclear Information System (INIS)

    Objective: In the present study the authors observed the effects of low dose radiation (LDR) on cAMP, cGMP and Fos expressions in mouse hypothalamus. Methods: The changes of cAMP and cGMP contents as well as Fos protein expression in hypothalamus were determined using competitive protein binding assay, radioimmunoassay and immuno-histochemistry, respectively. Results: The cAMP content in the hypothalamus increased rapidly after irradiation with 75 mGy X-rays. It reached its peak value at 15 min, and then returned to nearly normal value at 1 h, followed by small fluctuations. The changes of cGMP content were basically opposite to those of cAMP. The Fos protein was positive in a variety of neurons of hypothalamus after irradiation, and it reached the peak value at 8 h. Then it gradually returned to the control level 48 h after irradiation. Conclusion: These findings suggest that LDR could influence the neuronal signal transduction in mouse hypothalamus with modulating functions on the immune system

  18. Activation of µ-opioid receptors inhibits calcium-currents in the vestibular afferent neurons of the rat through a cAMP dependent mechanism

    Directory of Open Access Journals (Sweden)

    Enrique Soto

    2014-03-01

    Full Text Available Opioid receptors are expressed in the vestibular endorgans (afferent neurons and hair cells and are activated by the efferent system, which modulates the discharge of action potentials in the vestibular afferent neurons (VANs. In mammals, the VANs mainly express the µ opioid-receptor, but the function of the opioid receptor activation and the cellular mechanisms by which they exert their actions in these neurons are poorly studied. To determine the actions of the µ opioid receptor (MOR and the cell signaling mechanisms in the VANs, we made perforated patch-clamp recordings of VANs that were obtained from postnatal days 7 to 10 (P7-10 rats and then maintained in primary culture. The MOR agonist [D-Ala2, N-Me-Phe4, Gly5-ol]-enkephalin (DAMGO inhibited the total voltage-gated outward current; this effect was prevented by the perfusion of a Ca2+-free extracellular solution. We then studied the voltage-gated calcium current (Ica and found that DAMGO Met-enkephalin or endomorphine-1 inhibited the ICa in a dose-response fashion. The effects of DAMGO were prevented by the MOR antagonist (CTAP or by the pertussis toxin (PTX. The use of specific calcium channel blockers showed that MOR activation inhibited the T-, L- and N-type ICa. The use of various enzyme activators and inhibitors and of cAMP analogs allowed us to demonstrate that the MOR acts through a cAMP dependent signaling mechanism. In the current clamp experiments, MOR activation increased the duration and decreased the amplitude of the action potentials and modulated the discharge to the current injection. Pre-incubation with PTX occluded all MOR activation effects observed in the current clamp experiments.\tWe conclude that MOR activation inhibits the T-, L- and N-type ICa through the activation of a Gi/o protein that involves a decrease in AC-cAMP-PKA activity. The modulation of ICa may have an impact on the synaptic integration, excitability and neurotransmitter release from the VANs.

  19. MULTIPLE G PROTEINS COMPETE FOR BINDING WITH THE HUMAN GONADOTROPIN RELEASING HORMONE RECEPTOR

    OpenAIRE

    Knollman, Paul E.; Conn, P. Michael

    2008-01-01

    The GnRH receptor is coupled to G proteins of the families Gq and G11. Gq and G11. Coupling leads to intracellular signaling through the phospholipase C pathway. GnRHR coupling to other G proteins is controversial. This study provides evidence that G protein families Gs, Gi, Gq and G11 complete for binding with the GnRHR. We quantified interactions of over-expressed G proteins with GnRHR by a competitive binding approach, using measurements of second messengers, IP and cAMP. Transient co-tran...

  20. Beta-adrenergic stimulation of cFOS via protein kinase A is mediated by cAMP regulatory element binding protein (CREB)-dependent and tissue-specific CREB-independent mechanisms in corticotrope cells.

    Science.gov (United States)

    Boutillier, A L; Barthel, F; Roberts, J L; Loeffler, J P

    1992-11-25

    Catecholamines stimulate proopiomelanocortin (POMC) gene expression in corticotrope cells, but the molecular mechanisms of these effects are not known. While beta-adrenergic receptors stimulate the protein kinase A (PKA) system, the POMC promoter does not have classical cAMP-response elements (CREs). Therefore, we investigated the induction of the c-fos protooncogen, previously shown to increase POMC transcription in AtT20 cells. In this corticotrope-derived cell line, we show that activation of beta-receptors with isoprenaline (Iso) induces a transient rise in c-fos mRNA levels. Gel mobility shift assays with a labeled AP1 consensus sequence (TGACTCA) showed induction of specific binding activity after Iso treatment. Cotransfection experiments with dominant inhibitory PKA mutants and reporter genes containing c-fos promoter sequences showed that c-fos induction by Iso is entirely dependent on a functional PKA activity. Furthermore, we show that beta-receptor induction of c-fos in corticotrophs is mediated by at least two distinct cAMP-responsive sequences. cAMP regulatory element binding (CREB)-dependent induction is observed on the CRE located at -60 bp on the c-fos promoter. A region located in the vicinity of the dyad symetry element (-290) is also found to mediate tissue-specific cAMP induction. Transcriptional activation by this site, although sensitive to PKA antagonism, is not blocked by CREB mutants. PMID:1331087

  1. 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

    2013-01-01

    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 g

  2. Involvement of cAMP and protein kinase A in conidial differentiation by Erysiphe graminis f. sp. hordei.

    Science.gov (United States)

    Hall, A A; Bindslev, L; Rouster, J; Rasmussen, S W; Oliver, R P; Gurr, S J

    1999-11-01

    Erysiphe graminis f. sp. hordei, the causal agent of barley powdery mildew, is an obligate biotroph. On arrival on the host, a primary germ tube (PGT) emerges from the conidium. An appressorial germ tube (AGT) then appears, forms an appressorium, and effects host penetration. Such developmental precision may be due to multiple, plant-derived signals and to endogenous tactile and chemical signals. The transduction mechanism remains obscure. The isolation of an expressed sequence tag (EST) homologue of the catalytic subunit of cyclic AMP (cAMP)-dependent protein kinase A (PKA) enabled the corresponding gene to be characterized and the transcript to be identified in conidia and in PGT and AGT stage spores. cAMP-dependent PKA activity was detected in ungerminated conidia. These data suggest that PKA and cAMP are involved in conidial development. To substantiate this we exploited the responses of developing conidia to various surfaces, including exposure to the host leaf (fully inductive to AGT formation), cellulose membrane (semi-inductive), and glass (non-inductive). Assessment of fungal development, following application of exogenous cAMP or cAMP analogues, revealed that, at different concentrations and on different surfaces, cAMP either promoted or inhibited conidial differentiation. Various PKA inhibitors were tested for their effect on PKA activity and conidial development. A negative correlation was established between PKA inhibition in vitro and fungal development in vivo. Taken collectively, these data suggest that PKA and cAMP play a role in conidial differentiation in this obligate, plant-pathogenic fungus. PMID:10550894

  3. A Rapid and Efficient Immunoenzymatic Assay to Detect Receptor Protein Interactions: G Protein-Coupled Receptors

    OpenAIRE

    Elisa Zappelli; Simona Daniele; Abbracchio, Maria P.; Claudia Martini; Maria Letizia Trincavelli

    2014-01-01

    G protein-coupled receptors (GPCRs) represent one of the largest families of cell surface receptors, and are the target of at least one-third of the current therapeutic drugs on the market. Along their life cycle, GPCRs are accompanied by a range of specialized GPCR-interacting proteins (GIPs), which take part in receptor proper folding, targeting to the appropriate subcellular compartments and in receptor signaling tasks, and also in receptor regulation processes, such as desensitization and...

  4. Mlc is a transcriptional activator with a key role in integrating cyclic AMP receptor protein and integration host factor regulation of leukotoxin RNA synthesis in Aggregatibacter actinomycetemcomitans

    Science.gov (United States)

    Aggregatibacter actinomycetemcomitans, a periodontal pathogen, synthesizes leukotoxin (LtxA), a protein that helps the bacterium evade the host immune response. Transcription of the ltxA operon is induced during anaerobic growth. The cAMP receptor protein (CRP) indirectly increases ltxA expression...

  5. Melanocortin receptors and their accessory proteins

    OpenAIRE

    Cooray, Sadani N.; Clark, Adrian J.L.

    2010-01-01

    Abstract The melanocortin receptor family consists of 5 members which belong to the GPCR superfamily. Their specific ligands, the melanocortins are peptide hormones which are formed by the proteolytic cleavage of the proopiomelanocortin (POMC) protein. It is now recognised that certain GPCRs require accessory proteins for their function. Like these GPCRs the melanocortin receptor family is also known to be associated with accessory proteins that regulate their function. ...

  6. 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.

  7. Nesfatin-1 induces the phosphorylation levels of cAMP response element-binding protein for intracellular signaling in a neural cell line.

    Directory of Open Access Journals (Sweden)

    Emi Ishida

    Full Text Available Nesfatin-1 is a novel anorexic peptide that reduces the food intake of rodents when administered either intraventricularly or intraperitoneally. However, the molecular mechanism of intracellular signaling via Nesfatin-1 is yet to be resolved. In the current study, we investigated the ability of different neuronal cell lines to respond to Nesfatin-1 and further elucidated the signal transduction pathway of Nesfatin-1. To achieve this, we transfected several cell lines with various combinations of reporter vectors containing different kinds of response elements and performed reporter assays with Nesfatin-1, its active midsegment encoding 30 amino acid residues (M30 and M30-derived mutants. Notably, we found that both Nesfatin-1 as well as M30, significantly increased cAMP response element (CRE reporter activity in a mouse neuroblastoma cell line, NB41A3. An antagonist of Melanocortin 3/4 receptor, SHU9119, aborted the promoter activity, and a mutant M30, which exerts no anorexic effect in vivo did not induce the CRE reporter activity in NB41A3 cells. Western blotting analyses revealed that Nesfatin-1 and M30 significantly increased the phosphorylation levels of CRE-binding protein (CREB, without altering the intracellular cAMP levels. Further, our study showed that a mitogen-activated protein kinase (MAPK kinase inhibitor and an L-type Calcium (Ca(2+ channel blocker abolished the M30-induced CREB phosphorylation. Furthermore, the radio-receptor assay revealed that (125I-Nesfatin-1 binds in a saturable fashion to the membrane fractions of the mouse hypothalamus and NB41A3 cells, with Kd values of 0.79 nM and 0.17 nM, respectively. Collectively, our findings indicate the presence of a Nesfatin-1-specific receptor on the cell surface of NB41A3 cells and mouse hypothalamus. Our study highlights that Nesfatin-1, via its receptor, induces the phosphorylation of CREB, thus activating the intracellular signaling cascade in neurons.

  8. Evidence in vivo for autogenous control of the cyclic AMP receptor protein gene (crp) in Escherichia coli by divergent RNA.

    OpenAIRE

    Okamoto, K.; Hara, S.; Bhasin, R; Freundlich, M

    1988-01-01

    Control of crp expression in vivo was studied by using the cloned crp gene. The synthesis of the product of this gene, cyclic AMP (cAMP) receptor protein (CRP), was strongly reduced by exogenous cAMP. This regulation was completely abolished by the inactivation of a divergent promoter located within the crp promoter region. These data are consistent with our in vitro studies (Okamoto and Freundlich, Proc.Natl. Acad. Sci. USA 83:5000-5004, 1986), which showed that crp autoregulation is due to ...

  9. Modulation of opioid receptor function by protein-protein interactions.

    Science.gov (United States)

    Alfaras-Melainis, Konstantinos; Gomes, Ivone; Rozenfeld, Raphael; Zachariou, Venetia; Devi, Lakshmi

    2009-01-01

    Opioid receptors, MORP, DORP and KORP, belong to the family A of G protein coupled receptors (GPCR), and have been found to modulate a large number of physiological functions, including mood, stress, appetite, nociception and immune responses. Exogenously applied opioid alkaloids produce analgesia, hedonia and addiction. Addiction is linked to alterations in function and responsiveness of all three opioid receptors in the brain. Over the last few years, a large number of studies identified protein-protein interactions that play an essential role in opioid receptor function and responsiveness. Here, we summarize interactions shown to affect receptor biogenesis and trafficking, as well as those affecting signal transduction events following receptor activation. This article also examines protein interactions modulating the rate of receptor endocytosis and degradation, events that play a major role in opiate analgesia. Like several other GPCRs, opioid receptors may form homo or heterodimers. The last part of this review summarizes recent knowledge on proteins known to affect opioid receptor dimerization. PMID:19273296

  10. Autonomous and nonautonomous regulation of axis formation by antagonistic signaling via 7-span cAMP receptors and GSK3 in Dictyostelium

    OpenAIRE

    Ginsburg, Gail T.; Alan R. Kimmel

    1997-01-01

    Early during Dictyostelium development a fundamental cell-fate decision establishes the anteroposterior (prestalk/prespore) axis. Signaling via the 7-transmembrane cAMP receptor CAR4 is essential for creating and maintaining a normal pattern; car4-null alleles have decreased levels of prestalk-specific mRNAs but enhanced expression of prespore genes. car4− cells produce all of the signals required for prestalk differentiation but lack an extracellular factor necessary for prespore differentia...

  11. 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

  12. Synthetic FXR agonist GW4064 is a modulator of multiple G protein-coupled receptors.

    Science.gov (United States)

    Singh, Nidhi; Yadav, Manisha; Singh, Abhishek Kumar; Kumar, Harish; Dwivedi, Shailendra Kumar Dhar; Mishra, Jay Sharan; Gurjar, Anagha; Manhas, Amit; Chandra, Sharat; Yadav, Prem Narayan; Jagavelu, Kumaravelu; Siddiqi, Mohammad Imran; Trivedi, Arun Kumar; Chattopadhyay, Naibedya; Sanyal, Sabyasachi

    2014-05-01

    The synthetic nuclear bile acid receptor (farnesoid X receptor [FXR]) agonist GW4064 is extensively used as a specific pharmacological tool to illustrate FXR functions. We noticed that GW4064 activated empty luciferase reporters in FXR-deficient HEK-293T cells. We postulated that this activity of GW4064 might be routed through as yet unknown cellular targets and undertook an unbiased exploratory approach to identify these targets. Investigations revealed that GW4064 activated cAMP and nuclear factor for activated T-cell response elements (CRE and NFAT-RE, respectively) present on these empty reporters. Whereas GW4064-induced NFAT-RE activation involved rapid intracellular Ca(2+) accumulation and NFAT nuclear translocation, CRE activation involved soluble adenylyl cyclase-dependent cAMP accumulation and Ca(2+)-calcineurin-dependent nuclear translocation of transducers of regulated CRE-binding protein 2. Use of dominant negative heterotrimeric G-protein minigenes revealed that GW4064 caused activation of Gαi/o and Gq/11 G proteins. Sequential pharmacological inhibitor-based screening and radioligand-binding studies revealed that GW4064 interacted with multiple G protein-coupled receptors. Functional studies demonstrated that GW4064 robustly activated H1 and H4 and inhibited H2 histamine receptor signaling events. We also found that MCF-7 breast cancer cells, reported to undergo GW4064-induced apoptosis in an FXR-dependent manner, did not express FXR, and the GW4064-mediated apoptosis, also apparent in HEK-293T cells, could be blocked by selective histamine receptor regulators. Taken together, our results demonstrate identification of histamine receptors as alternate targets for GW4064, which not only necessitates cautious interpretation of the biological functions attributed to FXR using GW4064 as a pharmacological tool but also provides a basis for the rational designing of new pharmacophores for histamine receptor modulation. PMID:24597548

  13. Modulation of opioid receptor function by protein-protein interactions

    OpenAIRE

    Alfaras-Melainis, Konstantinos; Gomes, Ivone; Rozenfeld, Raphael; Zachariou, Venetia; Devi, Lakshmi,

    2009-01-01

    Opioid receptors, MORP, DORP and KORP, belong to the family A of G protein coupled receptors (GPCR), and have been found to modulate a large number of physiological functions, including mood, stress, appetite, nociception and immune responses. Exogenously applied opioid alkaloids produce analgesia, hedonia and addiction. Addiction is linked to alterations in function and responsiveness of all three opioid receptors in the brain. Over the last few years, a large number of studies identified pr...

  14. 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

    training camp. METHODS: In a randomized, double-blinded study, 18 elite cyclists consumed either a whey protein hydrolysate-carbohydrate beverage (PRO-CHO, 14 g protein/h and 69 g CHO/h) or an isocaloric carbohydrate beverage (CHO, 84 g/h) during each training session for six days (25-29 h cycling in total......). Diet and training were standardized and supervised. The diet was energy balanced and contained 1.7 g protein/kg/day. A 10-s peak power test and a 5-min all-out performance test were conducted before and after the first training session and repeated at day 6 of the camp. Blood and saliva samples were...... 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...

  15. 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.

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

    Directory of Open Access Journals (Sweden)

    Adam L Martin

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

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

    Science.gov (United States)

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

    2015-01-01

    The purpose of this study was to evaluate the extent of constitutive activity among orphan class-A G protein coupled receptors within the cAMP signaling pathway. Constitutive signaling was revealed by changes in gene expression under control of the cAMP response element. Gene expression was measured in Chinese hamster ovary cells transiently co-transfected with plasmids containing a luciferase reporter and orphan receptor. Criteria adopted for defining constitutive activation were: 1) 200% elevation over baseline reporter gene expression; 2) 40% inhibition of baseline expression; and 3) 40% inhibition of expression stimulated by 3 μM forskolin. Five patterns of activity were noted: 1) inhibition under both baseline and forskolin stimulated expression (GPR15, GPR17, GPR18, GPR20, GPR25, GPR27, GPR31, GPR32, GPR45, GPR57, GPR68, GPR83, GPR84, GPR132, GPR150, GPR176); 2) no effect on baseline expression, but inhibition of forskolin stimulated expression (GPR4, GPR26, GPR61, GPR62, GPR78, GPR101, GPR119); 3) elevation of baseline signaling coupled with inhibition of forskolin stimulated expression (GPR6, GPR12); 4) elevation of baseline signaling without inhibition of forskolin stimulated expression (GPR3, GPR21, GPR52, GPR65); and 5) no effect on expression (GPR1, GPR19, GPR22, GPR34, GPR35, GPR39, GPR63, GPR82, GPR85, GPR87). Constitutive activity was observed in 75% of the orphan class-A receptors examined (30 of 40). This constitutive signaling cannot be explained by simple overexpression of the receptor. Inhibition of cAMP mediated expression was far more common (65%) than stimulation of expression (15%). Orphan receptors that were closely related based on amino acid homology tended to have similar effects on gene expression. These results suggest that identification of inverse agonists may be a fruitful approach for categorizing these orphan receptors and targeting them for pharmacological intervention. PMID:26384023

  18. Receptor Proteins in Selective Autophagy

    Directory of Open Access Journals (Sweden)

    Christian Behrends

    2012-01-01

    Full Text Available Autophagy has long been thought to be an essential but unselective bulk degradation pathway. However, increasing evidence suggests selective autophagosomal turnover of a broad range of substrates. Bifunctional autophagy receptors play a key role in selective autophagy by tethering cargo to the site of autophagosomal engulfment. While the identity of molecular components involved in selective autophagy has been revealed at least to some extent, we are only beginning to understand how selectivity is achieved in this process. Here, we summarize the mechanistic and structural basis of receptor-mediated selective autophagy.

  19. cAMP prevents TNF-induced apoptosis through inhibiting DISC complex formation in rat hepatocytes

    Energy Technology Data Exchange (ETDEWEB)

    Bhattacharjee, Rajesh [Department of Surgery, University of Pittsburgh Medical Center, Pittsburgh, PA 15213 (United States); Xiang, Wenpei [Department of Surgery, University of Pittsburgh Medical Center, Pittsburgh, PA 15213 (United States); Family Planning Research Institute, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, People' s Republic of China (China); Wang, Yinna [Vascular Medicine Institute, University of Pittsburgh School of Medicine, 10051-5A BST 3, 3501 Fifth Avenue, Pittsburgh, PA 15261 (United States); Zhang, Xiaoying [Department of Medicine/Endocrinology Division, University of Pittsburgh Medical Center, 200 Lothrop St., Pittsburgh, PA 15213 (United States); Billiar, Timothy R., E-mail: billiartr@upmc.edu [Department of Surgery, University of Pittsburgh Medical Center, Pittsburgh, PA 15213 (United States)

    2012-06-22

    Highlights: Black-Right-Pointing-Pointer cAMP blocks cell death induced by TNF and actinomycin D in cultured hepatocytes. Black-Right-Pointing-Pointer cAMP blocks NF-{kappa}B activation induced by TNF and actinomycin D. Black-Right-Pointing-Pointer cAMP blocks DISC formation following TNF and actinomycin D exposure. Black-Right-Pointing-Pointer cAMP blocks TNF signaling at a proximal step. -- Abstract: Tumor necrosis factor {alpha} (TNF) is a pleiotropic proinflammatory cytokine that plays a role in immunity and the control of cell proliferation, cell differentiation, and apoptosis. The pleiotropic nature of TNF is due to the formation of different signaling complexes upon the binding of TNF to its receptor, TNF receptor type 1 (TNFR1). TNF induces apoptosis in various mammalian cells when the cells are co-treated with a transcription inhibitor like actinomycin D (ActD). When TNFR1 is activated, it recruits an adaptor protein, TNF receptor-associated protein with death domain (TRADD), through its cytoplasmic death effector domain (DED). TRADD, in turn, recruits other signaling proteins, including TNF receptor-associated protein 2 (TRAF2) and receptor-associated protein kinase (RIPK) 1, to form a complex. Subsequently, this complex combines with FADD and procaspase-8, converts into a death-inducing signaling complex (DISC) to induce apoptosis. Cyclic AMP (cAMP) is a second messenger that regulates various cellular processes such as cell proliferation, gene expression, and apoptosis. cAMP analogues are reported to act as anti-apoptotic agents in various cell types, including hepatocytes. We found that a cAMP analogue, dibutyryl cAMP (db-cAMP), inhibits TNF + ActD-induced apoptosis in rat hepatocytes. The protein kinase A (PKA) inhibitor KT-5720 reverses this inhibitory effect of cAMP on apoptosis. Cytoprotection by cAMP involves down-regulation of various apoptotic signal regulators like TRADD and FADD and inhibition of caspase-8 and caspase-3 cleavage. We also found

  20. G-protein coupling of cannabinoid receptors

    International Nuclear Information System (INIS)

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

  1. A new spore differentiation factor (SDF) secreted by Dictyostelium cells is phosphorylated by the cAMP dependent protein kinase.

    Science.gov (United States)

    Anjard, C; van Bemmelen, M; Véron, M; Reymond, C D

    1997-10-01

    Upon starvation, Dictyostelium discoideum unicellular amoebae form a multicellular organism leading to the development of a fruiting body containing spores. Single cells of sporogenous mutants, unlike wild type cells, are able to differentiate into spores under specific conditions. We show in this report that overexpression of the catalytic subunit of the cAMP dependent protein kinase (PKA), not only renders the cells sporogenous, but is also accompanied by the production/release of a diffusible spore differentiation factor (SDF). SDF is a small, thermostable phospho-polypeptide. In vitro dephosphorylation reduces SDF spore differentiation capacity, which can be regained in vitro by PKA phosphorylation. These results indicate that SDF is a PKA substrate and might be activated in vivo by this protein kinase. Since spore differentiation requires PKA catalytic subunit activation, we conclude that the response of prespore cells to SDF involves an intracellular pathway dependent on PKA. PMID:9373946

  2. 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.

  3. Expression of phosphorylated cAMP response element binding protein (p-CREB) in bladder afferent pathways in VIP-/- mice with cyclophosphamide (CYP)-induced cystitis

    DEFF Research Database (Denmark)

    Jensen, Dorthe G; Studeny, Simon; May, Victor;

    2008-01-01

    The expression of phosphorylated cAMP response element binding protein (p-CREB) in dorsal root ganglia (DRG) with and without cyclophosphamide (CYP)-induced cystitis (150 mg/kg, i.p; 48 h) was determined in VIP(-/-) and wild-type (WT) mice. p-CREB immunoreactivity (IR) was determined in bladder...

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

    DEFF Research Database (Denmark)

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

    2015-01-01

    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 ...

  5. Comparative study of somatostatin-human serum albumin fusion proteins and natural somatostatin on receptor binding, internalization and activation.

    Directory of Open Access Journals (Sweden)

    Ying Peng

    Full Text Available Albumin fusion technology, the combination of small molecular proteins or peptides with human serum albumin (HSA, is an effective method for improving the medicinal values of natural small molecular proteins or peptides. However, comparative studies between HSA-fusion proteins or peptides and the parent small molecules in biological and molecular mechanisms are less reported. In this study, we examined the binding property of two novel somatostatin-HSA fusion proteins, (SST142-HSA and (SST282-HSA, to human SSTRs in stably expressing SSTR1-5 HEK 293 cells; observed the regulation of receptor internalization and internalized receptor recycling; and detected the receptors activation of HSA fusion proteins in stably expressing SSTR2- and SSTR3-EGFP cells. We showed that both somatostatin-HSA fusion proteins had high affinity to all five SSTRs, stimulated the ERK1/2 phosphorylation and persistently inhibited the accumulation of forskolin-stimulated cAMP in SSTR2- and SSTR3-expressing cells; but were less potent than the synthetic somatostatin-14 (SST-14. Our experiments also showed that somatostatin-HSA fusion proteins did not induce the receptors internalization; rather, they accelerated the recycling of the internalized receptors induced by SST-14 to the plasma membrane. Our results indicated that somatostatin-HSA fusion proteins, different from SST-14, exhibit some particular properties in binding, regulating, and activating somatostatin receptors.

  6. 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.

  7. Antisense oligodeoxynucleotide-mediated disruption of hippocampal cAMP response element binding protein levels impairs consolidation of memory for water maze training

    OpenAIRE

    Guzowski, John F.; McGaugh, James L.

    1997-01-01

    Extensive evidence suggests that long term memory (LTM) formation is dependent on the activation of neuronal second messenger systems and requires protein synthesis. The cAMP response element binding protein (CREB) is a constitutively expressed regulatory transcription factor that couples changes in second messenger levels to changes in cellular transcription. Several recent studies suggest that CREB and related transcription factors regulate gene expression necessary for neuronal plasticity ...

  8. Allosteric Optical Control of a Class B G-Protein-Coupled Receptor.

    Science.gov (United States)

    Broichhagen, Johannes; Johnston, Natalie R; von Ohlen, Yorrick; Meyer-Berg, Helena; Jones, Ben J; Bloom, Stephen R; Rutter, Guy A; Trauner, Dirk; Hodson, David J

    2016-05-01

    Allosteric regulation promises to open up new therapeutic avenues by increasing drug specificity at G-protein-coupled receptors (GPCRs). However, drug discovery efforts are at present hampered by an inability to precisely control the allosteric site. Herein, we describe the design, synthesis, and testing of PhotoETP, a light-activated positive allosteric modulator of the glucagon-like peptide-1 receptor (GLP-1R), a class B GPCR involved in the maintenance of glucose homeostasis in humans. PhotoETP potentiates Ca(2+) , cAMP, and insulin responses to glucagon-like peptide-1 and its metabolites following illumination of cells with blue light. PhotoETP thus provides a blueprint for the production of small-molecule class B GPCR allosteric photoswitches, and may represent a useful tool for understanding positive cooperativity at the GLP-1R. PMID:27059784

  9. Computational modeling of acrylodan-labeled cAMP dependent protein kinase catalytic subunit unfolding.

    Science.gov (United States)

    Kuznetsov, Aleksei; Kivi, Rait; Järv, Jaak

    2016-04-01

    Structure of the cAMP-dependent protein kinase catalytic subunit, where the asparagine residue 326 was replaced with acrylodan-cystein conjugate to implement this fluorescence reporter group into the enzyme, was modeled by molecular dynamics (MD) method and the positioning of the dye molecule in protein structure was characterized at temperatures 300K, 500K and 700K. It was found that the acrylodan moiety, which fluorescence is very sensitive to solvating properties of its microenvironment, was located on the surface of the native protein at 300K that enabled its partial solvation with water. At high temperatures the protein structure significantly changed, as the secondary and tertiary structure elements were unfolded and these changes were sensitively reflected in positioning of the dye molecule. At 700K complete unfolding of the protein occurred and the reporter group was entirely expelled into water. However, at 500K an intermediate of the protein unfolding process was formed, where the fluorescence reporter group was directed towards the protein interior and buried in the core of the formed molten globule state. This different positioning of the reporter group was in agreement with the two different shifts of emission spectrum of the covalently bound acrylodan, observed in the unfolding process of the protein. PMID:26896699

  10. 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

  11. 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.

  12. G-protein-coupled receptors: past, present and future

    OpenAIRE

    Hill, Stephen J

    2006-01-01

    The G-protein-coupled receptor (GPCR) family represents the largest and most versatile group of cell surface receptors. Drugs active at these receptors have therapeutic actions across a wide range of human diseases ranging from allergic rhinitis to pain, hypertension and schizophrenia. This review provides a brief historical overview of the properties and signalling characteristics of this important family of receptors.

  13. 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

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

    International Nuclear Information System (INIS)

    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

  15. Coupling of guanine nucleotide inhibitory protein to somatostatin receptors on pancreatic acinar membranes

    International Nuclear Information System (INIS)

    Guanine nucleotides and pertussis toxin were used to investigate whether somatostatin receptors interact with the guanine nucleotide inhibitory protein (NI) on pancreatic acinar membranes in the rat. Guanine nucleotides reduced 125I-[Tyr1]somatostatin binding to acinar membranes up to 80%, with rank order of potency being 5'-guanylyl imidodiphosphate [Gpp(NH)p]>GTP>TDP>GMP. Scatchard analysis revealed that the decrease in somatostatin binding caused by Gpp(NH)p was due to the decrease in the maximum binding capacity without a significant change in the binding affinity. The inhibitory effect of Gpp(NH)p was partially abolished in the absence of Mg2+. When pancreatic acini were treated with 1 μg/ml pertussis toxin for 4 h, subsequent 125I-[Tyr1]somatostatin binding to acinar membranes was reduced. Pertussis toxin treatment also abolished the inhibitory effect of somatostatin on vasoactive intestinal peptide-stimulated increase in cellular content of adenosine 3',5'-cyclic monophosphate (cAMP) in the acini. The present results suggest that 1) somatostatin probably functions in the pancreas to regulate adenylate cyclase enzyme system via Ni, 2) the extent of modification of Ni is correlated with the ability of somatostatin to inhibit cAMP accumulation in acini, and 3) guanine nucleotides also inhibit somatostatin binding to its receptor

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

    OpenAIRE

    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.

    2012-01-01

    The relaxin hormone is involved in a variety of biological functions including female reproduction and parturition, 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) RXFP1, relaxin family receptor 1, is a cognate relaxin receptor that mainly signals through cyclic AMP second messenger. While agonists of the ...

  17. Expression of Tas1 Taste Receptors in Mammalian Spermatozoa: Functional Role of Tas1r1 in Regulating Basal Ca2+ and cAMP Concentrations in Spermatozoa

    OpenAIRE

    Meyer, Dorke; Voigt, Anja; Widmayer, Patricia; Borth, Heike; Huebner, Sandra; Breit, Andreas; Marschall, Susan; de Angelis, Martin Hrabé; Boehm, Ulrich; Meyerhof, Wolfgang; Gudermann, Thomas; Boekhoff, Ingrid

    2012-01-01

    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....

  18. Melanocortin receptor accessory proteins in adrenal disease and obesity

    OpenAIRE

    Jackson, David S.; Ramachandrappa, Shwetha; Clark, Adrian J; Chan, Li F.

    2015-01-01

    Melanocortin receptor accessory proteins (MRAPs) are regulators of the melanocortin receptor family. MRAP is an essential accessory factor for the functional expression of the MC2R/ACTH receptor. The importance of MRAP in adrenal gland physiology is demonstrated by the clinical condition familial glucocorticoid deficiency type 2. The role of its paralog melanocortin-2-receptor accessory protein 2 (MRAP2), which is predominantly expressed in the hypothalamus including the paraventricular nucle...

  19. Regulation of cessation of respiration and killing by cyclic 3',5'-adenosine monophosphate and its receptor protein after far-ultraviolet irradiation of Escherichia coli

    International Nuclear Information System (INIS)

    When Escherichia coli B/r cultures are irradiated with ultraviolet light (UV) (254 nm), those cells that are killed stop respiring by 60 min after irradiation. Post-UV treatment with cyclic adenosine 3',5'-adenosine monophosphate (cAMP) causes more cells to stop respiring and to die. We have studied these effects at a UV fluence of 52 I/m2 in a a wild-type E. coli K 12 strain and in mutants defective in cAMP metabolism. Strain CA 8,000 has crp+ and cya+ genes for the cAMP receptor protein (CRP) (required for transcription of operons regulated by cAMP) and for adenylate cyclase, respectively; CA 7901 is crp-; and CA 8306 is a cya deletion (Δ). The wild-type culture showed a small transient cessation of respiration, and addition of cAMP caused cessation to be nearly complete. The crp- culture showed no evidence of cessation of respiration, and cAMP had no effect. The Δ cya mutant also showed no cessation of respiration, but cAMP (5 mM) caused as complete inhibition as in the wild type. cAMP caused a 10-fold loss in viability of UV-irradiated wild-type and Δ cya liquid cultures but had no effect on the cpr- culture. Respiration and viability changes were also studied in a double mutant, CA8404 Δ cya crp*, which has an altered CRP that is, with respect to the lac operon, independent of cAMP. The respiration response to UV was similar to that of the wild-type culture, and both respiration and viability of cells in liquid culture were sensitive to cAMP. The survival data, obtained by plating immediately after irradiation, show the wild type, Δ cya strains, and Δ cya crp* to be equally sensitive and the crp- strain to be more resistant. We conclude that cessation of respiration and cell killing after UV irradiation are regulated by cAMP and the CRP. (orig.)

  20. 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

  1. Identification of a G protein-coupled receptor for pheromone biosynthesis activating neuropeptide from pheromone glands of the moth Helicoverpa zea

    OpenAIRE

    Choi, Man-Yeon; Fuerst, Emily-Jean; Rafaeli, Ada; Jurenka, Russell

    2003-01-01

    Pheromone biosynthesis-activating neuropeptide (PBAN), a peptide produced by the subesophageal ganglion, is used by a variety of moths to regulate pheromone production. PBAN acts directly on pheromone gland cells by using calcium and cAMP as second messengers. We have identified a gene encoding a G protein-coupled receptor (GPCR) from pheromone glands of the female moth Helicoverpa zea. The gene was identified based on sequence identity to a group of GPCRs from Drosoph...

  2. Topology of cAMP dependent protein kinase and A-kinase anchor proteins in mammalian mitochondria

    Czech Academy of Sciences Publication Activity Database

    Nuzzi, R.; Sardanelli, A. M.; Dobrová, Zuzana; Signorile, A.; De Rasmo, D.; Papa, S.

    Milano, 2005, s. 57. ISSN 0021-2938. [SIB 2005. Riccione (IT), 27.09.2005-30.09.2005] Institutional research plan: CEZ:AV0Z50200510 Keywords : protein kinase * mammalian mitochondria Subject RIV: EE - Microbiology, Virology

  3. Yeast Ste2 receptors as tools for study of mammalian protein kinases and adaptors involved in receptor trafficking

    OpenAIRE

    2006-01-01

    Background Mammalian receptors that couple to effectors via heterotrimeric G proteins (e.g., beta 2-adrenergic receptors) and receptors with intrinsic tyrosine kinase activity (e.g., insulin and IGF-I receptors) constitute the proximal points of two dominant cell signaling pathways. Receptors coupled to G proteins can be substrates for tyrosine kinases, integrating signals from both pathways. Yeast cells, in contrast, display G protein-coupled receptors (e.g., alpha-factor pheromone receptor ...

  4. Influence of Gα protein subtype and expression level on receptor phenotypes generated from calcitonin receptor and RAMP interaction

    International Nuclear Information System (INIS)

    Full text: The calcitonin receptor (CTR) is a class II G protein-coupled receptor (GPCR). Co-expression of CTR with receptor activity modifying protein (RAMP) 1, 2 or 3 yields multiple amylin receptor phenotypes that vary in pharmacology dependent upon CTR isoform and cellular background. In COS-7 cells, co-expression of hCTRI1- with either RAMP1 or-3 generates amylin receptors (125I-rat amylin binding = 482 fmol/106 cells), while RAMP2 (85 fmol/106 cells) has no significant effect. To examine the potential role of Gα subtype and expression level in RAMP modulation of CTR, individual Gα subtypes (Gαs, Gαo, Gαi, or Gαq) were cloned either 5' or 3' of hCTRI1- in the pCIN bicistronic vector. Cells transfected with these constructs express Gα and CTR, in either a higher or lower Gα:hCTR stoichiometry, depending on the relative position of their cloning. In COS-7 cells, augmented Gαs:hCTRI1- potentiated the RAMP2 interaction with hCTR to yield an amylin receptor (125I-rat amylin binding = 604 fmol/106 cells). Increased amylin binding in the presence of higher Gαs was accompanied by enhanced potency of rat amylin (EC50 = ∼ 1 nM υ 80 nM for hCTR+RAMP1 only) in stimulating cAMP production. Strikingly, enhanced expression of Gα1, or Gαo (but not Gαs or Gαq) resulted in a significant loss of 125I-salmon CT binding (423 fmol/106 cells compared with 26,900 fmol/1066 cells when hCTR is overexpressed relative to Gαi, or Gαo), both in the presence and absence of RAMPs, while amylin binding in the presence of RAMPs was maintained. Transfection of Gαi or Gαo, but not Gαs, into CHO-K1 cells that express an endogenous CTR also led to lower 125I-sCT binding. The data suggest that Gα protein subtype and level can have profound effects on CTR phenotypes. These findings may have implications for other GPCRs as well. Copyright (2001) Australasian Society of Clinical and Experimental Pharmacologists and Toxicologists

  5. Neurobiology of corticotropin releasing factor (CRF) receptors and CRF-binding protein: implications for the treatment of CNS disorders.

    Science.gov (United States)

    Behan, D P; Grigoriadis, D E; Lovenberg, T; Chalmers, D; Heinrichs, S; Liaw, C; De Souza, E B

    1996-09-01

    The actions of CRF in the brain and in the periphery are mediated through multiple binding sites. There are three receptors, CRF1, CRF2 alpha and CRF2 beta, which encode 411, 415 and 431 amino acid proteins and transduce signals via the stimulation of intracellular cAMP production. The recent identification of high-affinity non-peptide CRF receptor antagonists should allow for rapid progress in drug development of CRF receptor antagonists. In addition to the receptors, the actions of CRF in brain and in the periphery can also be modulated by a binding protein of 322 amino acids. Ligands of CRF-BP, such as CRF (6-33) can elevate brain levels of 'free' CRF and improve learning and memory without stress-like side effects of CRF receptor agonists. Urocortin, a mammalian CRF-related peptide with close sequence homology to fish urotensin, interacts with CRF1, CRF2 receptors and with CRF-BP. These data indicate that CRF receptor antagonists may be useful for the treatment of the disease states where CRF is elevated such as anxiety and depression, anorexia nervosa and stroke and that ligand inhibitors of CRF-BP may be used to elevate brain levels of 'free' urocortin and other CRF-related peptides. PMID:9118350

  6. G Protein-Coupled Receptors in Cancer

    Science.gov (United States)

    Bar-Shavit, Rachel; Maoz, Myriam; Kancharla, Arun; Nag, Jeetendra Kumar; Agranovich, Daniel; Grisaru-Granovsky, Sorina; Uziely, Beatrice

    2016-01-01

    Despite the fact that G protein-coupled receptors (GPCRs) are the largest signal-conveying receptor family and mediate many physiological processes, their role in tumor biology is underappreciated. Numerous lines of evidence now associate GPCRs and their downstream signaling targets in cancer growth and development. Indeed, GPCRs control many features of tumorigenesis, including immune cell-mediated functions, proliferation, invasion and survival at the secondary site. Technological advances have further substantiated GPCR modifications in human tumors. Among these are point mutations, gene overexpression, GPCR silencing by promoter methylation and the number of gene copies. At this point, it is imperative to elucidate specific signaling pathways of “cancer driver” GPCRs. Emerging data on GPCR biology point to functional selectivity and “biased agonism”; hence, there is a diminishing enthusiasm for the concept of “one drug per GPCR target” and increasing interest in the identification of several drug options. Therefore, determining the appropriate context-dependent conformation of a functional GPCR as well as the contribution of GPCR alterations to cancer development remain significant challenges for the discovery of dominant cancer genes and the development of targeted therapeutics. PMID:27529230

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

    DEFF Research Database (Denmark)

    Brighton, Cheryl A.; Rievaj, Juraj; Kuhre, Rune E.;

    2015-01-01

    -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......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......-cells, we observed that taurodeoxycholate (TDCA) and taurolithocholate (TLCA) increased intracellular cAMP and Ca2+. In primary intestinal cultures, TDCA was a more potent GLP-1 secretagogue than taurocholate (TCA) and TLCA, correlating with a stronger Ca2+ response to TDCA. Using small-volume Ussing...

  8. Quantifying Agonist Activity at G Protein-coupled Receptors

    OpenAIRE

    Ehlert, Frederick J.; Suga, Hinako; Griffin, Michael T.

    2011-01-01

    When an agonist activates a population of G protein-coupled receptors (GPCRs), it elicits a signaling pathway that culminates in the response of the cell or tissue. This process can be analyzed at the level of a single receptor, a population of receptors, or a downstream response. Here we describe how to analyze the downstream response to obtain an estimate of the agonist affinity constant for the active state of single receptors.

  9. Structural insights into G-protein-coupled receptor activation☆

    OpenAIRE

    Weis, William I.; Kobilka, Brian K.

    2008-01-01

    G-protein-coupled receptors (GPCRs) are the largest family of eukaryotic plasma membrane receptors, and are responsible for the majority of cellular responses to external signals. GPCRs share a common architecture comprising seven transmembrane (TM) helices. Binding of an activating ligand enables the receptor to catalyze the exchange of GTP for GDP in a heterotrimeric G protein. GPCRs are in a conformational equilibrium between inactive and activating states. Crystallographic and spectroscop...

  10. Rethinking the roles of CRP, cAMP, and sugar-mediated global regulation in the Vibrionaceae.

    Science.gov (United States)

    Colton, Deanna M; Stabb, Eric V

    2016-02-01

    Many proteobacteria modulate a suite of catabolic genes using the second messenger cyclic 3', 5'-AMP (cAMP) and the cAMP receptor protein (CRP). Together, the cAMP-CRP complex regulates target promoters, usually by activating transcription. In the canonical model, the phosphotransferase system (PTS), and in particular the EIIA(Glc) component for glucose uptake, provides a mechanistic link that modulates cAMP levels depending on glucose availability, resulting in more cAMP and activation of alternative catabolic pathways when glucose is unavailable. Within the Vibrionaceae, cAMP-CRP appears to play the classical role in modulating metabolic pathways; however, it also controls functions involved in natural competence, bioluminescence, pheromone signaling, and colonization of animal hosts. For this group of marine bacteria, chitin is an ecologically relevant resource, and chitin's monomeric sugar N-acetylglucosamine (NAG) supports robust growth while also triggering regulatory responses. Recent studies with Vibrio fischeri indicate that NAG and glucose uptake share EIIA(Glc), yet the responses of cAMP-CRP to these two carbon sources are starkly different. Moreover, control of cAMP levels appears to be more dominantly controlled by export and degradation. Perhaps more surprisingly, although CRP may require cAMP, its activity can be controlled in response to glucose by a mechanism independent of cAMP levels. Future studies in this area promise to shed new light on the role of cAMP and CRP. PMID:26215147

  11. The human N-formylpeptide receptor. Characterization of two cDNA isolates and evidence for a new subfamily of G-protein-coupled receptors

    International Nuclear Information System (INIS)

    Two variants of the human N-formylpeptide chemoattractant receptor have been isolated from a CDM8 expression library prepared from mRNA of human myeloid HL-60 cells differentiated to the granulocyte phenotype with Bt2cAMP. Both recombinant receptors, fMLP-R26 and fMLP-R98, are 350 amino acids long (Mr 38 420); they differ from each other by two residue changes at positions 101 and 346 and by significant differences in the 5' and 3' untranslated regions. Both clones were able to transfer to COS-7 cells the capacity to specificaly bind a new and highly efficient hydrophilic derivative of N-formyl-Met-Leu-Phe-Lys, referred to as fMLPK-Pep12. Photolabeling experiments revealed that the glycosylated form of the fMLP receptor in COS cells has a molecular weight (Mr 50 000-70 000) similar to that observed for the native receptor in differentiated HL-60 cells. Northern blot analysis revealed a major transcript of 1.6-1.7 kb and two minor hybridization signals of 2.3 and 3.1 kb, suggesting a related family of receptors. The complex hybridization pattern obtained with restricted genomic DNA was consistent with either two genes encoding fMLP receptor isoforms or a single gene with at least one intron in the coding sequence. Sequence comparison established that the fMLP receptor belongs to the G-protein-coupled receptor superfamily. The structural similarities observed with RDC1, a receptor isolated from a dog thyroid cDNA library, which shares weak homologies with other members of the family, suggests that the fMLP receptor is representative of a new subfamily

  12. Active site coupling in PDE:PKA complexes promotes resetting of mammalian cAMP signaling.

    Science.gov (United States)

    Krishnamurthy, Srinath; Moorthy, Balakrishnan Shenbaga; Xin Xiang, Lim; Xin Shan, Lim; Bharatham, Kavitha; Tulsian, Nikhil Kumar; Mihalek, Ivana; Anand, Ganesh S

    2014-09-16

    Cyclic 3'5' adenosine monophosphate (cAMP)-dependent-protein kinase (PKA) signaling is a fundamental regulatory pathway for mediating cellular responses to hormonal stimuli. The pathway is activated by high-affinity association of cAMP with the regulatory subunit of PKA and signal termination is achieved upon cAMP dissociation from PKA. Although steps in the activation phase are well understood, little is known on how signal termination/resetting occurs. Due to the high affinity of cAMP to PKA (KD ∼ low nM), bound cAMP does not readily dissociate from PKA, thus begging the question of how tightly bound cAMP is released from PKA to reset its signaling state to respond to subsequent stimuli. It has been recently shown that phosphodiesterases (PDEs) can catalyze dissociation of bound cAMP and thereby play an active role in cAMP signal desensitization/termination. This is achieved through direct interactions with the regulatory subunit of PKA, thereby facilitating cAMP dissociation and hydrolysis. In this study, we have mapped direct interactions between a specific cyclic nucleotide phosphodiesterase (PDE8A) and a PKA regulatory subunit (RIα isoform) in mammalian cAMP signaling, by a combination of amide hydrogen/deuterium exchange mass spectrometry, peptide array, and computational docking. The interaction interface of the PDE8A:RIα complex, probed by peptide array and hydrogen/deuterium exchange mass spectrometry, brings together regions spanning the phosphodiesterase active site and cAMP-binding sites of RIα. Computational docking combined with amide hydrogen/deuterium exchange mass spectrometry provided a model for parallel dissociation of bound cAMP from the two tandem cAMP-binding domains of RIα. Active site coupling suggests a role for substrate channeling in the PDE-dependent dissociation and hydrolysis of cAMP bound to PKA. This is the first instance, to our knowledge, of PDEs directly interacting with a cAMP-receptor protein in a mammalian system, and

  13. 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...... are believed to activate (agonists) or inhibit (competitive antagonists) receptor signalling by binding the receptor at the same site as the endogenous agonist, the orthosteric site. In contrast, allosteric ligands modulate receptor function by binding to different regions in the receptor, allosteric...... 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....

  14. A Novel Fic (Filamentation Induced by cAMP) Protein from Clostridium difficile Reveals an Inhibitory Motif-independent Adenylylation/AMPylation Mechanism.

    Science.gov (United States)

    Dedic, Emil; Alsarraf, Husam; Welner, Ditte Hededam; Østergaard, Ole; Klychnikov, Oleg I; Hensbergen, Paul J; Corver, Jeroen; van Leeuwen, Hans C; Jørgensen, René

    2016-06-17

    Filamentation induced by cAMP (Fic) domain proteins have been shown to catalyze the transfer of the AMP moiety from ATP onto a protein target. This type of post-translational modification was recently shown to play a crucial role in pathogenicity mediated by two bacterial virulence factors. Herein we characterize a novel Fic domain protein that we identified from the human pathogen Clostridium difficile The crystal structure shows that the protein adopts a classical all-helical Fic fold, which belongs to class II of Fic domain proteins characterized by an intrinsic N-terminal autoinhibitory α-helix. A conserved glutamate residue in the inhibitory helix motif was previously shown in other Fic domain proteins to prevent proper binding of the ATP γ-phosphate. However, here we demonstrate that both ATP binding and autoadenylylation activity of the C. difficile Fic domain protein are independent of the inhibitory motif. In support of this, the crystal structure of a mutant of this Fic protein in complex with ATP reveals that the γ-phosphate adopts a conformation unique among Fic domains that seems to override the effect of the inhibitory helix. These results provide important structural insight into the adenylylation reaction mechanism catalyzed by Fic domains. Our findings reveal the presence of a class II Fic domain protein in the human pathogen C. difficile that is not regulated by autoinhibition and challenge the current dogma that all class I-III Fic domain proteins are inhibited by the inhibitory α-helix. PMID:27076635

  15. Histamine-induced inhibition of leukotriene biosynthesis in human neutrophils: involvement of the H2 receptor and cAMP

    OpenAIRE

    Flamand, Nicolas; Plante, Hendrick; Picard, Serge; Laviolette, Michel; Borgeat, Pierre

    2004-01-01

    Histamine is generally regarded as a pro-inflammatory mediator in diseases such as allergy and asthma. A growing number of studies, however, suggest that this autacoid is also involved in the downregulation of human polymorphonuclear leukocyte (PMN) functions and inflammatory responses through activation of the Gs-coupled histamine H2 receptor.We report here that histamine inhibits thapsigargin- and ligand (PAF and fMLP)-induced leukotriene (LT) biosynthesis in human PMN in a dose-dependent m...

  16. Activation of P2X7 and P2Y11 purinergic receptors inhibits migration and normalizes tumor-derived endothelial cells via cAMP signaling.

    Science.gov (United States)

    Avanzato, D; Genova, T; Fiorio Pla, A; Bernardini, M; Bianco, S; Bussolati, B; Mancardi, D; Giraudo, E; Maione, F; Cassoni, P; Castellano, I; Munaron, L

    2016-01-01

    Purinergic signaling is involved in inflammation and cancer. Extracellular ATP accumulates in tumor interstitium, reaching hundreds micromolar concentrations, but its functional role on tumor vasculature and endothelium is unknown. Here we show that high ATP doses (>20 μM) strongly inhibit migration of endothelial cells from human breast carcinoma (BTEC), but not of normal human microvascular EC. Lower doses (1-10 mm result ineffective. The anti-migratory activity is associated with cytoskeleton remodeling and is significantly prevented by hypoxia. Pharmacological and molecular evidences suggest a major role for P2X7R and P2Y11R in ATP-mediated inhibition of TEC migration: selective activation of these purinergic receptors by BzATP mimics the anti-migratory effect of ATP, which is in turn impaired by their pharmacological or molecular silencing. Downstream pathway includes calcium-dependent Adenilyl Cyclase 10 (AC10) recruitment, cAMP release and EPAC-1 activation. Notably, high ATP enhances TEC-mediated attraction of human pericytes, leading to a decrease of endothelial permeability, a hallmark of vessel normalization. Finally, we provide the first evidence of in vivo P2X7R expression in blood vessels of murine and human breast carcinoma. In conclusion, we have identified a purinergic pathway selectively acting as an antiangiogenic and normalizing signal for human tumor-derived vascular endothelium. PMID:27586846

  17. Heterotrimeric G proteins precouple with G protein-coupled receptors in living cells

    OpenAIRE

    Nobles, M.; Benians, A.; Tinker, A

    2005-01-01

    Using fluorescence resonance energy transfer (FRET) microscopy, we investigate how heterotrimeric G proteins interact with G protein-coupled receptors (GPCRs). In the absence of receptor activation, the alpha 2A adrenergic and muscarinic M4 receptors are present on the cell membrane as dimers. Furthermore, there is an interaction between the G protein subunits alpha o, beta 1, and gamma 2 and a number of GPCRs including M4, a2A, the adenosine All receptor, and the dopamine D2 receptor under r...

  18. The cAMP signaling system inhibits the repair of γ-ray-induced DNA damage by promoting Epac1-mediated proteasomal degradation of XRCC1 protein in human lung cancer cells

    International Nuclear Information System (INIS)

    Highlights: ► cAMP signaling system inhibits repair of γ-ray-induced DNA damage. ► cAMP signaling system inhibits DNA damage repair by decreasing XRCC1 expression. ► cAMP signaling system decreases XRCC1 expression by promoting its proteasomal degradation. ► The promotion of XRCC1 degradation by cAMP signaling system is mediated by Epac1. -- Abstract: Cyclic AMP is involved in the regulation of metabolism, gene expression, cellular growth and proliferation. Recently, the cAMP signaling system was found to modulate DNA-damaging agent-induced apoptosis by regulating the expression of Bcl-2 family proteins and inhibitors of apoptosis. Thus, we hypothesized that the cAMP signaling may modulate DNA repair activity, and we investigated the effects of the cAMP signaling system on γ-ray-induced DNA damage repair in lung cancer cells. Transient expression of a constitutively active mutant of stimulatory G protein (GαsQL) or treatment with forskolin, an adenylyl cyclase activator, augmented radiation-induced DNA damage and inhibited repair of the damage in H1299 lung cancer cells. Expression of GαsQL or treatment with forskolin or isoproterenol inhibited the radiation-induced expression of the XRCC1 protein, and exogenous expression of XRCC1 abolished the DNA repair-inhibiting effect of forskolin. Forskolin treatment promoted the ubiquitin and proteasome-dependent degradation of the XRCC1 protein, resulting in a significant decrease in the half-life of the protein after γ-ray irradiation. The effect of forskolin on XRCC1 expression was not inhibited by PKA inhibitor, but 8-pCPT-2′-O-Me-cAMP, an Epac-selective cAMP analog, increased ubiquitination of XRCC1 protein and decreased XRCC1 expression. Knockdown of Epac1 abolished the effect of 8-pCPT-2′-O-Me-cAMP and restored XRCC1 protein level following γ-ray irradiation. From these results, we conclude that the cAMP signaling system inhibits the repair of γ-ray-induced DNA damage by promoting the ubiquitin

  19. 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...... resulted in prolonged signaling and enabled detection of weak partial agonists and/or ligands with low potency, which is highly advantageous in large HTS settings and hit identification. In addition, the assay enabled detection of β2AR inverse agonists and PDE inhibitors. High signal-to-noise ratios were...

  20. The melanocortin receptors and their accessory proteins

    OpenAIRE

    Ramachandrappa, Shwetha; Gorrigan, Rebecca J.; Clark, Adrian J.L.; Chan, Li F.

    2013-01-01

    The five melanocortin receptors (MCRs) named MC1R–MC5R have diverse physiological roles encompassing pigmentation, steroidogenesis, energy homeostasis and feeding behavior 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, anta...

  1. The melanocortin receptors and their accessory proteins

    OpenAIRE

    LiChan

    2013-01-01

    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, antagonis...

  2. 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......, there is increasing evidence that the receptor is involved in regulation of inflammation, metabolism and endocrine functions. GPRC6A could thus be an interesting target for new drugs in these therapeutic areas....

  3. 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

    signaling mediated by RTKs must be tightly regulated by interacting proteins including protein-tyrosine phosphatases and ubiquitin ligases. The suppressors of cytokine signaling (SOCS) family proteins are well-known negative regulators of cytokine receptors signaling consisting of eight structurally similar...... 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...

  4. Prolactin receptor and signal transduction to milk protein genes

    Energy Technology Data Exchange (ETDEWEB)

    Djiane, J.; Daniel, N.; Bignon, C. [Unite d`Endocrinologie Moleculaire, Jouy en Josas (France)] [and others

    1994-06-01

    After cloning of the mammary gland prolactin (PRL) receptor cDNA, a functional assay was established using co-transfection of PRL receptor cDNA together with a milk protein promoter/chloramphenicol acetyl transferase (CAT) construct in Chinese hamster ovary (CHO) cells. Different mutants of the PRL receptor were tested in this CAT assay to delimit the domains in the receptor necessary for signal transduction to milk protein genes. In CHO cells stably transfected with PRL receptor cDNA, high numbers of PRL receptor are expressed. By metabolic labeling and immunoprecipitation, expressed PRL receptor was identified as a single species of 100 kDa. Using these cells, we analyzed the effects of PRL on intracellular free Ca{sup ++} concentration. PRL stimulates Ca{sup ++} entry and induces secondary Ca{sup ++} mobilization. The entry of Ca{sup ++} is a result of an increase in K{sup +} conductance that hyperpolarizes the membranes. We have also analyzed tyrosine phosphorylation induced by PRL. In CHO cells stably transfected with PRL receptor cDNA, PRL induced a very rapid and transient tyrosine phosphorylation of a 100-kDa protein which is most probably the PRL receptor. The same finding was obtained in mammary membranes after PRL injection to lactating rabbits. Whereas tyrosine kinase inhibitors genistein and lavendustin were without effect, PRL stimulation of milk protein gene promoters was partially inhibited by 2 {mu}M herbimycin in CHO cells co-transfected with PRL receptor cDNA and the {Beta} lactoglobulin CAT construct. Taken together these observations indicate that the cytoplasmic domain of the PRL receptor interacts with one or several tyrosine kinases, which may represent early postreceptor events necessary for PRL signal transduction to milk protein genes. 14 refs., 4 figs.

  5. Structure and Function of Serotonin G protein Coupled Receptors

    OpenAIRE

    McCorvy, John D.; Roth, Bryan L.

    2015-01-01

    Serotonin receptors are prevalent throughout the nervous system and the periphery, and remain one of the most lucrative and promising drug discovery targets for disorders ranging from migraine headaches to neuropsychiatric disorders such as schizophrenia and depression. There are 14 distinct serotonin receptors, of which 13 are G protein coupled receptors (GPCRs), which are targets for approximately 40% of the approved medicines. Recent crystallographic and biochemical evidence has provided a...

  6. 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.

  7. Oligomeric forms of G protein-coupled receptors (GPCRs)

    OpenAIRE

    Palczewski, Krzysztof

    2010-01-01

    Oligomerization is a general characteristic of cell membrane receptors that is shared by G protein-coupled receptors (GPCRs) together with their G protein partners. Recent studies of these complexes, both in vivo and in purified reconstituted forms, unequivocally support this contention for GPCRs, perhaps with only rare exceptions. As evidence has evolved from experimental cell lines to more relevant in vivo studies and from indirect biophysical approaches to well defined isolated complexes o...

  8. Semiotic Selection of Mutated or Misfolded Receptor Proteins

    DEFF Research Database (Denmark)

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

    2013-01-01

    contention that the plasma membrane acts as the locus where several contextual cues may be integrated. As such it allows the semiotic selection of those receptor configurations that provide cells with the minimum essential requirements for agency. The occurrence of protein misfolding makes it impossible for...... 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....

  9. Regulation of ATP-sensitive K+ channels in insulinoma cells: Activation by somatostatin and protein kinase C and the role of cAMP

    International Nuclear Information System (INIS)

    The actions of somatostatin and of the phorbol ester 4β-phorbol 12-myristate 13-acetate (PMA) were studied in rat insulinoma (RINm5F) cells by electrophysiological and 86Rb+ flux techniques. Both PMA and somatostatin hyperpolarize insulinoma cells by activating ATP-sensitive K+ channels. The presence of intracellular GTP is required for the somatostatin effects. PMA- and somatostatin-induced hyperpolarization and channel activity are inhibited by the sulfonylurea glibenclamide. Glibenclamide-sensitive 86Rb+ efflux from insulinoma cells is stimulated by somatostatin in a dose-dependent manner (half maximal effect at 0.7 nM) and abolished by pertussis toxin pretreatment. Mutual roles of a GTP-binding protein, of protein kinase C, and of cAMP in the regulation of ATP-sensitive K+ channels are discussed

  10. Changes of phosphorylation of cAMP response element binding protein in rat nucleus accumbens after chronic ethanol intake: naloxone reversal

    Institute of Scientific and Technical Information of China (English)

    LIJing; LIYue-Hua; YUANXiao-Ru

    2003-01-01

    AIM: To study the changes in the expression and phosphorylation of cAMP response element binding protein(CREB) in the rat nucleus accumbens after chronic ethanol intake and its withdrawal. METHODS: Ethanol wasgiven in drinking water at the concentration of 6 % (v/v), for one month. Changes in the levels of CREB andphospho-CREB (p-CREB) protein in the nucleus accumbens were measured by immunohistochemistry methods.RESULTS: Ethanol given to rats in drinking water decreased the level of p-CREB protein in the nucleus accumbens(-75 %) at the time of exposure to ethanol. The decrement of p-CREB protein in the nucleus accumbens remainedat 24 h (-35 %) and 72 h (-28 %) of ethanol withdrawal, which recovered toward control level after 7 d of ethanolwithdrawal. However, chronic ethanol, as well as ethanol withdrawal failed to produce any significant alteration inthe level of CREB protein in the nucleus accumbens. Naloxone (alone) treatment of rats had no effect on the levelsof CREB and p-CREB protein in the nucleus accumbens. However, when naloxone was administered concurrentlywith ethanol treatment, it antagonized the down-regulation of p-CREB protein in the nucleus accumbens (142 %) ofrats exposed to ethanol. CONCLUSION: A long-term intake of ethanol solution down-regulates the phosphoryla-tion of CREB in the nucleus accumbens, and those changes can be reversed by naloxone, which may be one kindof the molecular mechanisms associated with ethano1 dependence.

  11. G Protein-Coupled Receptor Rhodopsin: A Prospectus

    OpenAIRE

    Filipek, Sławomir; Stenkamp, Ronald E.; Teller, David C.; Palczewski, Krzysztof

    2002-01-01

    Rhodopsin is a retinal photoreceptor protein of bipartite structure consisting of the transmembrane protein opsin and a light-sensitive chromophore 11-cis-retinal, linked to opsin via a protonated Schiff base. Studies on rhodopsin have unveiled many structural and functional features that are common to a large and pharmacologically important group of proteins from the G protein-coupled receptor (GPCR) superfamily, of which rhodopsin is the best-studied member. In this work, we focus on struct...

  12. Multiple Facets of cAMP Signalling and Physiological Impact: cAMP Compartmentalization in the Lung

    Directory of Open Access Journals (Sweden)

    Martina Schmidt

    2012-11-01

    Full Text Available Therapies involving elevation of the endogenous suppressor cyclic AMP (cAMP are currently used in the treatment of several chronic inflammatory disorders, including chronic obstructive pulmonary disease (COPD. Characteristics of COPD are airway obstruction, airway inflammation and airway remodelling, processes encompassed by increased airway smooth muscle mass, epithelial changes, goblet cell and submucosal gland hyperplasia. In addition to inflammatory cells, airway smooth muscle cells and (myofibroblasts, epithelial cells underpin a variety of key responses in the airways such as inflammatory cytokine release, airway remodelling, mucus hypersecretion and airway barrier function. Cigarette smoke, being next to environmental pollution the main cause of COPD, is believed to cause epithelial hyperpermeability by disrupting the barrier function. Here we will focus on the most recent progress on compartmentalized signalling by cAMP. In addition to G protein-coupled receptors, adenylyl cyclases, cAMP-specific phospho-diesterases (PDEs maintain compartmentalized cAMP signalling. Intriguingly, spatially discrete cAMP-sensing signalling complexes seem also to involve distinct members of the A-kinase anchoring (AKAP superfamily and IQ motif containing GTPase activating protein (IQGAPs. In this review, we will highlight the interaction between cAMP and the epithelial barrier to retain proper lung function and to alleviate COPD symptoms and focus on the possible molecular mechanisms involved in this process. Future studies should include the development of cAMP-sensing multiprotein complex specific disruptors and/or stabilizers to orchestrate cellular functions. Compartmentalized cAMP signalling regulates important cellular processes in the lung and may serve as a therapeutic target.

  13. σ1 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

    2010-01-01

    σ 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 μ opioid receptor, we now demonstrate a similar modulation of opioid function, as assessed by guanosine 5′-O-(3-[35S]thio)triphosphate ([35S]GTPγS) binding, by σ1 receptors. σ Ligands do not compete opioid receptor binding. Administered alone, neither σ agonists nor antagonists significantly stimulated [35S]GTPγS binding. Yet σ receptor selective antagonists, but not agonists, shifted the EC50 of opioid-induced stimulation of [35S]GTPγS 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 [35S]GTPγS binding. σ1 Receptors physically associate with μ opioid receptors, as shown by coimmunoprecipitation studies in transfected HEK cells, implying a direct interaction between the proteins. Thus, σ receptors modulate opioid transduction without influencing opioid receptor binding. RNA interference knockdown of σ1 in BE(2)-C cells also potentiated μ opioid-induced stimulation of [35S]GTPγS binding. These modulatory actions are not limited to μ and δ opioid receptors. In mouse brain membrane preparations, σ1-selective antagonists also potentiated both opioid receptor and muscarinic acetylcholine receptor-mediated stimulation of [35S]GTPγS binding, suggesting a broader role for σ receptors in modulating G-protein-coupled receptor signaling. PMID:20089882

  14. 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. PMID:20089882

  15. Effects of dopamine 1 receptor agonists on the content of cAMP in canine renal arteries%DA1受体激动剂对犬肾动脉cAMP生成量的影响

    Institute of Scientific and Technical Information of China (English)

    朱琳; 冯羡菊; 薛敬礼; 赵荣瑞

    2001-01-01

    Aim:To study the effects of DA1 receptor agonists, Fenoldopam(FODA) on the content of cAMP in canine renal arteries. Methods :Used radioimmunoassay technique to determine the content of cAMP in canine renal artery after given FODA and SCH23390. Results:FODA could increase the cAMP formation, and SCH23390 blocked the efficacy of FODA. Conclusion:The relaxing responses of renal arteries to DA receptor agonists may be related to the change of cAMP content.%目的:研究多巴胺1(DA1)受体激动剂非诺多泮(fenoldopam,FODA)对犬肾动脉cAMP含量的影响。方法:利用放射免疫分析技术,测定FODA对犬肾动脉DA1受体cAMP生成量的影响。结果:FODA可呈浓度依赖性激活肾动脉腺苷酸环化酶活性,增加cAMP生成量,选择性DA1受体阻断剂SCH23390能够显著减少FODA所引起的肾动脉cAMP生成量。结论:DA1受体激动剂对肾血管的舒张反应可能与cAMP生成量的变化有着密切关系。

  16. Serial femtosecond crystallography datasets from G protein-coupled receptors.

    Science.gov (United States)

    White, Thomas A; Barty, Anton; Liu, Wei; Ishchenko, Andrii; Zhang, Haitao; Gati, Cornelius; Zatsepin, Nadia A; Basu, Shibom; Oberthür, Dominik; Metz, Markus; Beyerlein, Kenneth R; Yoon, Chun Hong; Yefanov, Oleksandr M; James, Daniel; Wang, Dingjie; Messerschmidt, Marc; Koglin, Jason E; Boutet, Sébastien; Weierstall, Uwe; Cherezov, Vadim

    2016-01-01

    We describe the deposition of four datasets consisting of X-ray diffraction images acquired using serial femtosecond crystallography experiments on microcrystals of human G protein-coupled receptors, grown and delivered in lipidic cubic phase, at the Linac Coherent Light Source. The receptors are: the human serotonin receptor 2B in complex with an agonist ergotamine, the human δ-opioid receptor in complex with a bi-functional peptide ligand DIPP-NH2, the human smoothened receptor in complex with an antagonist cyclopamine, and finally the human angiotensin II type 1 receptor in complex with the selective antagonist ZD7155. All four datasets have been deposited, with minimal processing, in an HDF5-based file format, which can be used directly for crystallographic processing with CrystFEL or other software. We have provided processing scripts and supporting files for recent versions of CrystFEL, which can be used to validate the data. PMID:27479354

  17. 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...

  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. 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. PMID:25485460

  20. Identification of novel viral receptors with cell line expressing viral receptor-binding protein.

    Science.gov (United States)

    Mei, Mei; Ye, Jianqiang; Qin, Aijian; Wang, Lin; Hu, Xuming; Qian, Kun; Shao, Hongxia

    2015-01-01

    The viral cell receptors and infection can be blocked by the expression of the viral receptor-binding protein. Thus, the viral cell receptor is an attractive target for anti-viral strategies, and the identification of viral cell receptor is critical for better understanding and controlling viral disease. As a model system for viral entry and anti-retroviral approaches, avian sarcoma/leukosis virus (ASLV, including the A-J ten subgroups) has been studied intensively and many milestone discoveries have been achieved based on work with ASLV. Here, we used a DF1 cell line expressed viral receptor-binding protein to efficiently identify chicken Annexin A2 (chANXA2) as a novel receptor for retrovirus ALV-J (avian leukosis virus subgroup J). Our data demonstrate that antibodies or siRNA to chANXA2 significantly inhibited ALV-J infection and replication, and over-expression of chANXA2 permitted the entry of ALV-J into its non-permissible cells. Our findings have not only identified chANXA2 as a novel biomarker for anti-ALV-J, but also demonstrated that cell lines with the expression of viral receptor-binding protein could be as efficient tools for isolating functional receptors to identify novel anti-viral targets. PMID:25604889

  1. Cooperation between cAMP signalling and sulfonylurea in insulin secretion.

    Science.gov (United States)

    Shibasaki, T; Takahashi, T; Takahashi, H; Seino, S

    2014-09-01

    Although glucose is physiologically the most important regulator of insulin secretion, glucose-induced insulin secretion is modulated by hormonal and neural inputs to pancreatic β-cells. Most of the hormones and neurotransmitters evoke intracellular signals such as cAMP, Ca²⁺ , and phospholipid-derived molecules by activating G protein-coupled receptors (GPCRs). In particular, cAMP is a key second messenger that amplifies insulin secretion in a glucose concentration-dependent manner. The action of cAMP on insulin secretion is mediated by both protein kinase A (PKA)-dependent and Epac2A-dependent mechanisms. Many of the proteins expressed in β-cells are phosphorylated by PKA in vitro, but only a few proteins in which PKA phosphorylation directly affects insulin secretion have been identified. On the other hand, Epac2A activates the Ras-like small G protein Rap in a cAMP-dependent manner. Epac2A is also directly activated by various sulfonylureas, except for gliclazide. 8-pCPT-2'-O-Me-cAMP, an Epac-selective cAMP analogue, and glibenclamide, a sulfonylurea, synergistically activate Epac2A and Rap1, whereas adrenaline, which suppresses cAMP production in pancreatic β-cells, blocks activation of Epac2A and Rap1 by glibenclamide. Thus, cAMP signalling and sulfonylurea cooperatively activate Epac2A and Rap1. This interaction could account, at least in part, for the synergistic effects of incretin-related drugs and sulfonylureas in insulin secretion. Accordingly, clarification of the mechanism of Epac2A activation may provide therapeutic strategies to improve insulin secretion in diabetes. PMID:25200305

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

    Science.gov (United States)

    Fridlyand, Leonid E.; Philipson, Louis H.

    2016-01-01

    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 second messenger

  3. Identification of G protein-coupled receptors required for vitellogenin uptake into the oocytes of the red flour beetle, Tribolium castaneum.

    Science.gov (United States)

    Bai, Hua; Palli, Subba Reddy

    2016-01-01

    Previous studies suggested that a membrane receptor might be involved in mediating vitellogenin (Vg) uptake and juvenile hormone (JH)-regulated remodeling of follicular epithelium (also called 'patency'). G protein-coupled receptor (GPCR) family is one of the largest membrane receptor protein families and controls many key physiological processes. To investigate the role of GPCRs in insect reproduction and juvenile hormone-regulated Vg uptake, we performed a comprehensive RNA interference (RNAi) screen targeting GPCRs in the red flour beetle, Tribolium castaneum. Out of 112 GPCRs tested, knockdown of 41 GPCRs resulted in a reduction in fecundity. Interestingly, RNAi against two GPCRs (a Rhodopsin-like receptor and a Dopamine D2-like receptor) led to a significant reduction in Vg accumulation in developing oocytes. Functional assays of these two GPCRs showed that JH triggers a dose-dependent inhibition of intracellular cAMP levels in HEK293 cells expressing Tribolium Dopamine D2-like receptor. These data suggest that Dopamine D2-like receptor plays crucial roles in regulating Vg uptake and is a promising candidate membrane receptor mediating JH regulation of patency in the red flour beetle. PMID:27277501

  4. Eosinophil viability is increased by acidic pH in a cAMP- and GPR65-dependent manner.

    Science.gov (United States)

    Kottyan, Leah C; Collier, Ann R; Cao, Khanh H; Niese, Kathryn A; Hedgebeth, Megan; Radu, Caius G; Witte, Owen N; Khurana Hershey, Gurjit K; Rothenberg, Marc E; Zimmermann, Nives

    2009-09-24

    The microenvironment of the lung in asthma is acidic, yet the effect of acidity on inflammatory cells has not been well established. We now demonstrate that acidity inhibits eosinophil apoptosis and increases cellular viability in a dose-dependent manner between pH 7.5 and 6.0. Notably, acidity induced eosinophil cyclic adenosine 5'-monophosphate (cAMP) production and enhanced cellular viability in an adenylate cyclase-dependent manner. Furthermore, we identify G protein-coupled receptor 65 (GPR65) as the chief acid-sensing receptor expressed by eosinophils, as GPR65-deficient eosinophils were resistant to acid-induced eosinophil cAMP production and enhanced viability. Notably, GPR65(-/-) mice had attenuated airway eosinophilia and increased apoptosis in 2 distinct models of allergic airway disease. We conclude that eosinophil viability is increased in acidic microenvironments in a cAMP- and GPR65-dependent manner. PMID:19641187

  5. Antigen receptor signaling: integration of protein tyrosine kinase functions.

    Science.gov (United States)

    Tamir, I; Cambier, J C

    1998-09-17

    Antigen receptors on T and B cells function to transduce signals leading to a variety of biologic responses minimally including antigen receptor editing, apoptotic death, developmental progression, cell activation, proliferation and survival. The response to antigen depends upon antigen affinity and valence, involvement of coreceptors in signaling and differentiative stage of the responding cell. The requirement that these receptors integrate signals that drive an array of responses may explain their evolved structural complexity. Antigen receptors are composed of multiple subunits compartmentalized to provide antigen recognition and signal transduction function. In lieu of on-board enzymatic activity these receptors rely on associated Protein Tyrosine Kinases (PTKs) for their signaling function. By aggregating the receptors, and hence their appended PTKs, antigens induce PTK transphosphorylation, activating them to phosphorylate the receptor within conserved motifs termed Immunoreceptor Tyrosine-based Activation Motifs (ITAMs) found in transducer subunits. The tyrosyl phosphorylated ITAMs then interact with Src Homology 2 (SH2) domains within the PTKs leading to their further activation. As receptor phosphorylation is amplified, other effectors, such as Shc, dock by virtue of SH2 binding, and serve, in-turn, as substrates for these PTKs. This sequence of events not only provides a signal amplification mechanism by combining multiple consecutive steps with positive feedback, but also allows for signal diversification by differential recruitment of effectors that provide access to distinct parallel downstream signaling pathways. The subject of antigen receptor signaling has been recently reviewed in depth (DeFranco, 1997; Kurosaki, 1997). Here we discuss the biochemical basis of antigen receptor signal transduction, using the B cell receptor (BCR) as a paradigm, with specific emphasis on the involved PTKs. We review several specific mechanisms by which responses

  6. 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...... melanogaster), indicating that the ability to bind TA have evolved at least twice in animal evolution. We collected in total over 100 vertebrate TA-receptor sequences, and our phylogenetic analysis shows that several TA-receptors have evolved rapidly with remarkable species variation and that the common...

  7. G protein-coupled receptors as promising cancer targets.

    Science.gov (United States)

    Liu, Ying; An, Su; Ward, Richard; Yang, Yang; Guo, Xiao-Xi; Li, Wei; Xu, Tian-Rui

    2016-07-01

    G protein-coupled receptors (GPCRs) regulate an array of fundamental biological processes, such as growth, metabolism and homeostasis. Specifically, GPCRs are involved in cancer initiation and progression. However, compared with the involvement of the epidermal growth factor receptor in cancer, that of GPCRs have been largely ignored. Recent findings have implicated many GPCRs in tumorigenesis, tumor progression, invasion and metastasis. Moreover, GPCRs contribute to the establishment and maintenance of a microenvironment which is permissive for tumor formation and growth, including effects upon surrounding blood vessels, signaling molecules and the extracellular matrix. Thus, GPCRs are considered to be among the most useful drug targets against many solid cancers. Development of selective ligands targeting GPCRs may provide novel and effective treatment strategies against cancer and some anticancer compounds are now in clinical trials. Here, we focus on tumor related GPCRs, such as G protein-coupled receptor 30, the lysophosphatidic acid receptor, angiotensin receptors 1 and 2, the sphingosine 1-phosphate receptors and gastrin releasing peptide receptor. We also summarize their tissue distributions, activation and roles in tumorigenesis and discuss the potential use of GPCR agonists and antagonists in cancer therapy. PMID:27000991

  8. 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; Bender, Julia; Bach, Anders; Jacky, Birgitte P S; Strømgaard, Kristian; Deussing, Jan M; Schwartz, Thue W; Martinez, Karen L

    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...... colocalization of the full-length proteins in cells and with previous studies, we suggest that the range of relevant interactions might extend to interactions with K i = 450 µM in the in vitro assays. Within this range, we identify novel PSD-95 interactions with the chemokine receptor CXCR2, the neuropeptide Y...... receptor Y2, and four of the somatostatin receptors (SSTRs). The interaction with SSTR1 was further investigated in mouse hippocampal neurons, where we found a clear colocalization between the endogenously expressed proteins, indicating a potential for further investigation of the role of this interaction...

  9. Cardiac cAMP: production, hydrolysis, modulation and detection

    OpenAIRE

    Boularan, Cédric; Gales, Céline

    2015-01-01

    Cyclic adenosine 3′,5′-monophosphate (cAMP) modulates a broad range of biological processes including the regulation of cardiac myocyte contractile function where it constitutes the main second messenger for β-adrenergic receptors' signaling to fulfill positive chronotropic, inotropic and lusitropic effects. A growing number of studies pinpoint the role of spatial organization of the cAMP signaling as an essential mechanism to regulate cAMP outcomes in cardiac physiology. Here, we will briefl...

  10. Thyroid expression of an A2 adenosine receptor transgene induces thyroid hyperplasia and hyperthyroidism.

    OpenAIRE

    Ledent, C; Dumont, J E; Vassart, G.; Parmentier, M

    1992-01-01

    Cyclic AMP (cAMP) is the major intracellular second messenger of thyrotropin (TSH) action on thyroid cells. It stimulates growth as well as the function and differentiation of cultured thyrocytes. The adenosine A2 receptor, which activates adenylyl cyclase via coupling to the stimulating G protein (Gs), has been shown to promote constitutive activation of the cAMP cascade when transfected into various cell types. In order to test whether the A2 receptor was able to function similarly in vivo ...

  11. The spv virulence operon of Salmonella typhimurium LT2 is regulated negatively by the cyclic AMP (cAMP)-cAMP receptor protein system.

    OpenAIRE

    O'Byrne, C P; Dorman, C J

    1994-01-01

    The cyclic AMP (cAMP) receptor protein (CRP) was found to play a role in the growth phase regulation of the spv operon on the high-molecular-weight virulence plasmid of Salmonella typhimurium LT2. By using a lacZ reporter transcriptional fusion to the spvB structural gene on the single-copy virulence plasmid, it was found that while spvB transcription was induced in stationary-phase cultures, the induced level of expression was lower than that reported for the spv system in other serovars of ...

  12. Lesbian camp: An unearthing.

    Science.gov (United States)

    Nielsen, Elly-Jean

    2016-01-01

    Camp-a sensibility, a style, and a form of artistic self-expression-is an elusive concept said to be in the eye of the beholder. To refute Susan Sontag's ( 1966 ) claims that camp is apolitical and not especially homosexual, a number of recent scholarly works have been geared toward revealing camp's fundamental gayness. With the odd footnote aside, lesbian camp has been collapsed into the category of gay male camp, if not eclipsed entirely. Despite the negligible efforts made to legitimize lesbian camp, there are numerous salient cultural examples one might draw on to illustrate, typify, and substantiate a lesbian camp sensibility. I lay the ground work for this scholarly exercise by outlining various definitions and critiques of camp, and by discussing its history and application to queer theory. Then, to unveil lesbian camp, three non-mutually exclusive categories are discussed: classic, erotic, and radical. By gathering various strands of inquiry, and various textual examples (e.g., photography, artistic performances, and literary tropes), this article attempts to reach a more inclusive and nuanced understanding of lesbian camp. PMID:26701773

  13. Transmembrane AMPA receptor regulatory proteins and AMPA receptor function in the cerebellum.

    OpenAIRE

    Coombs, I. D.; Cull-Candy, S. G.

    2009-01-01

    Heterogeneity among AMPA receptor (AMPAR) subtypes is thought to be one of the key postsynaptic factors giving rise to diversity in excitatory synaptic signaling in the CNS. Recently, compelling evidence has emerged that ancillary AMPAR subunits—the so-called transmembrane AMPA receptor regulatory proteins (TARPs)—also play a vital role in influencing the variety of postsynaptic signaling. This TARP family of molecules controls both trafficking and functional properties of AMPARs at most, if ...

  14. 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.

  15. 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.

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

    International Nuclear Information System (INIS)

    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

  17. Molecular analysis of the interaction between the intracellular loops of the human serotonin receptor type 6 (5-HT6) and the α subunit of GS protein

    International Nuclear Information System (INIS)

    The serotonin type 6 (5-HT6) receptor is a G-protein coupled receptor (GPCR) coupled to a stimulatory G-protein (GS). To identify the structural basis for the interaction of the 5-HT6 receptor with the GS protein, we have dissected the interaction between GST-fusion proteins containing the second intracellular loop (iL2), the third intracellular loop (iL3), or the C-terminal tail of the 5-HT6 receptor and the α subunit of GS (GαS). The direct interaction of iL3 and GαS was demonstrated by co-immunoprecipitation. Furthermore, the kinetic parameters of the interaction between iL3 and GαS were measured by surface plasmon resonance, and the apparent dissociation constant was determined to be 0.9 x 10-6 M. In contrast, the second intracellular loop and C-terminal tail regions showed negligible affinity to GαS. The critical residues within the iL3 region for the interaction with GαS were identified as conserved positively charged residues near the C-terminus of iL3 by measuring the cellular levels of cAMP produced in response to 5-HT stimulation of cells transfected with 5-HT6 receptor mutants

  18. Minireview: Nutrient Sensing by G Protein-Coupled Receptors

    OpenAIRE

    Wauson, Eric M; Lorente-Rodríguez, Andrés; Cobb, Melanie H.

    2013-01-01

    G protein-coupled receptors (GPCRs) are membrane proteins that recognize molecules in the extracellular milieu and transmit signals inside cells to regulate their behaviors. Ligands for many GPCRs are hormones or neurotransmitters that direct coordinated, stereotyped adaptive responses. Ligands for other GPCRs provide information to cells about the extracellular environment. Such information facilitates context-specific decision making that may be cell autonomous. Among ligands that are impor...

  19. Large-scale production and protein engineering of G protein-coupled receptors for structural studies

    OpenAIRE

    Milić, Dalibor; Veprintsev, Dmitry B.

    2015-01-01

    Structural studies of G protein-coupled receptors (GPCRs) gave insights into molecular mechanisms of their action and contributed significantly to molecular pharmacology. This is primarily due to technical advances in protein engineering, production and crystallization of these important receptor targets. On the other hand, NMR spectroscopy of GPCRs, which can provide information about their dynamics, still remains challenging due to difficulties in preparation of isotopically labeled recepto...

  20. Identification of EPAC (Exchange Protein Activated by cAMP) bioinformatically as a potential signalling biomarker in Cardiovascular Disease (CVD) and its molecular docking by a lead molecule.

    Science.gov (United States)

    Bala, Saranya; Pathak, Ravi Kant; Mishra, Vachaspati

    2011-01-01

    The present work delineates the combinatorial approach of firstly, creation of a centralized data-set comprising signalling proteins identified on the basis of altered expression, such as over-expression or repression of a set of signalling protein(s) leading to the cause of the disease, which is based on published reports screened through Pubmed and secondly, in the in silico creation of novel lead (drug) molecules and docking of identified signalling biomarkers using such drugs to investigate possibility of their future application in the model systems eventually. EPAC (Exchange Protein Activated by cAMP) emerges as a signalling biomarker in cases studied presently. Brefeldin, the known inhibitor of EPAC, though the mechanism yet unexplored, has been the molecule used as the pharmacophore for creation of lead drug molecule. Various modifications have been incorporated into the pharmacophore to increase the hydrophobic interactions for increasing the binding efficiency of the generated lead molecule. Side-chain modifications of the pharmacophore and refinement of data through firedock upon docking of EPAC with the modified pharmacophore yielded best results on the bases of atomic contact energy, van der Waal and partial electrostatic interactions as well as additional estimations of the binding free energy. Modifications of CH3 at C15 with COOH and H at C2 with OH in brefeldin showed the best docking results on the basis of protein-drug interaction parameters. The present work provides a clue in rational design of EPAC inhibitors which could be developed as drug lead in combating CVD. PMID:21738308

  1. 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...

  2. *607449 G PROTEIN-COUPLED RECEPTOR 74; GPR74 [OMIM

    Lifescience Database Archive (English)

    Full Text Available FIELD NO 607449 FIELD TI 607449 G PROTEIN-COUPLED RECEPTOR 74; GPR74 ;;NEUROPEPTIDE FF2; NPFF2;; ... with well-defined criteria for either longstanding lean ness or severe obesity. The results were confirmed ... gainst obesity in 2 samples selected for obese and lean ... phenotypes. In a population-based sample, it was a ...

  3. Interaction of G protein coupled receptors and cholesterol.

    Science.gov (United States)

    Gimpl, Gerald

    2016-09-01

    G protein coupled receptors (GPCRs) form the largest receptor superfamily in eukaryotic cells. Owing to their seven transmembrane helices, large parts of these proteins are embedded in the cholesterol-rich plasma membrane bilayer. Thus, GPCRs are always in proximity to cholesterol. Some of them are functionally dependent on the specific presence of cholesterol. Over the last years, enormous progress on receptor structures has been achieved. While lipophilic ligands other than cholesterol have been shown to bind either inside the helix bundle or at the receptor-lipid interface, the binding site of cholesterol was either a single transmembrane helix or a groove between two or more transmembrane helices. A clear preference for one of the two membrane leaflets has not been observed. Not surprisingly, many hydrophobic residues (primarily leucine and isoleucine) were found to be involved in cholesterol binding. In most cases, the rough β-face of cholesterol contacted the transmembrane helix bundle rather than the surrounding lipid matrix. The polar hydroxy group of cholesterol was localized near the water-membrane interface with potential hydrogen bonding to residues in receptor loop regions. Although a canonical motif, designated as CCM site, was detected as a specific cholesterol binding site in case of the β2AR, this site was not found to be occupied by cholesterol in other GPCRs possessing the same motif. Cholesterol-receptor interactions can increase the compactness of the receptor structure and are able to enhance the conformational stability towards active or inactive receptor states. Overall, all current data suggest a high plasticity of cholesterol interaction sites in GPCRs. PMID:27108066

  4. Recreation Summer Camps 2016

    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,...

  5. 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

  6. Regulation of C3a Receptor Signaling in Human Mast Cells by G Protein Coupled Receptor Kinases

    OpenAIRE

    Qiang Guo; Hariharan Subramanian; Kshitij Gupta; Hydar Ali

    2011-01-01

    BACKGROUND: The complement component C3a activates human mast cells via its cell surface G protein coupled receptor (GPCR) C3aR. For most GPCRs, agonist-induced receptor phosphorylation leads to receptor desensitization, internalization as well as activation of downstream signaling pathways such as ERK1/2 phosphorylation. Previous studies in transfected COS cells overexpressing G protein coupled receptor kinases (GRKs) demonstrated that GRK2, GRK3, GRK5 and GRK6 participate in agonist-induced...

  7. Cardiac cAMP: production, hydrolysis, modulation and detection.

    Science.gov (United States)

    Boularan, Cédric; Gales, Céline

    2015-01-01

    Cyclic adenosine 3',5'-monophosphate (cAMP) modulates a broad range of biological processes including the regulation of cardiac myocyte contractile function where it constitutes the main second messenger for β-adrenergic receptors' signaling to fulfill positive chronotropic, inotropic and lusitropic effects. A growing number of studies pinpoint the role of spatial organization of the cAMP signaling as an essential mechanism to regulate cAMP outcomes in cardiac physiology. Here, we will briefly discuss the complexity of cAMP synthesis and degradation in the cardiac context, describe the way to detect it and review the main pharmacological arsenal to modulate its availability. PMID:26483685

  8. 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...

  9. Direct regulation of the natural competence regulator gene tfoX by cyclic AMP (cAMP) and cAMP receptor protein (CRP) in Vibrios

    OpenAIRE

    Rui Wu; Meng Zhao; Jing Li; He Gao; Biao Kan; Weili Liang

    2015-01-01

    TfoX (Sxy) and CRP are two important competence activators. The link between tfoX and CRP has been shown in H. influenza but lacking evidence of direct interaction. Recently a Sxy-dependent CRP (CRP-S) site autoregulating Sxy was reported in E. coli. Here, we show that the cAMP-CRP complex transcriptionally regulates tfoX expression through multiple canonical CRP (CRP-N) sites in Vibrios. This conclusion is supported by an analysis of the tfoX mRNA levels and tfoX transcriptional reporter fus...

  10. 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.

  11. Computational methods for studying G protein-coupled receptors (GPCRs).

    Science.gov (United States)

    Kaczor, Agnieszka A; Rutkowska, Ewelina; Bartuzi, Damian; Targowska-Duda, Katarzyna M; Matosiuk, Dariusz; Selent, Jana

    2016-01-01

    The functioning of GPCRs is classically described by the ternary complex model as the interplay of three basic components: a receptor, an agonist, and a G protein. According to this model, receptor activation results from an interaction with an agonist, which translates into the activation of a particular G protein in the intracellular compartment that, in turn, is able to initiate particular signaling cascades. Extensive studies on GPCRs have led to new findings which open unexplored and exciting possibilities for drug design and safer and more effective treatments with GPCR targeting drugs. These include discovery of novel signaling mechanisms such as ligand promiscuity resulting in multitarget ligands and signaling cross-talks, allosteric modulation, biased agonism, and formation of receptor homo- and heterodimers and oligomers which can be efficiently studied with computational methods. Computer-aided drug design techniques can reduce the cost of drug development by up to 50%. In particular structure- and ligand-based virtual screening techniques are a valuable tool for identifying new leads and have been shown to be especially efficient for GPCRs in comparison to water-soluble proteins. Modern computer-aided approaches can be helpful for the discovery of compounds with designed affinity profiles. Furthermore, homology modeling facilitated by a growing number of available templates as well as molecular docking supported by sophisticated techniques of molecular dynamics and quantitative structure-activity relationship models are an excellent source of information about drug-receptor interactions at the molecular level. PMID:26928552

  12. 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 lipoprotein receptor-related protein 1 (LRP1). We show here that on sensory neurons LRP1 is also the receptor that binds and rapidly endocytoses smaller oligomeric forms of infectious prion fibrils, and recombinant PrP fibrils. Although LRP1 binds two molecules of most ligands independently to its receptor...... both prion and LRP1 biology....

  13. 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

    -alpha;-amino acid receptor G-protein-coupled receptor family C, group 6, subtype A (GPRC6A) and seven orphan receptors. Aside from the orphan receptors, the family C GPCRs are dimeric receptors characterized by a large extracellular Venus flytrap domain which bind the endogenous agonists. Except from the GABA(B1...

  14. Camp Joy: Embracing Diversity.

    Science.gov (United States)

    Krehbiel, Amy

    2001-01-01

    Camp Joy (Ohio) offers a racially integrated program to disadvantaged inner-city foster children. To attract quality minority staff, the camp recruits through former campers, word of mouth, a leader-in-training program, job and internship fairs, and networking with nearby colleges and social agencies. Staff training and the intrinsic rewards of…

  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...

  16. Camp's "Disneyland" Effect.

    Science.gov (United States)

    Renville, Gary

    1999-01-01

    Describes the positive mental, physical, and social growth impacts that the camping experience had on the author, and urges camp program evaluation to plan and implement such changes. Sidebar lists steps of effective evaluation: program goals and objectives, goals of evaluation, implementation of evaluation, data analysis, and findings and…

  17. VisionCamp

    DEFF Research Database (Denmark)

    Lassen, Astrid Heidemann; Løwe Nielsen, Suna

    2009-01-01

    . Baseret på forløbet VisionCamp 2008, der satte eksplicit fokus på at kombinere fagfeltet "supply chain management" og kreativitet, illustrerer artiklen camp-metodens relevans og peger på centrale del-elementer i entreprenante kompetencer (kreative kompetencer, teknisk/faglige kompetencer og relationelle...

  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

    2013-01-01

    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. PMID:23633472

  19. 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 and...... 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...

  20. Interaction of Hepatitis C virus proteins with pattern recognition receptors

    Directory of Open Access Journals (Sweden)

    Imran Muhammad

    2012-06-01

    Full Text Available Abstract Hepatitis C virus (HCV is an important human pathogen that causes acute and chronic hepatitis, cirrhosis and hepatocellular carcinoma worldwide. This positive stranded RNA virus is extremely efficient in establishing persistent infection by escaping immune detection or hindering the host immune responses. Recent studies have discovered two important signaling pathways that activate the host innate immunity against viral infection. One of these pathways utilizes members of Toll-like receptor (TLR family and the other uses the RNA helicase retinoic acid inducible gene I (RIG-I as the receptors for intracellular viral double stranded RNA (dsRNA, and activation of transcription factors. In this review article, we summarize the interaction of HCV proteins with various host receptors/sensors through one of these two pathways or both, and how they exploit these interactions to escape from host defense mechanisms. For this purpose, we searched data from Pubmed and Google Scholar. We found that three HCV proteins; Core (C, non structural 3/4 A (NS3/4A and non structural 5A (NS5A have direct interactions with these two pathways. Core protein only in the monomeric form stimulates TLR2 pathway assisting the virus to evade from the innate immune system. NS3/4A disrupts TLR3 and RIG-1 signaling pathways by cleaving Toll/IL-1 receptor domain-containing adapter inducing IFN-beta (TRIF and Cardif, the two important adapter proteins of these signaling cascades respectively, thus halting the defense against HCV. NS5A downmodulates the expressions of NKG2D on natural killer cells (NK cells via TLR4 pathway and impairs the functional ability of these cells. TLRs and RIG-1 pathways have a central role in innate immunity and despite their opposing natures to HCV proteins, when exploited together, HCV as an ever developing virus against host immunity is able to accumulate these mechanisms for near unbeatable survival.

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

    DEFF Research Database (Denmark)

    Milligan, Graeme; Ulven, Trond; Murdoch, Hannah; Hudson, Brian D

    It is becoming evident that nutrients and metabolic intermediates derived from such nutrients regulate cellular function by activating a number of cell-surface G-protein coupled receptors (GPCRs). Until now, members of the GPCR family have largely been considered as the molecular targets 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...... diseases, major challenges remain to exploit their potential for therapeutic purposes. Mostly, this is due to limited characterisation and validation of these receptors because of paucity of selective and high-potency/affinity pharmacological agents to define the detailed function and regulation of these...

  2. Monitoring endosomal trafficking of the G protein-coupled receptor somatostatin receptor 3

    Science.gov (United States)

    Tower-Gilchrist, Cristy; Styers, Melanie L.; Yoder, Bradley K.; Berbari, Nicolas F.; Sztul, Elizabeth

    2016-01-01

    Endocytic trafficking of G protein-coupled receptors (GPCRs) regulates the number of cell surface receptors available for activation by agonists and serves as one mechanism that controls the intensity and duration of signaling. Deregulation of GPCR-mediated signaling pathways results in a multitude of diseases, and thus extensive efforts have been directed toward understand the pathways and molecular events that regulate endocytic trafficking of these receptors. The general paradigms associated with internalization and recycling, as well as many of the key regulators involved in endosomal trafficking of GPCRs have been identified. This knowledge provides goalposts to facilitate the analysis of endosomal pathways traversed by previously uncharacterized GPCRs. Some of the most informative markers associated with GPCR transit are the Rab members of the Ras-related family of small GTPases. Individual Rabs show high selectivity for distinct endosomal compartments, and thus co-localization of a GPCR with a particular Rab informs on the internalization pathway traversed by the receptor. Progress in our knowledge of endosomal trafficking of GPCRs has been achieved through advances in our ability to tag GPCRs and Rabs with fluorescent proteins and perform live cell imaging of multiple fluorophores, allowing real-time observation of receptor trafficking between subcellular compartments in a cell culture model. PMID:24359959

  3. Monomeric G protein-coupled receptor rhodopsin in solution activates its G protein transducin at the diffusion limit

    OpenAIRE

    Ernst, Oliver P.; Gramse, Verena; Kolbe, Michael; Hofmann, Klaus Peter; Heck, Martin

    2007-01-01

    G protein-coupled receptors mediate biological signals by stimulating nucleotide exchange in heterotrimeric G proteins (Gαβγ). Receptor dimers have been proposed as the functional unit responsible for catalytic interaction with Gαβγ. To investigate whether a G protein-coupled receptor monomer can activate Gαβγ, we used the retinal photoreceptor rhodopsin and its cognate G protein transducin (Gt) to determine the stoichiometry of rhodopsin/Gt binding and the rate of catalyzed nucleotide exchan...

  4. 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

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

  5. 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; Lund, Anders Henrik; Tommerup, Niels; Nykjaer, A; Sottrup-Jensen, L; Gliemann, J; Petersen, C M

    1996-01-01

    characteristic of the low density lipoprotein receptor gene family receptors, and 3) six tandemly arranged fibronectin type III repeats also found in certain neural adhesion proteins. sorLA-1 may therefore be classified as a hybrid receptor. Northern blotting revealed specific mRNA transcripts in brain, spinal...

  6. 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.

  7. Structure and function of serotonin G protein-coupled receptors.

    Science.gov (United States)

    McCorvy, John D; Roth, Bryan L

    2015-06-01

    Serotonin receptors are prevalent throughout the nervous system and the periphery, and remain one of the most lucrative and promising drug discovery targets for disorders ranging from migraine headaches to neuropsychiatric disorders such as schizophrenia and depression. There are 14 distinct serotonin receptors, of which 13 are G protein-coupled receptors (GPCRs), which are targets for approximately 40% of the approved medicines. Recent crystallographic and biochemical evidence has provided a converging understanding of the basic structure and functional mechanics of GPCR activation. Currently, two GPCR crystal structures exist for the serotonin family, the 5-HT1B and 5-HT2B receptor, with the antimigraine and valvulopathic drug ergotamine bound. The first serotonin crystal structures not only provide the first evidence of serotonin receptor topography but also provide mechanistic explanations into functional selectivity or biased agonism. This review will detail the findings of these crystal structures from a molecular and mutagenesis perspective for driving rational drug design for novel therapeutics incorporating biased signaling. PMID:25601315

  8. Involvement of the G-protein-coupled receptor 4 in RANKL expression by osteoblasts in an acidic environment.

    Science.gov (United States)

    Okito, Asuka; Nakahama, Ken-Ichi; Akiyama, Masako; Ono, Takashi; Morita, Ikuo

    2015-03-01

    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. PMID:25668130

  9. Therapeutic antibodies directed at G protein-coupled receptors

    OpenAIRE

    Hutchings, Catherine J; Koglin, Markus; Marshall, Fiona H.

    2010-01-01

    G protein-coupled receptors (GPCRs) are one of the most important classes of targets for small molecule drug discovery, but many current GPCRs of interest are proving intractable to small molecule discovery and may be better approached with bio-therapeutics. GPCRs are implicated in a wide variety of diseases where antibody therapeutics are currently used. These include inflammatory diseases such as rheumatoid arthritis and Crohn disease, as well as metabolic disease and cancer. Raising antibo...

  10. 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.

  11. Treponema pallidum receptor binding proteins interact with fibronectin

    International Nuclear Information System (INIS)

    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 35S-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. Mechanisms of regulation and function of G-protein-coupled receptor kinases

    Institute of Scientific and Technical Information of China (English)

    Wen Yang; Shi-Hai Xia

    2006-01-01

    G-protein-coupled receptor kinases (GRKs) interact with the agonist-activated form of G-protein-coupled receptor (GPCR) to affect receptor phosphorylation and to initiate profound impairment of receptor signaling,or desensitization. GPCR forms the largest family of cell surface receptors, and defects in GRK function have the potential consequence to affect GPCR-stimulated biological responses in many pathological situations.

  13. G protein coupled receptors as targets for next generation pesticides.

    Science.gov (United States)

    Audsley, Neil; Down, Rachel E

    2015-12-01

    There is an on-going need for the discovery and development of new pesticides due to the loss of existing products through the continuing development of resistance, the desire for products with more favourable environmental and toxicological profiles and the need to implement the principles of integrated pest management. Insect G protein coupled receptors (GPCRs) have important roles in modulating biology, physiology and behaviour, including reproduction, osmoregulation, growth and development. Modifying normal receptor function by blocking or over stimulating its actions may either result in the death of a pest or disrupt its normal fitness or reproductive capacity to reduce pest populations. Hence GPCRs offer potential targets for the development of next generation pesticides providing opportunities to discover new chemistries for invertebrate pest control. Such receptors are important targets for pharmaceutical drugs, but are under-exploited by the agro-chemical industry. The octopamine receptor agonists are the only pesticides with a recognized mode of action, as described in the classification scheme developed by the Insecticide Resistance Action Committee, that act via a GPCR. The availability of sequenced insect genomes has facilitated the characterization of insect GPCRs, but the development and utilization of screening assays to identify lead compounds has been slow. Various studies using knock-down technologies or applying the native ligands and/or neuropeptide analogues to pest insects in vivo, have however demonstrated that modifying normal receptor function can have an insecticidal effect. This review presents examples of potential insect neuropeptide receptors that are potential targets for lead compound development, using case studies from three representative pest species, Tribolium castaneum, Acyrthosiphon pisum, and Drosophila suzukii. Functional analysis studies on T. castaneum suggest that GPCRs involved in growth and development (eclosion

  14. Loss of Function of the Melanocortin 2 Receptor Accessory Protein 2 Is Associated with Mammalian Obesity

    OpenAIRE

    Asai, M; Ramachandrappa, S.; Joachim, M.; Shen, Y.; Zhang, R.; Nuthalapati, N.; V. Ramanathan; Strochlic, D. E.; Ferket, P.; Linhart, K.; Ho, C.; Novoselova, T. V.; Garg, S.; Ridderstrale, M.; Marcus, C

    2013-01-01

    Melanocortin receptor accessory proteins (MRAPs) modulate signaling of melanocortin receptors in vitro. To investigate the physiological role of brain-expressed Melanocortin 2 Receptor Accessory Protein 2 (MRAP2), we characterized mice with whole body and brain-specific targeted deletion of Mrap2, both of which develop severe obesity at a young age. Mrap2 interacts directly with Melanocortin 4 Receptor (Mc4r), a protein previously implicated in mammalian obesity, and it enhances Mc4r-mediated...

  15. Human G protein-coupled receptor studies in Saccharomyces cerevisiae.

    Science.gov (United States)

    Liu, Rongfang; Wong, Winsy; IJzerman, Adriaan P

    2016-08-15

    G protein-coupled receptors (GPCRs) are one of the largest families of membrane proteins, with approximately 800 different GPCRs in the human genome. Signaling via GPCRs regulates many biological processes, such as cell proliferation, differentiation, and development. In addition, many receptors have a pivotal role in immunophysiology. Many hormones and neurotransmitters are ligands for these receptors, and hence it is not surprising that many drugs, either mimicking or blocking the action of the bodily substances, have been developed. It is estimated that 30-40% of current drugs on the market target GPCRs. Further identifying and elucidating the functions of GPCRs will provide opportunities for novel drug discovery, including for immunotherapy. The budding yeast Saccharomyces cerevisiae (S. cerevisiae) is a very important and useful platform in this respect. There are many advantages of using a yeast assay system, as it is cheap, safe and stable; it is also convenient for rapid feasibility and optimization studies. Moreover, it offers a "null" background when studying human GPCRs. New developments regarding human GPCRs expressed in a yeast platform are providing insight into GPCR activation and signaling, and facilitate agonist and antagonist identification. In this review we summarize the latest findings regarding human G-protein-coupled receptors in studies using S. cerevisiae, ever since the year 2005 when we last published a review on this topic. We describe 11 families of GPCRs in detail, while including the principles and developments of each yeast system applied to these different GPCRs and highlight and generalize the experimental findings of GPCR function in these systems. PMID:26920251

  16. A day in the life of a G protein-coupled receptor: the contribution to function of G protein-coupled receptor dimerization

    OpenAIRE

    Milligan, G.

    2007-01-01

    G protein-coupled receptors are one of the most actively studied families of proteins. However, despite the ubiquity of protein dimerization and oligomerization as a structural and functional motif in biology, until the last decade they were generally considered as monomeric, non-interacting polypeptides. For the metabotropic glutamate-like group of G protein-coupled receptors, it is now firmly established that they exist and function as dimers or, potentially, even within higher-order struct...

  17. 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.

  18. Role of G protein-coupled receptors in inflammation

    Institute of Scientific and Technical Information of China (English)

    Lei SUN; Richard DYE

    2012-01-01

    G protein-coupled receptors (GPCRs) play important roles in inflammation.Inflammatory cells such as polymorphonuclear leuko-cytes (PMN),monocytes and macrophages express a large number of GPCRs for classic chemoattractants and chemokines.These receptors are critical to the migration of phagocytes and their accumulation at sites of inflammation,where these cells can exacer-bate inflammation but also contribute to its resolution.Besides chemoattractant GPCRs,protease activated receptors (PARs) such as PAR1 are involved in the regulation of vascular endothelial permeability.Prostaglandin receptors play different roles in inflam-matory cell activation,and can mediate both proinflammatory and anti-inflammatory functions.Many GPCRs present in inflammatory cells also mediate transcription factor activation,resulting in the synthesis and secretion of inflammatory factors and,in some cases,molecules that suppress inflammation.An understanding of the signaling paradigms of GPCRs in inflammatory cells is likely to facilitate translational research and development of improved anti-inflammatory therapies.

  19. Secretin family (Class B) G protein-coupled receptors – from molecular to clinical perspectives

    OpenAIRE

    Poyner, David R; Hay, Debbie L

    2012-01-01

    Family B G protein-coupled receptors represent an important but under-researched group of receptors. This edition of the British Journal of Pharmacology considers the roles and pharmacology of a number of these receptors. Whilst common themes emerge, it is clear that more work is needed to understand the details of each receptor in order to properly exploit them therapeutically.

  20. 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 matri...

  1. 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...

  2. 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.

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

    OpenAIRE

    Jennifer R. Lynch; Jenny Yingzi Wang

    2016-01-01

    G protein-coupled receptors (GPCRs) are a large superfamily of cell-surface signaling proteins that bind extracellular ligands and transduce signals into cells via heterotrimeric G proteins. GPCRs are highly tractable drug targets. Aberrant expression of GPCRs and G proteins has been observed in various cancers and their importance in cancer stem cells has begun to be appreciated. We have recently reported essential roles for G protein-coupled receptor 84 (GPR84) and G protein subunit Gαq in ...

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

    OpenAIRE

    Xin Gao; Tongyu Wu; Kirby D. Johnson; Jamie L. Lahvic; Erik A. Ranheim; Leonard I. Zon; Emery H. Bresnick

    2016-01-01

    Summary 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 t...

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

    OpenAIRE

    Gao, Xin; Wu, Tongyu; Johnson, Kirby D.; Lahvic, Jamie L.; Ranheim, Erik A.; Zon, Leonard I.; Bresnick, Emery H.

    2016-01-01

    Summary 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 t...

  6. G-Protein-Coupled Lysophosphatidic Acid Receptors and Their Regulation of AKT Signaling

    OpenAIRE

    Anjum Riaz; Ying Huang; Staffan Johansson

    2016-01-01

    A hallmark of G-protein-coupled receptors (GPCRs) is their ability to recognize and respond to chemically diverse ligands. Lysophospholipids constitute a relatively recent addition to these ligands and carry out their biological functions by activating G-proteins coupled to a large family of cell-surface receptors. This review aims to highlight salient features of cell signaling by one class of these receptors, known as lysophosphatidic acid (LPA) receptors, in the context of phosphatidylinos...

  7. Receptor-type protein tyrosine phosphatases in cancer

    Directory of Open Access Journals (Sweden)

    Yu Du

    2015-02-01

    Full Text Available Protein tyrosine phosphatases (PTPs play an important role in regulating cell signaling events in coordination with tyrosine kinases to control cell proliferation, apoptosis, survival, migration, and invasion. Receptor-type protein tyrosine phosphatases (PTPRs are a subgroup of PTPs that share a transmembrane domain with resulting similarities in function and target specificity. In this review, we summarize genetic and epigenetic alterations including mutation, deletion, amplification, and promoter methylation of PTPRs in cancer and consider the consequences of PTPR alterations in different types of cancers. We also summarize recent developments using PTPRs as prognostic or predictive biomarkers and/or direct targets. Increased understanding of the role of PTPRs in cancer may provide opportunities to improve therapeutic approaches.

  8. Can Specific Protein-Lipid Interactions Stabilize an Active State of the Beta 2 Adrenergic Receptor?

    Science.gov (United States)

    Neale, Chris; Herce, Henry D; Pomès, Régis; García, Angel E

    2015-10-20

    G-protein-coupled receptors are eukaryotic membrane proteins with broad biological and pharmacological relevance. Like all membrane-embedded proteins, their location and orientation are influenced by lipids, which can also impact protein function via specific interactions. Extensive simulations totaling 0.25 ms reveal a process in which phospholipids from the membrane's cytosolic leaflet enter the empty G-protein binding site of an activated β2 adrenergic receptor and form salt-bridge interactions that inhibit ionic lock formation and prolong active-state residency. Simulations of the receptor embedded in an anionic membrane show increased lipid binding, providing a molecular mechanism for the experimental observation that anionic lipids can enhance receptor activity. Conservation of the arginine component of the ionic lock among Rhodopsin-like G-protein-coupled receptors suggests that intracellular lipid ingression between receptor helices H6 and H7 may be a general mechanism for active-state stabilization. PMID:26488656

  9. The MprF protein is required for lysinylation of phospholipids in listerial membranes and confers resistance to cationic antimicrobial peptides (CAMPs) on Listeria monocytogenes

    NARCIS (Netherlands)

    Thedieck, Kathrin; Hain, Torsten; Mohamed, Walid; Tindall, Brian J; Nimtz, Manfred; Chakraborty, Trinad; Wehland, Jürgen; Jänsch, Lothar

    2006-01-01

    Pathogenic bacteria have to cope with defence mechanisms mediated by adaptive and innate immunity of the host cells. Cationic antimicrobial peptides (CAMPs) represent one of the most effective components of the host innate immune response. Here we establish the function of Lmo1695, a member of the V

  10. Cyclic AMP (cAMP) Receptor Protein-cAMP Complex Regulates Heparosan Production in Escherichia coli Strain Nissle 1917

    OpenAIRE

    Yan, Huihui; Bao, Feifei; Zhao, Liping; Yu, Yanying; Tang, Jiaqin; Zhou, Xianxuan

    2015-01-01

    Heparosan serves as the starting carbon backbone for the chemoenzymatic synthesis of heparin, a widely used clinical anticoagulant drug. The availability of heparosan is a significant concern for the cost-effective synthesis of bioengineered heparin. The carbon source is known as the pivotal factor affecting heparosan production. However, the mechanism by which carbon sources control the biosynthesis of heparosan is unclear. In this study, we found that the biosynthesis of heparosan was influ...

  11. Oligomerization of opioid receptors with β2-adrenergic receptors: A role in trafficking and mitogen-activated protein kinase activation

    OpenAIRE

    Jordan, B. A.; Trapaidze, N.; Gomes, I; Nivarthi, R.; Devi, L.A.

    2000-01-01

    G-protein-coupled receptors (GPCRs) have recently joined the list of cell surface receptors that dimerize. Dimerization has been shown to alter the ligand-binding, signaling, and trafficking properties of these receptors. Recent studies have shown that GPCRs heterodimerize with closely related members, resulting in the modulation of their function. In this study, we have attempted to determine whether members of GPCR superfamilies that couple to different families of G...

  12. Simvastatin enhances bone morphogenetic protein receptor type II expression

    International Nuclear Information System (INIS)

    Statins confer therapeutic benefits in systemic and pulmonary vascular diseases. Bone morphogenetic protein (BMP) receptors serve essential signaling functions in cardiovascular development and skeletal morphogenesis. Mutations in BMP receptor type II (BMPR2) are associated with human familial and idiopathic pulmonary arterial hypertension, and pathologic neointimal proliferation of vascular endothelial and smooth muscle cells within small pulmonary arteries. In severe experimental pulmonary hypertension, simvastatin reversed disease and conferred a 100% survival advantage. Here, modulation of BMPR2 gene expression by simvastatin is characterized in human embryonic kidney (HEK) 293T, pulmonary artery smooth muscle, and lung microvascular endothelial cells (HLMVECs). A 1.4 kb BMPR2 promoter containing Egr-1 binding sites confers reporter gene activation in 293T cells which is partially inhibited by simvastatin. Simvastatin enhances steady-state BMPR2 mRNA and protein expression in HLMVEC, through posttranscriptional mRNA stabilization. Simvastatin induction of BMPR2 expression may improve BMP-BMPR2 signaling thereby enhancing endothelial differentiation and function

  13. Towards predictive docking at aminergic G-protein coupled receptors.

    Science.gov (United States)

    Jakubík, Jan; El-Fakahany, Esam E; Doležal, Vladimír

    2015-11-01

    G protein-coupled receptors (GPCRs) are hard to crystallize. However, attempts to predict their structure have boomed as a result of advancements in crystallographic techniques. This trend has allowed computer-aided molecular modeling of GPCRs. We analyzed the performance of four molecular modeling programs in pose evaluation of re-docked antagonists / inverse agonists to 11 original crystal structures of aminergic GPCRs using an induced fit-docking procedure. AutoDock and Glide were used for docking. AutoDock binding energy function, GlideXP, Prime MM-GB/SA, and YASARA binding function were used for pose scoring. Root mean square deviation (RMSD) of the best pose ranged from 0.09 to 1.58 Å, and median RMSD of the top 60 poses ranged from 1.47 to 3.83 Å. However, RMSD of the top pose ranged from 0.13 to 7.33 Å and ranking of the best pose ranged from the 1st to 60th out of 60 poses. Moreover, analysis of ligand-receptor interactions of top poses revealed substantial differences from interactions found in crystallographic structures. Bad ranking of top poses and discrepancies between top docked poses and crystal structures render current simple docking methods unsuitable for predictive modeling of receptor-ligand interactions. Prime MM-GB/SA optimized for 3NY9 by multiple linear regression did not work well at 3NY8 and 3NYA, structures of the same receptor with different ligands. However, 9 of 11 trajectories of molecular dynamics simulations by Desmond of top poses converged with trajectories of crystal structures. Key interactions were properly detected for all structures. This procedure also worked well for cross-docking of tested β2-adrenergic antagonists. Thus, this procedure represents a possible way to predict interactions of antagonists with aminergic GPCRs. PMID:26453085

  14. A modeling strategy for G-protein coupled receptors

    Directory of Open Access Journals (Sweden)

    Anna Kahler

    2016-03-01

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

  15. 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. PMID:25772509

  16. Ethanol regulation of adenosine receptor-stimulated cAMP levels in a clonal neural cell line: an in vitro model of cellular tolerance to ethanol.

    OpenAIRE

    Gordon, A S; Collier, K; Diamond, I.

    1986-01-01

    The acute and chronic neurologic effects of ethanol appear to be due to its interaction with neural cell membranes. Chronic exposure to ethanol induces changes in the membrane that lead to tolerance to the effects of ethanol. However, the actual membrane changes that account for tolerance to ethanol are not understood. We have developed a model cell culture system, using NG108-15 neuroblastoma-glioma hybrid cells, to study cellular tolerance to ethanol. We have found that adenosine receptor-s...

  17. Rehabilitating camp cities : community driven planning for urbanised refugee camps

    OpenAIRE

    Misselwitz, Philipp

    2009-01-01

    Focussing on Palestine refugee camps in the Near East, this dissertation aims to shed light on the potential relevance of urban planning to refugee camp environments worldwide. In particular, there is a focus on the role architects and urban planners can play in facilitating participatory planning processes as well as providing guidance and expertise in the development of a spatial vision for Camp Cities. Part I - The Urbanisation of Refugee Camps as a Global Challenge The first part o...

  18. Distinct in vitro interaction pattern of dopamine receptor subtypes with adaptor proteins involved in post-endocytotic receptor targeting

    DEFF Research Database (Denmark)

    Heydorn, Arne; Søndergaard, Birgitte P; Hadrup, Niels;

    2004-01-01

    The mechanisms underlying targeted sorting of endocytosed receptors for recycling to the plasma membrane or degradation in lysosomes are poorly understood. In this report, the C-terminal tails of the five dopamine receptors (D1-D5) were expressed as glutathione S-transferase (GST) fusion proteins...

  19. 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.

  20. Ligand binding and activation mechanism og the glucagon-like peptide-1 receptor

    DEFF Research Database (Denmark)

    Underwood, Christina Rye

    In recent years, G-protein coupled receptors (GPCRs) have become important drug targets, which makes elucidation of their molecular structure and functional domains increasingly important for designing new and better therapeutic agents. The Glucagon-Like Peptide-1 receptor (GLP-1R) is a GPCR. Its...... molecule-mediated activation of GLP-1R (Study II). A fully functional, cysteine-deprived and Cterminally truncated GLP-1R is developed and characterised in Study III. In Study IV, a cAMP biosensor is used to investigate the cAMP kinetics of GLP-1R upon stimulation with different receptor agonists...

  1. Dynamic correlation networks in human peroxisome proliferator-activated receptor-γ nuclear receptor protein.

    Science.gov (United States)

    Fidelak, Jeremy; Ferrer, Silvia; Oberlin, Michael; Moras, Dino; Dejaegere, Annick; Stote, Roland H

    2010-10-01

    Peroxisome proliferator-activated receptor-γ nuclear receptor (PPAR-γ) belongs to the superfamily of nuclear receptor proteins that function as ligand-dependent transcription factors and plays a specific physiological role as a regulator of lipid metabolism. A number of experimental studies have suggested that allostery plays an important role in the functioning of PPAR-γ. Here we use normal-mode analysis of PPAR-γ to characterize a network of dynamically coupled amino acids that link physiologically relevant binding surfaces such as the ligand-dependent activation domain AF-2 with the ligand binding site and the heterodimer interface. Multiple calculations were done in both the presence and absence of the agonist rosiglitazone, and the differences in dynamics were characterized. The global dynamics of the ligand binding domain were affected by the ligand, and in particular, changes to the network of dynamically correlated amino acids were observed with only small changes in conformation. These results suggest that changes in dynamic couplings can be functionally significant with respect to the transmission of allosteric signals. PMID:20496064

  2. Gc protein-derived macrophage-activating factor (GcMAF) stimulates cAMP formation in human mononuclear cells and inhibits angiogenesis in chick embryo chorionallantoic membrane assay

    OpenAIRE

    S. Pacini; G.Morucci; T.Punzi; Gulisano, M; Ruggiero, M

    2010-01-01

    Abstract: The effects of Gc protein-derived macrophage-activating factor (GcMAF) have been studied in cancer and other conditions where angiogenesis is deregulated. In this study, we demonstrate for the first time that the mitogenic response of human peripheral blood mononuclear cells (PBMCs) to GcMAF was associated with 3'-5'-cyclic adenosine monophosphate (cAMP) formation. The effect was dose dependent, and maximal stimulation was achieved using 0.1 ng/ml. Heparin inhibited the stimulatory ...

  3. CCI: A Worldwide Camping Community.

    Science.gov (United States)

    Fawver, Gary Keith

    1992-01-01

    Describes the efforts of Christian Camping International (CCI), an alliance of Christian camping associations from Australia, Canada, the Far East, Latin America, New Zealand, United States, South Africa, Japan, and Brazil. The purpose of CCI is to help develop effective Christian camps, conferences, and retreat ministries. (LP)

  4. Protein-protein Interactions of the Androgen Receptor in Living Cells

    OpenAIRE

    Royen, Martin

    2008-01-01

    markdownabstract__Abstract__ Natural androgens, testosterone (T) and its derivative dihydrotestosterone (DHT) play a crucial role in the development and maintenance of the male phenotype. Androgens are steroids that exert their function via the androgen receptor (AR), a ligand dependent transcription factor. The human AR gene, is located on the X chromosome, and contains 8 exons, coding for a 110 kDa, 919 amino acids protein (Brinkmann et al., 1989; Hughes and Deeb, 2006). In the classical mo...

  5. Characterization of alternative therapeutic sites on the androgen receptor and novel protein-protein associations

    OpenAIRE

    Rodríguez Carbó, Laia

    2016-01-01

    The androgen receptor (AR) is a ligand-activated transcription factor that plays a crucial role in the correct development, differentiation, and function of male reproductive organs. Alterations in the AR protein or in the AR signaling pathway result in pathologies such as prostate cancer (PCa), which is the fifth leading cause of cancer-related death in men in most western industrialized countries. AR represents the major clinical target in the treatment of PCa and, to date, all the FDA-...

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

    DEFF Research Database (Denmark)

    Prihandoko, Rudi; Alvarez-Curto, Elisa; Hudson, Brian D; Butcher, Adrian J; Ulven, Trond; Miller, Ashley M; Tobin, Andrew B; Milligan, Graeme

    2016-01-01

    phosphoacceptor sites to alanine completely prevented phosphorylation of mFFA4 but did not limit receptor coupling to extracellular signal regulated protein kinase 1 and 2 (ERK1/2) activation. Rather, an inhibitor of Gq/11proteins completely prevented receptor signaling to ERK1/2. By contrast, the recruitment of......It is established that long-chain free fatty acids includingω-3 fatty acids mediate an array of biologic responses through members of the free fatty acid (FFA) receptor family, which includes FFA4. However, the signaling mechanisms and modes of regulation of this receptor class remain unclear. Here...... arrestin 3, receptor internalization, and activation of Akt were regulated by mFFA4 phosphorylation. The analysis of mFFA4 phosphorylation-dependent signaling was extended further by selective mutations of the phosphoacceptor sites. Mutations within cluster 2 did not affect agonist activation of Akt but...

  7. 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. PMID:26280129

  8. 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

    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.

  9. 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; Pelicci, P G; Pouysségur, J; Van Obberghen-Schilling, 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....... 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 of the...

  10. GPCRdb: the G protein-coupled receptor database - an introduction.

    Science.gov (United States)

    Munk, C; Isberg, V; Mordalski, S; Harpsøe, K; Rataj, K; Hauser, A S; Kolb, P; Bojarski, A J; Vriend, G; Gloriam, D E

    2016-07-01

    GPCRs make up the largest family of human membrane proteins and of drug targets. Recent advances in GPCR pharmacology and crystallography have shed new light on signal transduction, allosteric modulation and biased signalling, translating into new mechanisms and principles for drug design. The GPCR database, GPCRdb, has served the community for over 20 years and has recently been extended to include a more multidisciplinary audience. This review is intended to introduce new users to the services in GPCRdb, which meets three overall purposes: firstly, to provide reference data in an integrated, annotated and structured fashion, with a focus on sequences, structures, single-point mutations and ligand interactions. Secondly, to equip the community with a suite of web tools for swift analysis of structures, sequence similarities, receptor relationships, and ligand target profiles. Thirdly, to facilitate dissemination through interactive diagrams of, for example, receptor residue topologies, phylogenetic relationships and crystal structure statistics. Herein, these services are described for the first time; visitors and guides are provided with good practices for their utilization. Finally, we describe complementary databases cross-referenced by GPCRdb and web servers with corresponding functionality. PMID:27155948

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

    Science.gov (United States)

    Horn, F; Weare, J; Beukers, M W; Hörsch, S; Bairoch, A; Chen, W; Edvardsen, O; Campagne, F; Vriend, G

    1998-01-01

    The GPCRDB is a G protein-coupled receptor (GPCR) database system aimed at the collection and dissemination of GPCR related data. It holds sequences, mutant data and ligand binding constants as primary (experimental) data. Computationally derived data such as multiple sequence alignments, three dimensional models, phylogenetic trees and two dimensional visualization tools are added to enhance the database's usefulness. The GPCRDB is an EU sponsored project aimed at building a generic molecular class specific database capable of dealing with highly heterogeneous data. GPCRs were chosen as test molecules because of their enormous importance for medical sciences and due to the availability of so much highly heterogeneous data. The GPCRDB is available via the WWW at http://www.gpcr.org/7tm PMID:9399852

  12. Exploring the stochastic dynamics of correlated movement of receptor proteins in plasma membranes in vivo

    International Nuclear Information System (INIS)

    Ligand-induced receptor dimerization plays a crucial role in the signaling process of living cells. In this study, we developed a theoretical model and performed single-molecule tracking to explore the correlated diffusion processes of liganded epidermal growth factor receptors prior to dimer formation. We disclosed that both an attractive potential between liganded receptor proteins in proximity and correlated fluctuations in the local environments of the proteins play an important role to produce the observed correlated movement of the receptors. This result can serve as the foundation to shed light on the way in which receptor functions are regulated in plasma membranes in vivo

  13. Exploring the stochastic dynamics of correlated movement of receptor proteins in plasma membranes in vivo

    Science.gov (United States)

    Huang, Jung Y.; Lin, Chien Y.

    2015-12-01

    Ligand-induced receptor dimerization plays a crucial role in the signaling process of living cells. In this study, we developed a theoretical model and performed single-molecule tracking to explore the correlated diffusion processes of liganded epidermal growth factor receptors prior to dimer formation. We disclosed that both an attractive potential between liganded receptor proteins in proximity and correlated fluctuations in the local environments of the proteins play an important role to produce the observed correlated movement of the receptors. This result can serve as the foundation to shed light on the way in which receptor functions are regulated in plasma membranes in vivo.

  14. Exploring the stochastic dynamics of correlated movement of receptor proteins in plasma membranes in vivo

    Energy Technology Data Exchange (ETDEWEB)

    Huang, Jung Y., E-mail: jyhuang@faculty.nctu.edu.tw [The T.K.B. Research Center of Photonics, Chiao Tung University, Hsinchu 300, Taiwan (China); Lin, Chien Y. [Department of Photonics, Chiao Tung University, Hsinchu 300, Taiwan (China)

    2015-12-14

    Ligand-induced receptor dimerization plays a crucial role in the signaling process of living cells. In this study, we developed a theoretical model and performed single-molecule tracking to explore the correlated diffusion processes of liganded epidermal growth factor receptors prior to dimer formation. We disclosed that both an attractive potential between liganded receptor proteins in proximity and correlated fluctuations in the local environments of the proteins play an important role to produce the observed correlated movement of the receptors. This result can serve as the foundation to shed light on the way in which receptor functions are regulated in plasma membranes in vivo.

  15. Involvement of the G-protein-coupled receptor 4 in RANKL expression by osteoblasts in an acidic environment

    International Nuclear Information System (INIS)

    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

  16. 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.

  17. G-protein-coupled Receptor Kinase-interacting Proteins Inhibit Apoptosis by Inositol 1,4,5-Triphosphate Receptor-mediated Ca2+ Signal Regulation*

    OpenAIRE

    Zhang, Songbai; Hisatsune, Chihiro; Matsu-ura, Toru; Mikoshiba, Katsuhiko

    2009-01-01

    The inositol 1,4,5-trisphosphate (IP3) receptor (IP3R) is an intracellular IP3-gated calcium (Ca2+) release channel and plays important roles in regulation of numerous Ca2+-dependent cellular responses. Many intracellular modulators and IP3R-binding proteins regulate the IP3R channel function. Here we identified G-protein-coupled receptor kinase-interacting proteins (GIT), GIT1 and GIT2, as novel IP3R-binding proteins. We found that both GIT1 and GIT2 directly bind to all three subtypes of IP...

  18. Procedure for Calculation of Potency and Efficacy for Ligands Acting on G(s)- and G (i)-Coupled Receptors

    DEFF Research Database (Denmark)

    Meier, Eddi; Schousboe, Arne; Belhage, Bo

    2012-01-01

    Structure activity relationship (SAR) analyses of pharmacological data of compounds constitute an important part of the discovery process in the design of new drug candidates with improved pharmacological properties. In particular G-Protein Coupled Receptors (GPCRs) associated with the cAMP secon...... measured signals (cpm or cps) using cAMP standard curves. The present work outlines the mathematical procedures by which such transformations are to be performed....

  19. Cross genome phylogenetic analysis of human and Drosophila G protein-coupled receptors: application to functional annotation of orphan receptors

    Directory of Open Access Journals (Sweden)

    Sowdhamini Ramanathan

    2005-08-01

    Full Text Available Abstract Background The cell-membrane G-protein coupled receptors (GPCRs are one of the largest known superfamilies and are the main focus of intense pharmaceutical research due to their key role in cell physiology and disease. A large number of putative GPCRs are 'orphans' with no identified natural ligands. The first step in understanding the function of orphan GPCRs is to identify their ligands. Phylogenetic clustering methods were used to elucidate the chemical nature of receptor ligands, which led to the identification of natural ligands for many orphan receptors. We have clustered human and Drosophila receptors with known ligands and orphans through cross genome phylogenetic analysis and hypothesized higher relationship of co-clustered members that would ease ligand identification, as related receptors share ligands with similar structure or class. Results Cross-genome phylogenetic analyses were performed to identify eight major groups of GPCRs dividing them into 32 clusters of 371 human and 113 Drosophila proteins (excluding olfactory, taste and gustatory receptors and reveal unexpected levels of evolutionary conservation across human and Drosophila GPCRs. We also observe that members of human chemokine receptors, involved in immune response, and most of nucleotide-lipid receptors (except opsins do not have counterparts in Drosophila. Similarly, a group of Drosophila GPCRs (methuselah receptors, associated in aging, is not present in humans. Conclusion Our analysis suggests ligand class association to 52 unknown Drosophila receptors and 95 unknown human GPCRs. A higher level of phylogenetic organization was revealed in which clusters with common domain architecture or cellular localization or ligand structure or chemistry or a shared function are evident across human and Drosophila genomes. Such analyses will prove valuable for identifying the natural ligands of Drosophila and human orphan receptors that can lead to a better understanding

  20. Conformational Fluctuations in G-Protein-Coupled Receptors

    Science.gov (United States)

    Brown, Michael F.

    2014-03-01

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

  1. Identification of the Receptor Binding Domain of the Mouse Mammary Tumor Virus Envelope Protein

    OpenAIRE

    Zhang, Yuanming; Rassa, John C.; deObaldia, Maria Elena; Albritton, Lorraine M.; Susan R Ross

    2003-01-01

    Mouse mammary tumor virus (MMTV) is a betaretrovirus that infects rodent cells and uses mouse transferrin receptor 1 for cell entry. To characterize the interaction of MMTV with its receptor, we aligned the MMTV envelope surface (SU) protein with that of Friend murine leukemia virus (F-MLV) and identified a putative receptor-binding domain (RBD) that included a receptor binding sequence (RBS) of five amino acids and a heparin-binding domain (HBD). Mutation of the HBD reduced virus infectivity...

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

    OpenAIRE

    Parker, Michael S.; Renu Sah; Ambikaipakan Balasubramaniam; Park, Edwards A.; Sallee, Floyd R.; Parker, Steven L.

    2014-01-01

    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 c...

  3. Instability of a Class A G Protein-Coupled Receptor Oligomer Interface

    OpenAIRE

    Fonseca, Jacqueline M.; Lambert, Nevin A.

    2009-01-01

    The quaternary structure of G protein-coupled receptors (GPCRs) can influence their trafficking and ability to transduce signals. GPCR oligomers are generally portrayed as long-lived entities, although the stability of these complexes has not been studied. Here we show that D2 dopamine receptor protomers interact transiently at a specific oligomer interface. Selective immobilization of cyan fluorescent protein-D2 receptors (C-D2Rs) in the plasma membrane failed to comp...

  4. Receptor-mediated inhibition of adenylate cyclase and stimulation of arachidonic acid release in 3T3 fibroblasts. Selective susceptibility to islet-activating protein, pertussis toxin

    International Nuclear Information System (INIS)

    Thrombin exhibited diverse effects on mouse 3T3 fibroblasts. It (a) decreased cAMP in the cell suspension, (b) inhibited adenylate cyclase in the Lubrol-permeabilized cell suspension in a GTP-dependent manner, increased releases of (c) arachidonic acid and (d) inositol from the cell monolayer prelabeled with these labeled compounds, (e) increased 45Ca2+ uptake into the cell monolayer, and (f) increased 86Rb+ uptake into the cell monolayer in a ouabain-sensitive manner. Most of the effects were reproduced by bradykinin, platelet-activating factor, and angiotensin II. The receptors for these agonists are thus likely to be linked to three separate effector systems: the adenylate cyclase inhibition, the phosphoinositide breakdown leading to Ca2+ mobilization and phospholipase A2 activation, and the Na,K-ATPase activation. Among the effects of these agonists, (a), (b), (c), and (e) were abolished, but (d) and (f) were not, by prior treatment of the cells with islet-activating protein (IAP), pertussis toxin, which ADP-ribosylates the Mr = 41,000 protein, the alpha-subunit of the inhibitory guanine nucleotide regulatory protein (Ni), thereby abolishing receptor-mediated inhibition of adenylate cyclase. The effects (a), (c), (d), and (e) of thrombin, but not (b), were mimicked by A23187, a calcium ionophore. The effects of A23187, in contrast to those of receptor agonists, were not affected by the treatment of cells with IAP. Thus, the IAP substrate, the alpha-subunit of Ni, or the protein alike, may play an additional role in signal transduction arising from the Ca2+-mobilizing receptors, probably mediating process(es) distal to phosphoinositide breakdown and proximal to Ca2+ gating

  5. Calcium mobilization from fish scales is mediated by parathyroid hormone related protein via the parathyroid hormone type 1 receptor.

    Science.gov (United States)

    Rotllant, J; Redruello, B; Guerreiro, P M; Fernandes, H; Canario, A V M; Power, D M

    2005-12-15

    The scales of bony fish represent a significant reservoir of calcium but little is known about their contribution, as well as of bone, to calcium balance and how calcium deposition and mobilization are regulated in calcified tissues. In the present study we report the action of parathyroid hormone-related protein (PTHrP) on calcium mobilization from sea bream (Sparus auratus) scales in an in vitro bioassay. Ligand binding studies of piscine 125I-(1-35(tyr))PTHrP to the membrane fraction of isolated sea bream scales revealed the existence of a single PTH receptor (PTHR) type. RT-PCR of fish scale cDNA using specific primers for two receptor types found in teleosts, PTH1R, and PTH3R, showed expression only of PTH1R. The signalling mechanisms mediating binding of the N-terminal amino acid region of PTHrP were investigated. A synthetic peptide (10(-8) M) based on the N-terminal 1-34 amino acid residues of Fugu rubripes PTHrP strongly stimulated cAMP synthesis and [3H]myo-inositol incorporation in sea bream scales. However, peptides (10(-8) M) with N-terminal deletions, such as (2-34), (3-34) and (7-34)PTHrP, were defective in stimulating cAMP production but stimulated [3H]myo-inositol incorporation. (1-34)PTHrP induced significant osteoclastic activity in scale tissue as indicated by its stimulation of tartrate-resistant acid phosphatase. In contrast, (7-34)PTHrP failed to stimulate the activity of this enzyme. This activity could also be abolished by the adenylyl cyclase inhibitor SQ-22536, but not by the phospholipase C inhibitor U-73122. The results of the study indicate that one mechanism through which N-terminal (1-34)PTHrP stimulates osteoclastic activity of sea bream scales, is through PTH1R and via the cAMP/AC intracellular signalling pathway. It appears, therefore, that fish scales can act as calcium stores and that (1-34)PTHrP regulates calcium mobilization from them; it remains to be established if this mechanism contributes to calcium homeostasis in vivo

  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

    International Nuclear Information System (INIS)

    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

    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.

  8. 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 was...... unknown. Here we identify endothelial protein C receptor (EPCR), which mediates the cytoprotective effects of activated protein C, as the endothelial receptor for DC8 and DC13 PfEMP1. We show that EPCR binding is mediated through the amino-terminal cysteine-rich interdomain region (CIDRα1) of DC8 and...... group A PfEMP1 subfamilies, and that CIDRα1 interferes with protein C binding to EPCR. This PfEMP1 adhesive property links P. falciparum cytoadhesion to a host receptor involved in anticoagulation and endothelial cytoprotective pathways, and has implications for understanding malaria pathology and the...

  9. 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

    2015-01-01

    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 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. PMID:26561036

  10. Computational Modeling for the Activation Cycle of G-proteins by G-protein-coupled Receptors

    CERN Document Server

    Bao, Yifei; Glavy, Joseph; White, Tommy; 10.4204/EPTCS.40.4

    2010-01-01

    In this paper, we survey five different computational modeling methods. For comparison, we use the activation cycle of G-proteins that regulate cellular signaling events downstream of G-protein-coupled receptors (GPCRs) as a driving example. Starting from an existing Ordinary Differential Equations (ODEs) model, we implement the G-protein cycle in the stochastic Pi-calculus using SPiM, as Petri-nets using Cell Illustrator, in the Kappa Language using Cellucidate, and in Bio-PEPA using the Bio-PEPA eclipse plug in. We also provide a high-level notation to abstract away from communication primitives that may be unfamiliar to the average biologist, and we show how to translate high-level programs into stochastic Pi-calculus processes and chemical reactions.

  11. A homogeneous single-label time-resolved fluorescence cAMP assay.

    Science.gov (United States)

    Martikkala, Eija; Rozwandowicz-Jansen, Anita; Hänninen, Pekka; Petäjä-Repo, Ulla; Härmä, Harri

    2011-03-01

    G-protein-coupled receptors (GPCRs) are an important class of pharmaceutical drug targets. Functional high-throughput GPCR assays are needed to test an increasing number of synthesized novel drug compounds and their function in signal transduction processes. Measurement of changes in the cyclic adenosine monophosphate (cAMP) concentration is a widely used method to verify GPCR activation in the adenylyl cyclase pathway. Here, a single-label time-resolved fluorescence and high-throughput screening (HTS)-feasible method was developed to measure changes in cAMP levels in HEK293(i) cells overexpressing either β(2)-adrenergic or δ-opioid receptors. In the quenching resonance energy transfer (QRET) technique, soluble quenchers reduce the signal of unbound europium(III)-labeled cAMP in solution, whereas the antibody-bound fraction is fluorescent. The feasibility of this homogeneous competitive assay was proven by agonist-mediated stimulation of receptors coupled to either the stimulatory G(s) or inhibitory G(i) proteins. The reproducibility of the assays was excellent, and Z' values exceeded 0.7. The dynamic range, signal-to-background ratio, and detection limit were compared with a commercial time-resolved fluorescence resonance energy transfer (TR-FRET) assay. In both homogeneous assays, similar assay parameters were obtained when adenylyl cyclase was stimulated directly by forskolin or via agonist-mediated activation of the G(s)-coupled β(2)AR. The advantage of using the single-label approach relates to the cost-effectiveness of the QRET system compared with the two-label TR-FRET assay as there is no need for labeling of two binding partners leading to reduced requirements for assay optimization. PMID:21343601

  12. 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

  13. 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. PMID:20835432

  14. Differential ligand-dependent protein–protein interactions between nuclear receptors and a neuronal-specific cofactor

    OpenAIRE

    Greiner, Erich F.; Kirfel, Jutta; Greschik, Holger; Huang, DongYa; Becker, Peter; Kapfhammer, Josef P.; Schüle, Roland

    2000-01-01

    Nuclear receptors are transcription factors that require multiple protein–protein interactions to regulate target gene expression. We have cloned a 27-kDa protein, termed NIX1 (neuronal interacting factor X 1), that directly binds nuclear receptors in vitro and in vivo. Protein–protein interaction between NIX1 and ligand-activated or constitutive active nuclear receptors, including retinoid-related orphan receptor β (RORβ) (NR1F2), strictly depends on the conserved receptor C-terminal activat...

  15. A Summer Camp

    Institute of Scientific and Technical Information of China (English)

    2012-01-01

    正This summer,I had some special days.I joined Dongzhou International Educational Exchange Summer Camp. First,I will tell you about our foreign teachers,they are Shrina and Rebecca. They are friendly and beautiful.They are students at Oxford University. We talked about many things:famous people,subjects in England,different jobs, our deal days,western star signs,what can we say in a restaurant and so on.

  16. Interprofessional Flight Camp.

    Science.gov (United States)

    Alfes, Celeste M; Rowe, Amanda S

    2016-01-01

    The Dorothy Ebersbach Academic Center for Flight Nursing in Cleveland, OH, holds an annual flight camp designed for master's degree nursing students in the acute care nurse practitioner program, subspecializing in flight nursing at the Frances Payne Bolton School of Nursing at Case Western Reserve University. The weeklong interprofessional training is also open to any health care provider working in an acute care setting and focuses on critical care updates, trauma, and emergency care within the critical care transport environment. This year, 29 graduate nursing students enrolled in a master's degree program from Puerto Rico attended. Although the emergency department in Puerto Rico sees and cares for trauma patients, there is no formal trauma training program. Furthermore, the country only has 1 rotor wing air medical transport service located at the Puerto Rico Medical Center in San Juan. Flight faculty and graduate teaching assistants spent approximately 9 months planning for their participation in our 13th annual flight camp. Students from Puerto Rico were extremely pleased with the learning experiences at camp and expressed particular interest in having more training time within the helicopter flight simulator. PMID:27021671

  17. Chronic exercise improves repeated restraint stress-induced anxiety and depression through 5HT1A receptor and cAMP signaling in hippocampus

    OpenAIRE

    Kim, Mun Hee; Leem, Yea Hyun

    2014-01-01

    [Purpose] Mood disorders such as anxiety and depression are prevalent psychiatric illness, but the role of 5HT1A in the anti-depressive effects of exercise has been rarely known yet. We investigated whether long-term exercise affected a depressive-like behavior and a hippocampal 5HT1A receptor-mediated cAMP/PKA/CREB signaling in depression mice model. [Methods] To induce depressive behaviors, mice were subjected to 14 consecutive days of restraint stress (2 hours/day). Depression-like behavio...

  18. Protein kinase C phosphorylation sensitizes but does not activate the capsaicin receptor transient receptor potential vanilloid 1 (TRPV1)

    OpenAIRE

    Bhave, Gautam; HU, HUI-JUAN; Glauner, Kathi S.; Zhu, Weiguo; Wang, Haibin; Brasier, D. J.; Oxford, Gerry S; Gereau, Robert W.

    2003-01-01

    Protein kinase C (PKC) modulates the function of the capsaicin receptor transient receptor potential vanilloid 1 (TRPV1). This modulation manifests as increased current when the channel is activated by capsaicin. In addition, studies have suggested that phosphorylation by PKC might directly gate the channel, because PKC-activating phorbol esters induce TRPV1 currents in the absence of applied ligands. To test whether PKC both modulates and gates the TRPV1 function by direct phosphorylation, w...

  19. 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.

  20. 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...

  1. 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

  2. Regulation of G protein-coupled receptors by palmitoylation and cholesterol

    OpenAIRE

    Goddard Alan D; Watts Anthony

    2012-01-01

    Abstract Due to their membrane location, G protein-coupled receptors (GPCRs) are subject to regulation by soluble and integral membrane proteins as well as membrane components, including lipids and sterols. GPCRs also undergo a variety of post-translational modifications, including palmitoylation. A recent article by Zheng et al. in BMC Cell Biology demonstrates cooperative roles for receptor palmitoylation and cholesterol binding in GPCR dimerization and G protein coupling, underlining the c...

  3. Maturation of steroid receptors: an example of functional cooperation among molecular chaperones and their associated proteins.

    Science.gov (United States)

    Kimmins, S; MacRae, T H

    2000-04-01

    The selective modulation of transcription exerted by steroids depends upon recognition of signalling molecules by properly folded cytoplasmic receptors and their subsequent translocation into the nucleus. These events require a sequential and dynamic series of protein-protein interactions in order to fashion receptors that bind stably to steroids. Central to receptor maturation, therefore, are several molecular chaperones and their accessory proteins; Hsp70, Hsp40, and hip modulate the 3-dimensional conformation of steroid receptors, permitting reaction via hop with Hsp90, arguably the central protein in the process. Binding to Hsp90 leads to dissociation of some proteins from the receptor complex while others are recruited. Notably, p23 stabilizes receptors in a steroid binding state, and the immunophilins, principally CyP40 and Hsp56, arrive late in receptor complex assembly. In this review, the functions of molecular chaperones during steroid receptor maturation are explored, leading to a general mechanistic model indicative of chaperone cooperation in protein folding. PMID:11147968

  4. Bioluminescent Ligand-Receptor Binding Assays for Protein or Peptide Hormones.

    Science.gov (United States)

    Liu, Ya-Li; Guo, Zhan-Yun

    2016-01-01

    Bioluminescence has been widely used in biomedical research due to its high sensitivity, low background, and broad linear range. In recent studies, we applied bioluminescence to ligand-receptor binding assays for some protein or peptide hormones based on a newly developed small monomeric Nanoluciferase (NanoLuc) reporter that has the so far brightest bioluminescence. The conventional ligand-receptor binding assays rely on radioligands that have drawbacks, such as radioactive hazards and short shelf lives. In contrast, the novel bioluminescent binding assays use the NanoLuc-based protein or peptide tracers that are safe, stable, and ultrasensitive. Thus, the novel bioluminescent ligand-receptor binding assay would be applied to more and more protein or peptide hormones for ligand-receptor interaction studies in future. In the present article, we provided detailed protocols for setting up the novel bioluminescent ligand-receptor binding assays using two representative protein hormones as examples. PMID:27424896

  5. 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.

    Science.gov (United States)

    Zuloaga, R; Fuentes, E N; Molina, A; Valdés, J A

    2013-10-18

    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 IP3/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. PMID:24064350

  6. 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

    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...

  7. The G Protein-Coupled Estrogen Receptor Agonist G-1 Inhibits Nuclear Estrogen Receptor Activity and Stimulates Novel Phosphoproteomic Signatures.

    Science.gov (United States)

    Smith, L Cody; Ralston-Hooper, Kimberly J; Ferguson, P Lee; Sabo-Attwood, Tara

    2016-06-01

    Estrogen exerts cellular effects through both nuclear (ESR1 and ESR2) and membrane-bound estrogen receptors (G-protein coupled estrogen receptor, GPER); however, it is unclear if they act independently or engage in crosstalk to influence hormonal responses. To investigate each receptor's role in proliferation, transcriptional activation, and protein phosphorylation in breast cancer cells (MCF-7), we employed selective agonists for ESR1 propyl-pyrazole-triol (PPT), ESR2 diarylpropionitrile (DPN), and GPER (G-1) and also determined the impact of xenoestrogens bisphenol-A (BPA) and genistein on these effects. As anticipated, 17β-estradiol (E2), PPT, DPN, BPA, and genistein each enhanced proliferation and activation of an ERE-driven reporter gene whereas G-1 had no significant impact. However, G-1 significantly reduced E2-, PPT-, DPN-, BPA-, and genistein-induced proliferation and ERE activation at doses greater than 500 nM indicating that G-1 mediated inhibition is not ESR isotype specific. As membrane receptors initiate cascades of phosphorylation events, we performed a global phosphoproteomic analysis on cells exposed to E2 or G-1 to identify potential targets of receptor crosstalk via downstream protein phosphorylation targets. Of the 211 phosphorylated proteins identified, 40 and 13 phosphoproteins were specifically modified by E2 and G-1, respectively. Subnetwork enrichment analysis revealed several processes related to cell cycle were specifically enriched by G-1 compared with E2. Further there existed a number of newly identified proteins that were specifically phosphorylated by G-1. These phosphorylation networks highlight specific proteins that may modulate the inhibitory effects of G-1 and suggest a novel role for interference with nuclear receptor activity driven by E2 and xenoestrogens. PMID:27026707

  8. 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,

  9. DMPD: Macrophage-stimulating protein and RON receptor tyrosine kinase: potentialregulators of macrophage inflammatory activities. [Dynamic Macrophage Pathway CSML Database

    Lifescience Database Archive (English)

    Full Text Available 12472665 Macrophage-stimulating protein and RON receptor tyrosine kinase: potential...:545-53. (.png) (.svg) (.html) (.csml) Show Macrophage-stimulating protein and RON receptor tyrosine kinase: poten...le Macrophage-stimulating protein and RON receptor tyrosine kinase: potentialregulators of macrophage inflam

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

    Science.gov (United States)

    Gao, Xin; Wu, Tongyu; Johnson, Kirby D; Lahvic, Jamie L; Ranheim, Erik A; Zon, Leonard I; Bresnick, Emery H

    2016-03-01

    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. PMID:26905203

  11. 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.

  12. Transcriptional activation of peroxisome proliferator-activated receptor-γ requires activation of both protein kinase A and Akt during adipocyte differentiation

    International Nuclear Information System (INIS)

    Research highlights: → Elevated cAMP activates both PKA and Epac. → PKA activates CREB transcriptional factor and Epac activates PI3K/Akt pathway via Rap1. → Akt modulates PPAR-γ transcriptional activity in concert with CREB. -- Abstract: Peroxisome proliferator-activated receptor-γ (PPAR-γ) is required for the conversion of pre-adipocytes. However, the mechanism underlying activation of PPAR-γ is unclear. Here we showed that cAMP-induced activation of protein kinase A (PKA) and Akt is essential for the transcriptional activation of PPAR-γ. 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-γ was markedly enhanced by 3-isobutyl-1-methylxanthine (IBMX), but not insulin and dexamethasone. In addition, IBMX-induced PPAR-γ 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-γ transcriptional activity. These results indicate that both PKA and Akt signaling pathways are required for transcriptional activation of PPAR-γ, suggesting post-translational activation of PPAR-γ might be critical step for adipogenic gene expression.

  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. Protein-protein interactions in crystals of the human receptor-type protein tyrosine phosphatase ICA512 ectodomain.

    Directory of Open Access Journals (Sweden)

    María E Primo

    Full Text Available ICA512 (or IA-2 is a transmembrane protein-tyrosine phosphatase located in secretory granules of neuroendocrine cells. Initially, it was identified as one of the main antigens of autoimmune diabetes. Later, it was found that during insulin secretion, the cytoplasmic domain of ICA512 is cleaved and relocated to the nucleus, where it stimulates the transcription of the insulin gene. The role of the other parts of the receptor in insulin secretion is yet to be unveiled. The structures of the intracellular pseudocatalytic and mature extracellular domains are known, but the transmembrane domain and several intracellular and extracellular parts of the receptor are poorly characterized. Moreover the overall structure of the receptor remains to be established. We started to address this issue studying by X-ray crystallography the structure of the mature ectodomain of ICA512 (ME ICA512 and variants thereof. The variants and crystallization conditions were chosen with the purpose of exploring putative association interfaces, metal binding sites and all other structural details that might help, in subsequent works, to build a model of the entire receptor. Several structural features were clarified and three main different association modes of ME ICA512 were identified. The results provide essential pieces of information for the design of new experiments aimed to assess the structure in vivo.

  15. Functional somatostatin receptors on a rat pancreatic acinar cell line

    International Nuclear Information System (INIS)

    Somatostatin receptors from a rat pancreatic acinar cell line, AR4-2J, were characterized biochemically, structurally, and functionally. Binding of 125I-[Tyr11]Somatostatin to AR4-2J cells was saturable, exhibiting a single class of high-affinity binding sites with a maximal binding capacity of 258 ± 20 fmol/106 cells. Somatostatin receptor structure was analyzed by covalently cross-linking 125I-[Tyr11]somatostatin to its plasma membrane receptors. Gel electrophoresis and autoradiography of cross-linked proteins revealed a peptide containing the somatostatin receptor. Somatostatin inhibited vasoactive intestinal peptide (VIP)-stimulated adenosine 3',5'-cyclic monophosphate (cAMP) formation in a dose-dependent manner. The concentration of somatostatin that caused half-maximal inhibition of cAMP formation was close to the receptor affinity for somatostatin. Pertussis toxin pretreatment of AR4-2J cells prevented somatostatin inhibition of VIP-stimulated cAMP formation as well as somatostatin binding. The authors conclude that AR4-2J cells exhibit functional somatostatin receptors that retain both specificity and affinity of the pancreatic acinar cell somatostatin receptors and act via the pertussis toxin-sensitive guanine nucleotide-binding protein Ni to inhibit adenylate cyclase

  16. Evolution of the thyrotropin receptor: a G protein coupled receptor with an intrinsic capacity to dimerize.

    Science.gov (United States)

    Kaczur, Viktória; Puskás, László G; Takács, Mária; Rácz, István András; Szendroi, Andrea; Tóth, Sára; Nagy, Zsuzsanna; Szalai, Csaba; Balázs, Csaba; Falus, András; Knudsen, Bjarne; Farid, Nadir R

    2003-04-01

    The rapidly escalating number of genome sequences has emphasized the basic tenants of the schema of life. By the same token comparisons according to specialized function or niche within nature expose genomic strategies to optimize the use of resources and ensure biological success. Increasing complexity may result from diversification, shuffling, and re-arrangement of an otherwise limited functional genomic complement. To further test the concept of relative structural plasticity of the TSH receptor we sequenced the TSHR gene of two Old World monkey species Macaca mulatta and Cercopithecus aethiops, evolutionary removed from Homo sapiens by >20Myr. Both genes encoded a protein of 764 residues. This structure was 99% homologous between the two species of Old World monkeys while C. aethiops was 97% and M. mulatta was 96% homologous to H. sapiens. TSHR sequence comparisons were sought for an additional eight mammals as well as four (two Salmon, Tilapia, and Sea Bass) from teleosts. The amino-acid sequences of the 14 TSH receptors were similar. The most variable sequences were those of the intracellular tail and the distal cysteine-rich C-terminus flanking region of the ectodomain, whereas the trans-membrane domain was most preserved. Some sequences were decidedly H. sapiens specific, while others were primate specific or showed the changes expected of evolutionary descent. Others, however, exhibited "cross-species polymorphism," sometimes at quite remarkable evolutionary distances. As opposed to H. sapiens the sequence differences may have subtle influences on TSHR function or may affect long-range compensation for radical changes in adducts. The two Old World monkeys share with other lower mammals the absence of a glycosylation site at 113-115. Sea Bass and Tilapia have four glycosylation sites, whereas the two salmon receptors have only three. Changes in some critical residues raise questions about variation in function: thus S281 is conserved in all mammals and an

  17. 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.

  18. Antipeptide antibody that specifically inhibits insulin receptor autophosphorylation and protein kinase activity

    International Nuclear Information System (INIS)

    Two site-specific antibodies that immunoprecipitate the human insulin receptor have been prepared by immunizing rabbits with chemically synthesized peptides derived from the cDNA-predicted amino acid sequence of the β subunit of the proreceptor. Antibodies to the carboxyl terminus (AbP5) and to a domain around tyrosine-960 (AbP4) specifically recognize the β subunit of the receptor on immunoblots. Both antibodies immunoprecipitated 125I-labeled insulin-receptor complexes and the autophosphorylated receptor. Although neither antibody inhibited insulin binding to the receptor, both insulin-dependent autophosphorylation and exogenous substrate phosphorylation were inhibited by AbP4. Inhibition by AbP4 was dependent upon the phosphorylation state of the receptor; it was not detected when the receptor was autophosphorylated prior to addition of AbP4. AbP4 did not inhibit activity of the related epidermal growth factor (EGF)-receptor tyrosine protein kinase nor did it inhibit the activity of cAMP-dependent kinase or protein kinase C. The observation that an antibody directed to residues 952-967 of the proreceptor neutralizes the protein kinase activity of the β subunit suggest that this region may play a critical role in the function of the hormone-dependent, protein tyrosine-specific kinase activity of the insulin receptor

  19. A Camp in the Desert

    OpenAIRE

    Heise, Aaron Kent

    2006-01-01

    A house is pulled apart into its separate rooms and joined in the out-of-doors. This collection of rooms is recognized as a camp. This move is in agreement with the site of the house, which is the foothills of the Rincon Mountains, twenty miles east of Tucson, Arizona, and bordering along Saguaro National Monument. The collection of structures that make up the buildings of the camp are joined by a path that encircles the camp, and also describes the active life of the camp. The design o...

  20. 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.

  1. Direct Modulation of Heterotrimeric G Protein-coupled Signaling by a Receptor Kinase Complex.

    Science.gov (United States)

    Tunc-Ozdemir, Meral; Urano, Daisuke; Jaiswal, Dinesh Kumar; Clouse, Steven D; Jones, Alan M

    2016-07-01

    Plants and some protists have heterotrimeric G protein complexes that activate spontaneously without canonical G protein-coupled receptors (GPCRs). In Arabidopsis, the sole 7-transmembrane regulator of G protein signaling 1 (AtRGS1) modulates the G protein complex by keeping it in the resting state (GDP-bound). However, it remains unknown how a myriad of biological responses is achieved with a single G protein modulator. We propose that in complete contrast to G protein activation in animals, plant leucine-rich repeat receptor-like kinases (LRR RLKs), not GPCRs, provide this discrimination through phosphorylation of AtRGS1 in a ligand-dependent manner. G protein signaling is directly activated by the pathogen-associated molecular pattern flagellin peptide 22 through its LRR RLK, FLS2, and co-receptor BAK1. PMID:27235398

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

    DEFF Research Database (Denmark)

    Chung, Ka Young; Rasmussen, Søren Gøgsig Faarup; Liu, Tong; Li, Sheng; DeVree, Brian T; Chae, Pil Seok; Calinski, Diane; Kobilka, Brian K; Woods, Virgil L; Sunahara, Roger K

    2011-01-01

    G protein-coupled receptors represent the largest family of membrane receptors that instigate signalling through nucleotide exchange on heterotrimeric G proteins. Nucleotide exchange, or more precisely, GDP dissociation from the G protein α-subunit, is the key step towards G protein activation and...... initiation of downstream signalling cascades. Despite a wealth of biochemical and biophysical studies on inactive and active conformations of several heterotrimeric G proteins, the molecular underpinnings of G protein activation remain elusive. To characterize this mechanism, we applied peptide amide......), we provide a rationale for a mechanism of nucleotide exchange, whereby the receptor perturbs the structure of the amino-terminal region of the α-subunit of Gs and consequently alters the 'P-loop' that binds the β-phosphate in GDP. As with the Ras family of small-molecular-weight G proteins, P...

  3. Absence of serum growth hormone binding protein in patients with growth hormone receptor deficiency (Laron dwarfism).

    OpenAIRE

    Daughaday, W H; Trivedi, B

    1987-01-01

    It has recently been recognized that human serum contains a protein that specifically binds human growth hormone (hGH). This protein has the same restricted specificity for hGH as the membrane-bound GH receptor. To determine whether the GH-binding protein is a derivative of, or otherwise related to, the GH receptor, we have examined the serum of three patients with Laron-type dwarfism, a condition in which GH refractoriness has been attributed to a defect in the GH receptor. The binding of 12...

  4. Immunochemical strategy for quantification of G-coupled olfactory receptor proteins on natural nanovesicles.

    Science.gov (United States)

    Sanmartí-Espinal, Marta; Galve, Roger; Iavicoli, Patrizia; Persuy, Marie-Annick; Pajot-Augy, Edith; Marco, M-Pilar; Samitier, Josep

    2016-03-01

    Cell membrane proteins are involved in a variety of biochemical pathways and therefore constitute important targets for therapy and development of new drugs. Bioanalytical platforms and binding assays using these membrane protein receptors for drug screening or diagnostic require the construction of well-characterized liposome and lipid bilayer arrays that act as support to prevent protein denaturation during biochip processing. Quantification of the protein receptors in the lipid membrane arrays is a key issue in order to produce reproducible and well-characterized chips. Herein, we report a novel immunochemical analytical approach for the quantification of membrane proteins (i.e., G-protein-coupled receptor, GPCR) in nanovesicles (NVs). The procedure allows direct determination of tagged receptors (i.e., c-myc tag) without any previous protein purification or extraction steps. The immunochemical method is based on a microplate ELISA format and quantifies this tag on proteins embedded in NVs with detectability in the picomolar range, using protein bioconjugates as reference standards. The applicability of the method is demonstrated through the quantification of the c-myc-olfactory receptor (OR, c-myc-OR1740) in the cell membrane NVs. The reported method opens the possibility to develop well-characterized drug-screening platforms based on G-coupled proteins embedded on membranes. PMID:26724468

  5. Inhibition of WNT Signaling by G Protein-Coupled Receptor (GPCR) Kinase 2 (GRK2)

    OpenAIRE

    WANG, LIMING; Gesty-Palmer, Diane; Fields, Timothy A.; Spurney, Robert F.

    2009-01-01

    Activation of Wnt signaling pathways causes release and stabilization of the transcription regulator β-catenin from a destruction complex composed of axin and the adenomatous polyposis coli (APC) protein (canonical signaling pathway). Assembly of this complex is facilitated by a protein-protein interaction between APC and a regulator of G protein signaling (RGS) domain in axin. Because G protein-coupled receptor kinase 2 (GRK2) has a RGS domain that is closely related to the RGS domain in axi...

  6. Profiling Carbohydrate-Receptor Interaction with Recombinant Innate Immunity Receptor-Fc Fusion Proteins*

    OpenAIRE

    Hsu, Tsui-Ling; Cheng, Shih-Chin; Yang, Wen-Bin; Chin, See-Wen; Bo-hua CHEN; Huang, Ming-Ting; Hsieh, Shie-Liang; Wong, Chi-Huey

    2009-01-01

    The recognition of bacteria, viruses, fungi, and other microbes is controlled by host immune cells, which are equipped with many innate immunity receptors, such as Toll-like receptors, C-type lectin receptors, and immunoglobulin-like receptors. Our studies indicate that the immune modulating properties of many herbal drugs, for instance, the medicinal fungus Reishi (Ganoderma lucidum) and Cordyceps sinensis, could be attributed to their polysaccharide components. These polysaccharides specifi...

  7. Application of G Protein-Coupled Receptor-Heteromer Identification Technology to Monitor β-Arrestin Recruitment to G Protein-Coupled Receptor Heteromers

    OpenAIRE

    Heng B See; Seeber, Ruth M.; Kocan, Martina; Eidne, Karin A.; Pfleger, Kevin D.G.

    2011-01-01

    Understanding the role of G protein-coupled receptor (GPCR; also known as a 7 transmembrane receptor) heteromerization in the physiology and pathophysiology of cellular function has now become a major research focus. However, there is currently a lack of cell-based assays capable of profiling the specific functional consequences of heteromerization in a ligand-dependent manner. Understanding the pharmacology specifically associated with heteromer function in contrast to monomer or homomer fun...

  8. Full and partial agonists of thromboxane prostanoid receptor unveil fine tuning of receptor superactive conformation and G protein activation.

    Directory of Open Access Journals (Sweden)

    Valérie Capra

    Full Text Available The intrahelical salt bridge between E/D(3.49 and R(3.50 within the E/DRY motif on helix 3 (H3 and the interhelical hydrogen bonding between the E/DRY and residues on H6 are thought to be critical in stabilizing the class A G protein-coupled receptors in their inactive state. Removal of these interactions is expected to generate constitutively active receptors. This study examines how neutralization of E(3.49/6.30 in the thromboxane prostanoid (TP receptor alters ligand binding, basal, and agonist-induced activity and investigates the molecular mechanisms of G protein activation. We demonstrate here that a panel of full and partial agonists showed an increase in affinity and potency for E129V and E240V mutants. Yet, even augmenting the sensitivity to detect constitutive activity (CA with overexpression of the receptor or the G protein revealed resistance to an increase in basal activity, while retaining fully the ability to cause agonist-induced signaling. However, direct G protein activation measured through bioluminescence resonance energy transfer (BRET indicates that these mutants more efficiently communicate and/or activate their cognate G proteins. These results suggest the existence of additional constrains governing the shift of TP receptor to its active state, together with an increase propensity of these mutants to agonist-induced signaling, corroborating their definition as superactive mutants. The particular nature of the TP receptor as somehow "resistant" to CA should be examined in the context of its pathophysiological role in the cardiovascular system. Evolutionary forces may have favored regulation mechanisms leading to low basal activity and selected against more highly active phenotypes.

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

    DEFF Research Database (Denmark)

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

    2012-01-01

    , GPCR oligomerization has been extensively studied using methods like bioluminescence resonance energy transfer (BRET) and today, receptor-receptor interactions within the GPCR superfamily is a well-established phenomenon. Evidence of the impact of GPCR oligomerization on, e.g., ligand binding, receptor...

  10. 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.

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

    DEFF Research Database (Denmark)

    Rasmussen, Søren Gøgsig Faarup; 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

    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...

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

    DEFF Research Database (Denmark)

    Steen, Anne; Larsen, Olav; Thiele, Stefanie;

    2014-01-01

    -switches based on recently published 7TM crystals and molecular dynamics studies. All three forms of biased signaling are abundant in the chemokine system. This challenges our understanding of "classic" redundancy inevitably ascribed to this system, where multiple chemokines bind to the same receptor and where a......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...... absolute, i.e., full versus no activation. Here we discuss biased signaling in the chemokine system, including the structural basis for biased signaling in chemokine receptors, as well as in class A 7TM receptors in general. This includes overall helical movements and the contributions of micro...

  13. Basic evidence for class A G-protein-coupled receptor heteromerization

    Directory of Open Access Journals (Sweden)

    Rafael eFranco

    2016-03-01

    Full Text Available Cell membrane receptors rarely work on isolation, often they form oligomeric complexes with other receptor molecules and they may directly interact with different proteins of the signal transduction machinery. For a variety of reasons, rhodopsin-like class A G-protein-coupled receptors (GPCRs seem an exception to the general rule of receptor-receptor direct interaction. In fact, controversy surrounds their potential to form homo- hetero-dimers/oligomers with other class A GPCRs; in a sense, the field is going backwards instead of forward. This review focuses on the convergent, complementary and telling evidence showing that homo- and heteromers of class A GPCRs exist in transfected cells and, more importantly, in natural sources. It is time to decide between questioning the occurrence of heteromers or, alternatively, facing the vast scientific and technical challenges that class A receptor-dimer/oligomer existence pose to Pharmacology and to Drug Discovery.

  14. 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...

  15. 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

    International Nuclear Information System (INIS)

    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 IP3/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

  16. 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.

  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. PMID:27378756

  18. Cell-free protein synthesis and purification of human dopamine D2 receptor long isoform.

    Science.gov (United States)

    Basu, Dipannita; Castellano, Jessica M; Thomas, Nancy; Mishra, Ram K

    2013-01-01

    The human dopamine D2 receptor long isoform (D2L) has significant implications in neurological and neuropsychiatric disorders such as Parkinson's disease and schizophrenia. Detailed structural knowledge of this receptor is limited owing to its highly hydrophobic nature, which leads to protein aggregation and host toxicity when expressed in cellular systems. The newly emerging field of cell-free protein expression presents numerous advantages to overcome these challenges. This system utilizes protein synthesis machinery and exogenous DNA to synthesize functional proteins outside of intact cells. This study utilizes two different cell-free systems for the synthesis of human dopamine D2L receptor. These include the Escherichia coli lysate-based system and the wheat-germ lysate-based system. The bacterial cell-free method used pET 100/D-TOPO vector to synthesize hexa-histidine-tagged D2L receptor using a dialysis bag system; the resulting protein was purified using nickel-nitrilotriacetic acid affinity resin. The wheat germ system used pEU-glutathione-S-transferase (GST) vector to synthesize GST-tagged D2L receptor using a bilayer translation method; the resulting protein was purified using a GST affinity resin. The presence and binding capacity of the synthesized D2L receptor was confirmed by immunoblotting and radioligand competition assays, respectively. Additionally, in-gel protein sequencing via Nano LC-MS/MS was used to confirm protein synthesis via the wheat germ system. The results showed both systems to synthesize microgram quantities of the receptor. Improved expression of this highly challenging protein can improve research and understanding of the human dopamine D2L receptor. PMID:23424095

  19. G-protein coupled receptors of the renin-angiotensin system: new targets against breast cancer?

    OpenAIRE

    Rodrigues-Ferreira, Sylvie; Nahmias, Clara

    2015-01-01

    G-protein coupled receptors (GPCRs) constitute the largest family of membrane receptors, with high potential for drug discovery. These receptors can be activated by a panel of different ligands including ions, hormones, small molecules, and vasoactive peptides. Among those, angiotensins [angiotensin II (AngII) and angiotensin 1–7] are the major biologically active products of the classical and alternative renin-angiotensin system (RAS). These peptides bind and activate three different subtype...

  20. Mechanisms Governing the Activation and Trafficking of Yeast G Protein-coupled Receptors

    OpenAIRE

    Stefan, Christopher J.; Overton, Mark C.; Blumer, Kendall J.

    1998-01-01

    We have addressed the mechanisms governing the activation and trafficking of G protein-coupled receptors (GPCRs) by analyzing constitutively active mating pheromone receptors (Ste2p and Ste3p) of the yeast Saccharomyces cerevisiae. Substitution of the highly conserved proline residue in transmembrane segment VI of these receptors causes constitutive signaling. This proline residue may facilitate folding of GPCRs into native, inactive conformations, and/or mediate a...

  1. G protein-coupled receptors as new therapeutic targets for type 2 diabetes

    OpenAIRE

    Reimann, Frank; Gribble, Fiona M.

    2015-01-01

    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 ...

  2. 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 als...... head. Furthermore, we identified a gene sequence in the genomic database from the malaria mosquito Anopheles gambiae that very likely codes for a crustacean cardioactive peptide receptor....

  3. Tyrosine-specific protein kinase activity is associated with the purified insulin receptor.

    OpenAIRE

    Kasuga, M.; Fujita-Yamaguchi, Y; Blithe, D L; Kahn, C. R.

    1983-01-01

    Highly purified human placental insulin receptors were obtained by sequential affinity chromatography on wheat germ agglutinin and insulin-agarose. The preparation had an insulin binding capacity of 4,700 pmol/mg of protein approaching theoretical purity. The purified receptor revealed three major bands of Mr 135,000, 95,000, and 52,000 in NaDodSO4/polyacrylamide gel electrophoresis after reduction by dithiothreitol. All three bands were immunoprecipitated by anti-insulin-receptor antibodies....

  4. Activation of the A2A adenosine G-protein-coupled receptor by conformational selection.

    Science.gov (United States)

    Ye, Libin; Van Eps, Ned; Zimmer, Marco; Ernst, Oliver P; Prosser, R Scott

    2016-05-12

    Conformational selection and induced fit are two prevailing mechanisms to explain the molecular basis for ligand-based activation of receptors. G-protein-coupled receptors are the largest class of cell surface receptors and are important drug targets. A molecular understanding of their activation mechanism is critical for drug discovery and design. However, direct evidence that addresses how agonist binding leads to the formation of an active receptor state is scarce. Here we use (19)F nuclear magnetic resonance to quantify the conformational landscape occupied by the adenosine A2A receptor (A2AR), a prototypical class A G-protein-coupled receptor. We find an ensemble of four states in equilibrium: (1) two inactive states in millisecond exchange, consistent with a formed (state S1) and a broken (state S2) salt bridge (known as 'ionic lock') between transmembrane helices 3 and 6; and (2) two active states, S3 and S3', as identified by binding of a G-protein-derived peptide. In contrast to a recent study of the β2-adrenergic receptor, the present approach allowed identification of a second active state for A2AR. Addition of inverse agonist (ZM241385) increases the population of the inactive states, while full agonists (UK432097 or NECA) stabilize the active state, S3', in a manner consistent with conformational selection. In contrast, partial agonist (LUF5834) and an allosteric modulator (HMA) exclusively increase the population of the S3 state. Thus, partial agonism is achieved here by conformational selection of a distinct active state which we predict will have compromised coupling to the G protein. Direct observation of the conformational equilibria of ligand-dependent G-protein-coupled receptor and deduction of the underlying mechanisms of receptor activation will have wide-reaching implications for our understanding of the function of G-protein-coupled receptor in health and disease. PMID:27144352

  5. Sweet Taste Receptor Expressed in Pancreatic β-Cells Activates the Calcium and Cyclic AMP Signaling Systems and Stimulates Insulin Secretion

    OpenAIRE

    Nakagawa, Yuko; Nagasawa, Masahiro; Yamada, Satoko; Hara, Akemi; Mogami, Hideo; Nikolaev, Viacheslav O.; Lohse, Martin J.; Shigemura, Noriatsu; Ninomiya, Yuzo; Kojima, Itaru

    2009-01-01

    Background Sweet taste receptor is expressed in the taste buds and enteroendocrine cells acting as a sugar sensor. We investigated the expression and function of the sweet taste receptor in MIN6 cells and mouse islets. Methodology/Principal Findings The expression of the sweet taste receptor was determined by RT–PCR and immunohistochemistry. Changes in cytoplasmic Ca2+ ([Ca2+]c) and cAMP ([cAMP]c) were monitored in MIN6 cells using fura-2 and Epac1-camps. Activation of protein kinase C was mo...

  6. Summer Camp, July 2016

    CERN Document Server

    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...

  7. Estrogen receptor-associated proteins: possible mediators of hormone-induced transcription.

    Science.gov (United States)

    Halachmi, S; Marden, E; Martin, G; MacKay, H; Abbondanza, C; Brown, M

    1994-06-01

    The estrogen receptor is a transcription factor which, when bound to estradiol, binds DNA and regulates expression of estrogen-responsive genes. A 160-kilodalton estrogen receptor-associated protein, ERAP160, was identified that exhibits estradiol-dependent binding to the receptor. Mutational analysis of the receptor shows that its ability to activate transcription parallels its ability to bind ERAP160. Antiestrogens are unable to promote ERAP160 binding and can block the estrogen-dependent interaction of the receptor and ERAP160 in a dose-dependent manner. This evidence suggests that ERAP160 may mediate estradiol-dependent transcriptional activation by the estrogen receptor. Furthermore, the ability of antiestrogens to block estrogen receptor-ERAP160 complex formation could account for their therapeutic effects in breast cancer. PMID:8197458

  8. Reduced expression of epidermal growth factor receptor related protein in gastric cancer

    OpenAIRE

    Moon, W. S.; Tarnawski, A S; Chai, J.; Yang, J. T.; Majumdar, A.P.N.

    2005-01-01

    Objectives: The recently cloned epidermal growth factor receptor related protein (ERRP) has been proposed to be a negative regulator of the epidermal growth factor receptor (EGFR). Because of the causal involvement of EGFR and its ligands in gastric cancer growth, we investigated expression of ERRP and cell proliferation in human gastric cancer.

  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. 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.

  11. Encountering Child Abuse at Camp.

    Science.gov (United States)

    Durall, John K.

    1997-01-01

    Defines child abuse, including the three categories: physical, sexual, and psychological. Presents characteristics and behaviors of each type of abuse, and long-term effects. Discusses how to handle abuse that occurs at camp, and the effects on the camp. Sidebars present abuse statistics, 15 activities that promote psychological wellness, and 8…

  12. Explaining the Value of Camp.

    Science.gov (United States)

    Chenery, Mary Faeth

    1994-01-01

    Overviews the philosophy and theory of camp experiences and discusses the special benefits of camps, including experiences that can lead to significant life-changing outcomes, sound educational goals, a sense of psychological and physical safety, and helping children to deal with social problems such as the degradation of the environment and human…

  13. Common and distinct mechanisms of activation of rhodopsin and other G protein-coupled receptors

    OpenAIRE

    Sumire Nakamura; Takeshi Itabashi; Daisuke Ogawa; Tetsuji Okada

    2013-01-01

    Detailed and systematic examination of high-resolution structural data is a rational strategy for understanding the function of biological macromolecules. G protein-coupled receptors (GPCRs) are an exceptionally valuable superfamily of proteins for such analysis. The most intriguing question is how a variety of extracellular stimuli evoke structural changes in the intracellular surface of the receptors. The recent active-like crystal structures of GPCRs provide information for uncovering comm...

  14. Gustatory sensory cells express a receptor responsive to protein breakdown products (GPR92)

    OpenAIRE

    Haid, Désirée; Widmayer, Patricia; Voigt, Anja; Chaudhari, Nirupa; Boehm, Ulrich; Breer, Heinz

    2013-01-01

    The ingestion of dietary protein is of vital importance for the maintenance of fundamental physiological processes. The taste modality umami, with its prototype stimulus, glutamate, is considered to signal the protein content of food. Umami was thought to be mediated by the heterodimeric amino acid receptor, T1R1+T1R3. Based on knockout studies, additional umami receptors are likely to exist. In addition to amino acids, certain peptides can also elicit and enhance umami taste suggesting that ...

  15. The arylpiperazine derivatives N-(4-cyanophenylmethyl)-4-(2-diphenyl)-1-piperazinehexanamide and N-benzyl-4-(2-diphenyl)-1-piperazinehexanamide exert a long-lasting inhibition of human serotonin 5-HT7 receptor binding and cAMP signaling.

    Science.gov (United States)

    Atanes, Patricio; Lacivita, Enza; Rodríguez, Javier; Brea, José; Burgueño, Javier; Vela, José Miguel; Cadavid, María Isabel; Loza, María Isabel; Leopoldo, Marcello; Castro, Marián

    2013-12-01

    We performed a detailed in vitro pharmacological characterization of two arylpiperazine derivatives, compound N-(4-cyanophenylmethyl)-4-(2-diphenyl)-1-piperazinehexanamide (LP-211) previously identified as a high-affinity brain penetrant ligand for 5-hydroxytryptamine (serotonin) type 7 (5-HT7) receptors, and its analog N-benzyl-4-(2-diphenyl)-1-piperazinehexanamide (MEL-9). Both ligands exhibited competitive displacement of [(3)H]-(2R)-1-[(3-hydroxyphenyl)sulfonyl]-2-[2-(4-methyl-1-piperidinyl)ethyl]pyrrolidine ([(3)H]-SB-269970) radioligand binding and insurmountable antagonism of 5-carboxamidotryptamine (5-CT)-stimulated cyclic adenosine monophosphate (cAMP) signaling in human embryonic kidney (HEK293) cells stably expressing human 5-HT7 receptors. They also inhibited forskolin-stimulated adenylate cyclase activity in 5-HT7-expressing HEK293 cells but not in the parental cell line. The compounds elicited long-lasting (at least 24 h) concentration-dependent inhibition of radioligand binding at 5-HT7-binding sites in whole-cell radioligand binding assays, after pretreatment of the cells with the compounds and subsequent compound removal. In cAMP assays, pretreatment of cells with the compounds rendered 5-HT7 receptors unresponsive to 5-CT and also rendered 5-HT7-expressing HEK293 cells unresponsive to forskolin. Compound 1-(2-biphenyl)piperazine (RA-7), a known active metabolite of LP-211 present in vivo, was able to partially inhibit 5-HT7 radioligand binding in a long-lasting irreversible manner. Hence, LP-211 and MEL-9 were identified as high-affinity long-acting inhibitors of human 5-HT7 receptor binding and function in cell lines. The detailed in vitro characterization of these two pharmacological tools targeting 5-HT7 receptors may benefit the study of 5-HT7 receptor function and it may lead to the development of novel selective pharmacological tools with defined functional properties at 5-HT7 receptors. PMID:25505568

  16. G protein coupled receptor kinase 2 interacting protein 1 (GIT1) is a novel regulator of mitochondrial biogenesis in heart

    OpenAIRE

    Pang, Jinjiang; Xu, Xiangbin; Getman, Michael R.; Shi, Xi; Belmonte, Stephen L.; Michaloski, Heidi; Mohan, Amy; Blaxall, Burns C.; Berk, Bradford C.

    2011-01-01

    G-protein-coupled receptor (GPCR)-kinase interacting protein-1 (GIT1) is a multi-function scaffold protein. However, little is known about its physiological role in the heart. Here we sought to identify the cardiac function of GIT1. Global GIT1 knockout (KO) mice were generated and exhibited significant cardiac hypertrophy that progressed to heart failure. Electron microscopy revealed that the hearts of GIT1 KO mice demonstrated significant morphological abnormities in mitochondria, including...

  17. Involvement of hippocampal AMPA glutamate receptor changes and the cAMP/protein kinase A/CREB-P signalling pathway in memory consolidation of an avoidance task in rats

    Directory of Open Access Journals (Sweden)

    Bernabeu R.

    1997-01-01

    Full Text Available Training in step-down inhibitory avoidance (0.3-mA footshock is followed by biochemical changes in rat hippocampus that strongly suggest an involvement of quantitative changes in glutamate AMPA receptors, followed by changes in the dopamine D1 receptor/cAMP/protein kinase A (PKA/CREB-P signalling pathway in memory consolidation. AMPA binding to its receptor and levels of the AMPA receptor-specific subunit GluR1 increase in the hippocampus within the first 3 h after training (20-70%. Binding of the specific D1 receptor ligand, SCH23390, and cAMP levels increase within 3 or 6 h after training (30-100%. PKA activity and CREB-P levels show two peaks: a 35-40% increase 0 h after training, and a second increase 3-6 h later (35-60%. The results correlate with pharmacological findings showing an early post-training involvement of AMPA receptors, and a late involvement of the D1/cAMP/PKA/CREB-P pathway in memory consolidation of this task

  18. 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. PMID:24014093

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

    International Nuclear Information System (INIS)

    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 A2A and D2 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 D2R-CFP or A2AR-YFP. These two types of cells were co-cultured, and FRET analysis demonstrated simultaneously positive cells to the D2R-CFP and A2AR-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 A2AR positive MVs were treated with the adenosine A2A receptor agonist CGS-21680. The significant increase in cAMP accumulation clearly demonstrated that A2ARs were functionally competent in target cells. These findings demonstrate that A2A receptors capable of recognizing and decoding extracellular signals can be safely transferred via MVs from source to target cells.

  20. 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.

  1. The multiligand α2-macroglobulin receptor/low density lipoprotein receptor-related protein

    DEFF Research Database (Denmark)

    Gliemann, Jørgen; Nykjær, Anders; Petersen, Claus Munck;

    1994-01-01

    The fusion of separate lines of research has greatly helped in elucidating the function of the giant members of the low density lipoprotein (LDL) receptor (LDLR) supergene family. The cDNA encoding a large protein structurally closely related to LDLR, and hence named LDLR-related protein (LRP), was...... cloned by Herz et al. in 1988.'Evidence was provided demonstrating that LRP can function as a receptor for chylomicron remnants@-migrating very low density lipoproteins (P-VLDL) rich in apolipoprotein E (apoE)?' The a2-macroglobulin (a2M) receptor (a2MR) was purified from rat livep and human p l a~e n t...... from the observation that affinity-purified a2MR/LRP contains a 40-kDa5.8 or 39-kDa6.' protein, designated a2MRAP, in addition to the a2MFULRP a- and P-chains. cDNA cloning" disclosed the 323-residue protein as both the human homologue of mouse heparin binding protein 44 (see reference 11) and...

  2. Whole proteome identification of plant candidate G-protein coupled receptors in Arabidopsis, rice, and poplar: computational prediction and in-vivo protein coupling

    OpenAIRE

    Gookin, Timothy E; Kim, Junhyong; Assmann, Sarah M.

    2008-01-01

    Background The classic paradigm of heterotrimeric G-protein signaling describes a heptahelical, membrane-spanning G-protein coupled receptor that physically interacts with an intracellular Gα subunit of the G-protein heterotrimer to transduce signals. G-protein coupled receptors comprise the largest protein superfamily in metazoa and are physiologically important as they sense highly diverse stimuli and play key roles in human disease. The heterotrimeric G-protein signaling mechanism is conse...

  3. Oligomerization of a Cargo Receptor Directs Protein Sorting into COPII-coated Transport Vesicles

    OpenAIRE

    Sato, Ken; Nakano, Akihiko

    2003-01-01

    Secretory proteins are transported from the endoplasmic reticulum (ER) to the Golgi complex in vesicles coated with coat protein complex II (COPII). The incorporation of certain transport molecules (cargo) into the COPII vesicles is thought to be mediated by cargo receptors. Here we show that Emp47p, a type-I membrane protein, is specifically required for the transport of an integral membrane protein, Emp46p, from the ER. Exit of Emp46p from the ER was saturable and de...

  4. 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

  5. A Computational Modeling and Simulation Approach to Investigate Mechanisms of Subcellular cAMP Compartmentation.

    Science.gov (United States)

    Yang, Pei-Chi; Boras, Britton W; Jeng, Mao-Tsuen; Docken, Steffen S; Lewis, Timothy J; McCulloch, Andrew D; Harvey, Robert D; Clancy, Colleen E

    2016-07-01

    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 temporal

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

    DEFF Research Database (Denmark)

    Martinez, Karen L.; Meyer, Bruno H.; Hovius, Ruud; Lundstrom, Kenneth; Vogel, Horst

    2003-01-01

    of receptor function and in turn for the design and development of novel therapeutic compound. Here we show how ligand-receptor interaction can be investigated in situ with high sensitivity on sensor surfaces by total internal reflection fluorescence (TIRF) measurements. A generally applicable method...... 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...

  7. Brain-specific interleukin-1 receptor accessory protein in sleep regulation

    OpenAIRE

    Taishi, Ping; Davis, Christopher J.; Bayomy, Omar; Zielinski, Mark R.; Liao, Fan; Clinton, James M.; Smith, Dirk E.; Krueger, James M.

    2011-01-01

    Interleukin (IL)-1β is involved in several brain functions, including sleep regulation. It promotes non-rapid eye movement (NREM) sleep via the IL-1 type I receptor. IL-1β/IL-1 receptor complex signaling requires adaptor proteins, e.g., the IL-1 receptor brain-specific accessory protein (AcPb). We have cloned and characterized rat AcPb, which shares substantial homologies with mouse AcPb and, compared with AcP, is preferentially expressed in the brain. Furthermore, rat somatosensory cortex Ac...

  8. Receptor-G Protein Interaction Studied by Bioluminescence Resonance Energy Transfer: Lessons From Protease-Activated Receptor 1

    Directory of Open Access Journals (Sweden)

    Mohammed Akli eAYOUB

    2012-06-01

    Full Text Available Since its development, the bioluminescence resonance energy transfer (BRET approach has been extensively applied to study G protein-coupled receptors (GPCRs in real time and in live cells. One of the major aspects of GPCRs investigated in considerable details is their physical coupling to the heterotrimeric G proteins. As a result, new concepts have emerged, but few questions are still a matter of debate illustrating the complexity of GPCR-G protein interactions and coupling. Here, we summarized the recent advances on our understanding of GPCR-G protein coupling based on BRET approaches and supported by other FRET-based studies. We essentially focused on our recent studies in which we addressed the concept of preassembly versus the agonist-dependent interaction between the protease-activated receptor 1 (PAR1 and its cognate G proteins. We discussed the concept of agonist-induced conformational changes within the preassembled PAR1-G protein complexes as well as the critical question how the multiple coupling of PAR1 with two different G proteins, Gi1 and G12, but also -arrestin 1, can be regulated.

  9. The pro-apoptotic protein death-associated protein 3 (DAP3) interacts with the glucocorticoid receptor and affects the receptor function.

    Science.gov (United States)

    Hulkko, S M; Wakui, H; Zilliacus, J

    2000-08-01

    The yeast two-hybrid system was used to isolate cDNAs encoding proteins that interact with the glucocorticoid receptor (GR) ligand-binding domain in a ligand-dependent manner. One isolated cDNA encoded a fragment of death-associated protein 3 (DAP3), which has been implicated as a positive mediator of apoptosis. In vitro experiments showed that the full-length DAP3 also interacted with GR. The main interaction domain was mapped to the N-terminal region of DAP3 that had previously been shown to function in a dominant-negative fashion, protecting cells from apoptosis. Co-transfection experiments in COS-7 cells showed that DAP3 had a stimulatory effect on the ligand-induced transcriptional activation by GR and also increased the steroid-sensitivity. Furthermore, DAP3 formed a complex with several other nuclear receptors and some basic helix-loop-helix/Per-Arnt-Sim proteins, as well as with heat-shock protein 90 (hsp90) (Arnt is the aryl-hydrocarbon-receptor nuclear translocator, and Per and Sim are the Drosophila proteins Period and Single-minded). The results suggest that DAP3 could have an important role in GR action, possibly by modulating the cytoplasmic GR-hsp90 complex. Since glucocorticoids can induce apoptosis, the pro-apoptotic DAP3 protein may be involved in this function of GR. PMID:10903152

  10. Biased signaling through G-protein-coupled PROKR2 receptors harboring missense mutations.

    Science.gov (United States)

    Sbai, Oualid; Monnier, Carine; Dodé, Catherine; Pin, Jean-Philippe; Hardelin, Jean-Pierre; Rondard, Philippe

    2014-08-01

    Various missense mutations in the gene coding for prokineticin receptor 2 (PROKR2), a G-protein-coupled receptor, have been identified in patients with Kallmann syndrome. However, the functional consequences of these mutations on the different signaling pathways of this receptor have not been studied. We first showed that the wild-type PROKR2 can activate different G-protein subtypes (Gq, Gs, and Gi/o) and recruit β-arrestins in transfected HEK-293 cells. We then examined, for each of these signaling pathways, the effects of 9 mutations that did not significantly impair cell surface targeting or ligand binding of the receptor. Four mutant receptors showing defective Gq signaling (R85C, R85H, R164Q, and V331M) could still recruit β-arrestins on ligand activation, which may cause biased signaling in vivo. Conversely, the R80C receptor could activate the 3 types of G proteins but could not recruit β-arrestins. Finally, the R268C receptor could recruit β-arrestins and activate the Gq and Gs signaling pathways but could not activate the Gi/o signaling pathway. Our results validate the concept that mutations in the genes encoding membrane receptors can bias downstream signaling in various ways, possibly leading to pathogenic and, perhaps in some cases, protective (e.g., R268C) effects. PMID:24830383

  11. Cloning and characterization of a human orphan family C G-protein coupled receptor GPRC5D

    DEFF Research Database (Denmark)

    Bräuner-Osborne, H; Jensen, A A; Sheppard, P O;

    2001-01-01

    Recently three orphan G-protein coupled receptors, RAIG1, GPRC5B and GPRC5C, with homology to members of family C (metabotropic glutamate receptor-like) have been identified. Using the protein sequences of these receptors as queries we identified overlapping expressed sequence tags which were...

  12. 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.

  13. D1 dopamine receptor-induced cyclic AMP-dependent protein kinase phosphorylation and potentiation of striatal glutamate receptors.

    Science.gov (United States)

    Price, C J; Kim, P; Raymond, L A

    1999-12-01

    Dopamine receptor activation regulates cyclic AMP levels and is critically involved in modulating neurotransmission in the striatum. Others have shown that alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA)-type glutamate receptor-mediated current is potentiated by cyclic AMP-dependent protein kinase (PKA) activation. We made whole-cell patch clamp recordings from cultured striatal neurons and tested whether D1-type dopamine receptor activation affected AMPA receptor-mediated currents. After a 5-min exposure to the D1 agonist SKF 81297 (1 microM), kainate-evoked current amplitude was enhanced in approximately 75% of cells to 121+/-2.5% of that recorded prior to addition of drug. This response was inhibited by the D1 antagonist SCH 23390 and mimicked by activators of PKA. Moreover, by western blot analysis using an antibody specific for the phosphorylated PKA site Ser845 of GluR1, we observed a marked increase in phosphorylated GluR1 following a 10-min exposure of striatal neurons to 1 microM SKF 81297. Our data demonstrate that activation of D1-type dopamine receptors on striatal neurons promotes phosphorylation of AMPA receptors by PKA as well as potentiation of current amplitude. These results elucidate one mechanism by which dopamine can modulate neurotransmission in the striatum. PMID:10582604

  14. High-resolution crystal structure of an engineered human beta2-adrenergic G protein-coupled receptor

    DEFF Research Database (Denmark)

    Cherezov, Vadim; Rosenbaum, Daniel M; Hanson, Michael A;

    2007-01-01

    Heterotrimeric guanine nucleotide-binding protein (G protein)-coupled receptors constitute the largest family of eukaryotic signal transduction proteins that communicate across the membrane. We report the crystal structure of a human beta2-adrenergic receptor-T4 lysozyme fusion protein bound to t...... very similar to that of retinal in rhodopsin, structural differences in the ligand-binding site and other regions highlight the challenges in using rhodopsin as a template model for this large receptor family....

  15. 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.; Frimurer, T.M.

    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...... screening and to design and optimize drug candidates. However, low sequence identity between receptors, conformational flexibility, and chemical diversity of ligands present an enormous challenge to molecular modeling approaches. It is our hypothesis that rapid Monte-Carlo sampling of protein backbone 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...

  16. The mapping of yeast's G-protein coupled receptor with an atomic force microscope

    Science.gov (United States)

    Takenaka, Musashi; Miyachi, Yusuke; Ishii, Jun; Ogino, Chiaki; Kondo, Akihiko

    2015-03-01

    An atomic force microscope (AFM) can measure the adhesion force between a sample and a cantilever while simultaneously applying a rupture force during the imaging of a sample. An AFM should be useful in targeting specific proteins on a cell surface. The present study proposes the use of an AFM to measure the adhesion force between targeting receptors and their ligands, and to map the targeting receptors. In this study, Ste2p, one of the G protein-coupled receptors (GPCRs), was chosen as the target receptor. The specific force between Ste2p on a yeast cell surface and a cantilever modified with its ligand, α-factor, was measured and found to be approximately 250 pN. In addition, through continuous measuring of the cell surface, a mapping of the receptors on the cell surface could be performed, which indicated the differences in the Ste2p expression levels. Therefore, the proposed AFM system is accurate for cell diagnosis.

  17. 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...... characterized the G protein-coupled receptors expressed in gastric Sst-RFP-positive cells and probed their effects on SST secretion in primary cell cultures. Surprisingly, besides SST, amylin and PYY were also highly enriched in the SST cells. Several receptors found to regulate SST secretion were highly...... receptor were all positive regulators. 4) The neuropeptide receptors for calcitonin gene-related peptide, adrenomedullin, and vasoactive intestinal peptide acted as positive regulators, no effect was observed using galanin and nociceptin although transcripts for the corresponding receptors appeared highly...

  18. Basic Pharmacological and Structural Evidence for Class A G-Protein-Coupled Receptor Heteromerization

    Science.gov (United States)

    Franco, Rafael; Martínez-Pinilla, Eva; Lanciego, José L.; Navarro, Gemma

    2016-01-01

    Cell membrane receptors rarely work on isolation, often they form oligomeric complexes with other receptor molecules and they may directly interact with different proteins of the signal transduction machinery. For a variety of reasons, rhodopsin-like class A G-protein-coupled receptors (GPCRs) seem an exception to the general rule of receptor–receptor direct interaction. In fact, controversy surrounds their potential to form homo- hetero-dimers/oligomers with other class A GPCRs; in a sense, the field is going backward instead of forward. This review focuses on the convergent, complementary and telling evidence showing that homo- and heteromers of class A GPCRs exist in transfected cells and, more importantly, in natural sources. It is time to decide between questioning the occurrence of heteromers or, alternatively, facing the vast scientific and technical challenges that class A receptor-dimer/oligomer existence pose to Pharmacology and to Drug Discovery. PMID:27065866

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

    OpenAIRE

    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 environment, which subsequently results in the initiation of downstream signaling (Kobe and Kajava, 2001; Torii, 2005). The membrane-associated receptors often form in multimeric complexes that con...

  20. Identification of nucleolin as new ErbB receptors- interacting protein.

    Directory of Open Access Journals (Sweden)

    Ayelet Di Segni

    Full Text Available BACKGROUND: The ErbB receptor tyrosine kinases are major contributors to malignant transformation. These receptors are frequently overexpressed in a variety of human carcinomas. The role of the ErbB receptors and their ligands in carcinomas and the mechanism by which their overexpression leads to cancer development is still unclear. Ligand binding to specific ErbB receptor is followed by receptor dimerization, phosphorylation and recruitment of SH2 containing cytoplasmic proteins, which initiate the cascade of signaling events. Nevertheless, increasing data suggest that there are non-phosphorylated receptor-substrate interactions that may affect ErbB-mediated responses. METHODOLOGY/PRINCIPAL FINDINGS: In the present study, using GST-ErbB4 fusion protein pull down assay and mass spectroscopic analysis, we have found the ErbB receptors interact with nucleolin via their cytoplasmic tail. Nucleolin is a ubiquitous, nonhistone, nucleolar, multifunctional phosphoprotein that is also overexpressed in cancer cells. Our results demonstrate that overexpression of ErbB1 and nucleolin may lead to receptor dimerization, phosphorylation and to anchorage independent growth. CONCLUSIONS/SIGNIFICANCE: The oncogenic potential of ErbB depends on receptor levels and activation. Our results suggest that nucleolin may affect ErbB dimerization and activation leading to enhanced cell growth.

  1. Eosinophil viability is increased by acidic pH in a cAMP- and GPR65-dependent manner

    OpenAIRE

    Kottyan, Leah C.; Collier, Ann R.; Cao, Khanh H.; Niese, Kathryn A; Hedgebeth, Megan; Radu, Caius G.; Owen N Witte; Gurjit K Khurana Hershey; Rothenberg, Marc E.; Zimmermann, Nives

    2009-01-01

    The microenvironment of the lung in asthma is acidic, yet the effect of acidity on inflammatory cells has not been well established. We now demonstrate that acidity inhibits eosinophil apoptosis and increases cellular viability in a dose-dependent manner between pH 7.5 and 6.0. Notably, acidity induced eosinophil cyclic adenosine 5′-monophosphate (cAMP) production and enhanced cellular viability in an adenylate cyclase–dependent manner. Furthermore, we identify G protein-coupled receptor 65 (...

  2. 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

  3. 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

    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...... GPCRs and highlight the presence within GASPs of a novel protein-protein interaction motif that might represent a new target to investigate the involvement of GASPs in the modulation of the activity of GPCRs.......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...

  4. Mechanism of inhibition of growth hormone receptor signaling by suppressor of cytokine signaling proteins

    DEFF Research Database (Denmark)

    Hansen, J A; Lindberg, K; Hilton, D J; Nielsen, Jens Høiriis; Billestrup, N

    1999-01-01

    In this study we have investigated the role of suppressor of cytokine signaling (SOCS) proteins in GH receptor-mediated signaling. GH-induced transcription was inhibited by SOCS-1 and SOCS-3, while SOCS-2 and cytokine inducible SH2-containing protein (CIS) had no effect By using chimeric SOCS pro...

  5. Computational Exploration of a Protein Receptor Binding Space with Student Proposed Peptide Ligands

    Science.gov (United States)

    King, Matthew D.; Phillips, Paul; Turner, Matthew W.; Katz, Michael; Lew, Sarah; Bradburn, Sarah; Andersen, Tim; McDougal, Owen M.

    2016-01-01

    Computational molecular docking is a fast and effective "in silico" method for the analysis of binding between a protein receptor model and a ligand. The visualization and manipulation of protein to ligand binding in three-dimensional space represents a powerful tool in the biochemistry curriculum to enhance student learning. The…

  6. Metabotropic glutamate receptors inhibit microglial glutamate release

    Directory of Open Access Journals (Sweden)

    Gary Guo Li

    2012-08-01

    Full Text Available Pro-inflammatory stimuli evoke an export of glutamate from microglia that is sufficient to contribute to excitotoxicity in neighbouring neurons. Since microglia also express various glutamate receptors themselves, we were interested in the potential feedback of glutamate on this system. Several agonists of mGluRs (metabotropic glutamate receptors were applied to primary rat microglia, and the export of glutamate into their culture medium was evoked by LPS (lipopolysaccharide. Agonists of group-II and -III mGluR ACPD [(1S,3R-1-aminocyclopentane-1,3-dicarboxylic acid] and L-AP4 [L-(+-2-amino-4-phosphonobutyric acid] were both capable of completely blocking the glutamate export without interfering with the production of NO (nitric oxide; the group-I agonist tADA (trans-azetidine-2,4-dicarboxylic acid was ineffective. Consistent with the possibility of feedback, inhibition of mGluR by MSPG [(R,S-α-2-methyl-4sulfonophenylglycine] potentiated glutamate export. As the group-II and -III mGluR are coupled to Gαi-containing G-proteins and the inhibition of adenylate cyclase, we explored the role of cAMP in this effect. Inhibition of cAMP-dependent protein kinase [also known as protein kinase A (PKA] by H89 mimicked the effect of ACPD, and the mGluR agonist had its actions reversed by artificially sustaining cAMP through the PDE (phosphodiesterase inhibitor IBMX (isobutylmethylxanthine or the cAMP mimetic dbcAMP (dibutyryl cAMP. These data indicate that mGluR activation attenuates a potentially neurotoxic export of glutamate from activated microglia and implicate cAMP as a contributor to this aspect of microglial action.

  7. Involvement of protein kinases on the upregulation of endothelin receptors in rat basilar and mesenteric arteries

    DEFF Research Database (Denmark)

    Jamali, Roya; Edvinsson, Lars

    2006-01-01

    protein kinases (c-Jun N-terminal kinase [JNK], protein kinase C [PKC], and extracellular signal-regulated kinase [ERK1/2]) in ET(B) receptor upregulation after organ culture. Rat basilar and mesenteric arteries were incubated for 24 hrs in Dulbecco's modified Eagle's medium (DMEM) with or without the PKC...... were determined with a real-time polymerase chain reaction (PCR). The cellular localization and protein level of ET(B) receptors were evaluated by immunohistochemistry. The PKC and ERK1/2 inhibitors attenuated the contraction induced by S6c in the basilar arteries more than in the mesenteric arteries...

  8. 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......-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...

  9. delta-Opioid receptors exhibit high efficiency when activating trimeric G proteins in membrane domains.

    Science.gov (United States)

    Bourova, Lenka; Kostrnova, Alexandra; Hejnova, Lucie; Moravcova, Zuzana; Moon, Hyo-Eun; Novotny, Jiri; Milligan, Graeme; Svoboda, Petr

    2003-04-01

    Low-density membrane fragments (domains) were separated from the bulk of plasma membranes of human embryonic kidney (HEK)293 cells expressing a delta-opioid (DOP) receptor-Gi1alpha fusion protein by drastic homogenization and flotation on equilibrium sucrose density gradients. The functional activity of trimeric G proteins and capacity of the DOP receptor to stimulate both the fusion protein-linked Gi1alpha and endogenous pertussis-toxin sensitive G proteins was measured as d-Ala2, d-Leu5-enkephalin stimulated high-affinity GTPase or guanosine-5'-[gamma-35S]triphosphate ([35S]GTPgammaS) binding. The maximum d-Ala2-d-Leu5 enkephalin (DADLE)-stimulated GTPase was two times higher in low-density membrane fragments than in bulk of plasma membranes; 58 and 27 pmol/mg/min, respectively. The same difference was obtained for [35S]GTPgammaS binding. Contrarily, the low-density domains contained no more than half the DOP receptor binding sites (Bmax = 6.6 pmol/mg versus 13.6 pmol/mg). Thus, when corrected for expression levels of the receptor, low-density domains exhibited four times higher agonist-stimulated GTPase and [35S]GTPgammaS binding than the bulk plasma membranes. The regulator of G protein signaling RGS1, enhanced further the G protein functional activity but did not remove the difference between domain-bound and plasma membrane pools of G protein. The potency of the agonist in functional studies and the affinity of specific [3H]DADLE binding to the receptor were, however, the same in both types of membranes - EC50 = 4.5 +/- 0.1 x 10(-8) and 3.2 +/- 1.4 x 10(-8) m for GTPase; Kd = 1.2 +/- 0.1 and 1.3 +/- 0.1 nm for [3H]DADLE radioligand binding assay. Similar results were obtained when sodium bicarbonate was used for alkaline isolation of membrane domains. By contrast, detergent-insensitive membrane domains isolated following treatment of cells with Triton X100 exhibited no DADLE-stimulated GTPase or GTPgammaS binding. Functional coupling between the DOP receptor

  10. Role of Cbl-associated protein/ponsin in receptor tyrosine kinase signaling and cell adhesion

    Directory of Open Access Journals (Sweden)

    Ritva Tikkanen

    2012-10-01

    Full Text Available The Cbl-associated protein/ponsin (CAP is an adaptor protein that contains a so-called Sorbin homology (SoHo domain and three Src homology 3 (SH3 domains which are engaged in diverse protein-protein interactions. CAP has been shown to function in the regulation of the actin cytoskeleton and cell adhesion and to be involved in the differentiation of muscle cells and adipocytes. In addition, it participates in signaling pathways through several receptor tyrosine kinases such as insulin and neurotrophin receptors. In the last couple of years, several studies have shed light on the details of these processes and identified novel interaction partners of CAP. In this review, we summarize these recent findings and provide an overview on the function of CAP especially in cell adhesion and membrane receptor signaling.

  11. Comparison of work motivation in camp supervisors and camp counselors in Greek private camps

    Directory of Open Access Journals (Sweden)

    George Costa

    2008-06-01

    Full Text Available The purpose of this study was twofold. First, the study sought to better understand the work motivators that led camp supervisors and counselors to accept their job. Second, the study sought to better understand the ranking and rating of 20 work motivators from supervisors and camp counselors. Responders to the research questionnaire (n=121 were camp supervisory staff and counselors, age 15-55, working in seven private camps in Greece. Two instruments were used to collect data. The first instrument collected demographic data while the second instrument focused on ranking and rating 20 work motivators. The study suggested that Herzberg's theory (Motivator / Hygiene does not apply on a full scale. The results suggested that supervisory staff indicated good working conditions, meeting other people and carrying out personal growth are important. In addition, they don't like travel and don't consider working in a camp, as a stable job. The camp counselors want to have fun in their job and the opportunity to work with youth. They also don't consider working in a camp, as a stable job and they don't accept the responsibility in proportion to their position.

  12. Putative hAPN receptor binding sites in SARS_CoV spike protein

    Institute of Scientific and Technical Information of China (English)

    YUXiao-Jing; LUOCheng; LinJian-Cheng; HAOPei; HEYou-Yu; GUOZong-Ming; QINLei; SUJiong; LIUBo-Shu; HUANGYin; NANPeng; LIChuan-Song; XIONGBin; LUOXiao-Min; ZHAOGuo-Ping; PEIGang; CHENKai-Xian; SHENXu; SHENJian-Hua; ZOUJian-Ping; HEWei-Zhong; SHITie-Liu; ZHONGYang; JIANGHua-Liang; LIYi-Xue

    2003-01-01

    AIM:To obtain the information of ligand-receptor binding between thd S protein of SARS_CoV and CD13, identify the possible interacting domains or motifs related to binding sites, and provide clues for studying the functions of SARS proteins and designing anti-SARS drugs and vaccines. METHODS: On the basis of comparative genomics, the homology search, phylogenetic analyses, and multi-sequence alignment were used to predict CD13 related interacting domains and binding sites sites in the S protein of SARS_CoV. Molecular modeling and docking simulation methods were employed to address the interaction feature between CD13 and S protein of SARS_CoV in validating the bioinformatics predictions. RESULTS:Possible binding sites in the SARS_CoV S protein to CD13 have been mapped out by using bioinformatics analysis tools. The binding for one protein-protein interaction pair (D757-R761 motif of the SARS_CoV S protein to P585-A653 domain of CD13) has been simulated by molecular modeling and docking simulation methods. CONCLUSION:CD13 may be a possible receptor of the SARS_CoV S protein which may be associated with the SARS infection. This study also provides a possible strategy for mapping the possible binding receptors of the proteins in a genome.

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

    DEFF Research Database (Denmark)

    Wands, Amberlyn M; Fujita, Akiko; McCombs, Janet E; Cervin, Jakob; Dedic, Benjamin; Rodriguez, Andrea C; Nischan, Nicole; Bond, Michelle R; Mettlen, Marcel; Trudgian, David C; Lemoff, Andrew; Quiding-Järbrink, Marianne; Gustavsson, Bengt; Steentoft, Catharina; Clausen, Henrik; Mirzaei, Hamid; Teneberg, Susann; Yrlid, Ulf; Kohler, Jennifer J

    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...... report that CTB binds cell surface glycoproteins. Relative contributions of gangliosides and glycoproteins to CTB binding depend on cell type, and CTB binds primarily to glycoproteins in colonic epithelial cell lines. Using a metabolically incorporated photocrosslinking sugar, we identified one CTB...

  14. Visualizing G protein-coupled receptors in action through confocal microscopy techniques.

    Science.gov (United States)

    Castillo-Badillo, Jean A; Cabrera-Wrooman, Alejandro; García-Sáinz, J Adolfo

    2014-05-01

    G protein-coupled receptors constitute one of the most abundant entities in cellular communication. Elucidation of their structure and function as well as of their regulation began 30-40 years ago and the advance has markedly increased during the last 15 years. They participate in a plethora of cell functions such as regulation of metabolic fluxes, contraction, secretion, differentiation, or proliferation, and in essentially all activities of our organism; these receptors are targets of a large proportion of prescribed and illegal drugs. Fluorescence techniques have been used to study receptors for many years. The experimental result was usually a two-dimensional (2D) micrograph. Today, the result can be a spatiotemporal (four-dimensional, 4D) movie. Advances in microscopy, fluorescent protein design, and computer-assisted analysis have been of great importance to increase our knowledge on receptor regulation and function and create opportunities for future research. In this review we briefly depict the state of the art of the G protein-coupled receptor field and the methodologies used to study G protein-coupled receptor location, trafficking, dimerization, and other types of receptor-protein interaction. Fluorescence techniques now permit the capture of receptor images with high resolution and, together with a variety of fluorescent dyes that color organelles (such as the plasma membrane or the nucleus) or the cytoskeleton, allow researchers to obtain a much clearer idea of what is taking place at the cellular level. These developments are changing the way we explore cell communication and signal transduction, permitting deeper understanding of the physiological and pathophysiological processes. PMID:24751328

  15. CD46 Measles Virus Receptor Polymorphisms Influence Receptor Protein Expression and Primary Measles Vaccine Responses in Naive Australian Children

    OpenAIRE

    Clifford, Holly D.; Hayden, Catherine M.; Khoo, Siew-Kim; Zhang, Guicheng; Le Souëf, Peter N.; Richmond, Peter

    2012-01-01

    Despite the availability of measles vaccines, infants continue to die from measles. Measles vaccine responses vary between individuals, and poor immunogenicity is likely to preclude protection against measles. CD46 is a ubiquitously expressed specific receptor for vaccine strains of measles virus. CD46 polymorphisms have not been functionally investigated but may affect CD46 protein expression, which in turn may mediate primary measles antibody responses in infants. In a cohort of children ag...

  16. Registration Day-Camp 2016

    CERN Multimedia

    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.

  17. Protein interactome mining defines melatonin MT1 receptors as integral component of presynaptic protein complexes of neurons.

    Science.gov (United States)

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

    2016-01-01

    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 MT1 and MT2 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, memory, sleep, and also in Alzheimer's disease and depression. However, little is known about the subcellular localization of melatonin receptors and the molecular aspects involved in neuronal functions of melatonin. Identification of protein complexes associated with GPCRs has been shown to be a valid approach to improve our understanding of their function. By combining proteomic and genomic approaches we built an interactome of MT1 and MT2 receptors, which comprises 378 individual proteins. Among the proteins interacting with MT1 , but not with MT2 , we identified several presynaptic proteins, suggesting a potential role of MT1 in neurotransmission. Presynaptic localization of MT1 receptors in the hypothalamus, striatum, and cortex was confirmed by subcellular fractionation experiments and immunofluorescence microscopy. MT1 physically interacts with the voltage-gated calcium channel Cav 2.2 and inhibits Cav 2.2-promoted Ca(2+) entry in an agonist-independent manner. In conclusion, we show that MT1 is part of the presynaptic protein network and negatively regulates Cav 2.2 activity, providing a first hint for potential synaptic functions of MT1. PMID:26514267

  18. Biophysical characterization of G-protein coupled receptor-peptide ligand binding

    OpenAIRE

    Langelaan, David N.; Ngweniform, Pascaline; Rainey, Jan K.

    2011-01-01

    G-protein coupled receptors (GPCRs) are ubiquitous membrane proteins allowing intracellular response to extracellular factors that range from photons of light to small molecules to proteins. Despite extensive exploitation of GRCRs as therapeutic targets, biophysical characterization of GPCR-ligand interactions remains challenging. In this minireview, we focus on techniques which have been successfully employed for structural and biophysical characterization of peptide ligands binding to their...

  19. Lactate Receptor Sites Link Neurotransmission, Neurovascular Coupling, and Brain Energy Metabolism

    DEFF Research Database (Denmark)

    Lauritzen, Knut H; Morland, Cecilie; Puchades, Maja; Holm-Hansen, Signe; Hagelin, Else Marie; Lauritzen, Fredrik; Attramadal, Håvard; Storm-Mathisen, Jon; Gjedde, Albert; Bergersen, Linda H

    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 by physiolog...... GPR81 receptors, can act as a volume transmitter that links neuronal activity, cerebral energy metabolism and energy substrate availability....

  20. Physical and functional interactions of human papillomavirus E2 protein with nuclear receptor coactivators

    International Nuclear Information System (INIS)

    In addition to the human papillomavirus (HPV)-induced immortalization of epithelial cells, which usually requires integration of the viral DNA into the host cell genome, steroid hormone-activated nuclear receptors (NRs) are thought to bind to specific DNA sequences within transcriptional regulatory regions on the long control region to either increase or suppress transcription of dependent genes. In this study, our data suggest that the NR coactivator function of HPV E2 proteins might be mediated through physical and functional interactions with not only NRs but also the NR coactivators GRIP1 (glucocorticoid receptor-interacting protein 1) and Zac1 (zinc-finger protein which regulates apoptosis and cell cycle arrest 1), reciprocally regulating their transactivation activities. GRIP1 and Zac1 both were able to act synergistically with HPV E2 proteins on the E2-, androgen receptor-, and estrogen receptor-dependent transcriptional activation systems. GRIP1 and Zac1 might selectively function with HPV E2 proteins on thyroid receptor- and p53-dependent transcriptional activation, respectively. Hence, the transcriptional function of E2 might be mediated through NRs and NR coactivators to regulate E2-, NR-, and p53-dependent transcriptional activations

  1. Spectral methods for study of the G-protein-coupled receptor rhodopsin. II. Magnetic resonance methods

    Science.gov (United States)

    Struts, A. V.; Barmasov, A. V.; Brown, M. F.

    2016-02-01

    This article continues our review of spectroscopic studies of G-protein-coupled receptors. Magnetic resonance methods including electron paramagnetic resonance (EPR) and nuclear magnetic resonance (NMR) provide specific structural and dynamical data for the protein in conjunction with optical methods (vibrational, electronic spectroscopy) as discussed in the accompanying article. An additional advantage is the opportunity to explore the receptor proteins in the natural membrane lipid environment. Solid-state 2H and 13C NMR methods yield information about both the local structure and dynamics of the cofactor bound to the protein and its light-induced changes. Complementary site-directed spin-labeling studies monitor the structural alterations over larger distances and correspondingly longer time scales. A multiscale reaction mechanism describes how local changes of the retinal cofactor unlock the receptor to initiate large-scale conformational changes of rhodopsin. Activation of the G-protein-coupled receptor involves an ensemble of conformational substates within the rhodopsin manifold that characterize the dynamically active receptor.

  2. cAMP反应元件结合蛋白在中枢神经系统疾病中的作用%The role of cAMP response element binding protein in central nervous system diseases

    Institute of Scientific and Technical Information of China (English)

    刘玲玲; 王力娜; 崔丽丽; 杨蕊; 李敏; 杜炜; 张祥建

    2010-01-01

    The cAMP response element binding protein (CREB) is expressed widely in the central nervous system. It is involved extensively in the pathophysiological processes, such as the cell structure, signal transduction, genetic transcription, synaptic transmission, and metabolism by regulating its downstream genes transcription. CREB is not only involved in the process of normal development of the brain, but also closely correlated with the central nervous system diseases, including ischemic stroke, Alzheimer's disease, Huntington's disease and drug addiction.%cAMP反应元件结合蛋白(cAMP-response element-binding protein,CREB)在中枢神经系统表达广泛,通过对其下游基冈的转录调控,广泛参与细胞结构、信号转导、基因转录、突触传递以及代谢等病理牛理学过程.CREB不仅参与脑的正常发育过程,而且还与缺血性卒中、阿尔茨海默病、亨廷顿病和药物成瘾等中枢神经系统疾病密切相关.

  3. 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

  4. 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.

  5. Insights into the structural biology of G-protein coupled receptors impacts drug design for central nervous system neurodegenerative processes

    OpenAIRE

    Dalet, Farfán-García Eunice; Guadalupe, Trujillo-Ferrara José; María del Carmen, Castillo-Hernández; Humberto, Guerra-Araiza Christian; Antonio, Soriano-Ursúa Marvin

    2013-01-01

    In the last few years, there have been important new insights into the structural biology of G-protein coupled receptors. It is now known that allosteric binding sites are involved in the affinity and selectivity of ligands for G-protein coupled receptors, and that signaling by these receptors involves both G-protein dependent and independent pathways. The present review outlines the physiological and pharmacological implications of this perspective for the design of new drugs to treat disord...

  6. Function of G-Protein-Coupled Estrogen Receptor-1 in Reproductive System Tumors

    Science.gov (United States)

    Qian, Hongyan; Xuan, Jingxiu; Liu, Yuan; Shi, Guixiu

    2016-01-01

    The G-protein-coupled estrogen receptor-1 (GPER-1), also known as GPR30, is a novel estrogen receptor mediating estrogen receptor signaling in multiple cell types. The progress of estrogen-related cancer is promoted by GPER-1 activation through mitogen-activated protein kinases (MAPK), phosphoinositide 3-kinase (PI3K), and phospholipase C (PLC) signaling pathways. However, this promoting effect of GPER-1 is nonclassic estrogen receptor (ER) dependent manner. In addition, clinical evidences revealed that GPER-1 is associated with estrogen resistance in estrogen-related cancer patients. These give a hint that GPER-1 may be a novel therapeutic target for the estrogen-related cancers. However, preclinical studies also found that GPER-1 activation of its special agonist G-1 inhibits cancer cell proliferation. This review aims to summarize the characteristics and complex functions of GPER-1 in cancers.

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

    DEFF Research Database (Denmark)

    Grundmann, Manuel; Tikhonova, Irina G; Hudson, Brian D; Smith, Nicola J; Mohr, Klaus; Ulven, Trond; Milligan, Graeme; Kenakin, Terry; Kostenis, Evi

    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...... binding mode with transient activation of a first receptor site followed by sustained activation of a second topographically distinct site. We identify 4-CMTB (2-(4-chlorophenyl)-3-methyl-N-(thiazol-2-yl)butanamide), previously classified as a pure allosteric agonist of the free fatty acid receptor 2, as...... mutational 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....

  8. Targeting G protein-coupled receptor kinases (GRKs) in Heart Failure

    OpenAIRE

    Brinks, Henriette; Koch, Walter J

    2010-01-01

    In the human body, over 1000 different G protein-coupled receptors (GPCRs) mediate a broad spectrum of extracellular signals at the plasma membrane, transmitting vital physiological features such as pain, sight, smell, inflammation, heart rate and contractility of muscle cells. Signaling through these receptors is primarily controlled and regulated by a group of kinases, the GPCR kinases (GRKs), of which only seven are known and thus, interference with these common downstream GPCR regulators ...

  9. Large-scale polymorphism discovery in macaque G-protein coupled receptors

    OpenAIRE

    Goswami, Dharmendra B; Ogawa, Lisa M; Ward, Joshua M.; Miller, Gregory M.; Vallender, Eric J.

    2013-01-01

    Background G-protein coupled receptors (GPCRs) play an inordinately large role in human health. Variation in the genes that encode these receptors is associated with numerous disorders across the entire spectrum of disease. GPCRs also represent the single largest class of drug targets and associated pharmacogenetic effects are modulated, in part, by polymorphisms. Recently, non-human primate models have been developed focusing on naturally-occurring, functionally-parallel polymorphisms in can...

  10. Large-scale polymorphism discovery in macaque G-protein coupled receptors

    OpenAIRE

    Goswami, Dharmendra B; Ogawa, Lisa M; Ward, Joshua M.; Miller, Gregory M.; Vallender, Eric J.

    2013-01-01

    Background: G-protein coupled receptors (GPCRs) play an inordinately large role in human health. Variation in the genes that encode these receptors is associated with numerous disorders across the entire spectrum of disease. GPCRs also represent the single largest class of drug targets and associated pharmacogenetic effects are modulated, in part, by polymorphisms. Recently, non-human primate models have been developed focusing on naturally-occurring, functionally-parallel polymorphisms in ca...

  11. Farnesoid X Receptor Inhibits the Transcriptional Activity of Carbohydrate Response Element Binding Protein in Human Hepatocytes

    OpenAIRE

    Caron, Sandrine; Huaman Samanez, Carolina; Dehondt, Hélène; Ploton, Maheul; Briand, Olivier; Lien, Fleur; Dorchies, Emilie; Dumont, Julie; Postic, Catherine; Cariou, Bertrand; Lefebvre, Philippe; Staels, Bart

    2013-01-01

    The glucose-activated transcription factor carbohydrate response element binding protein (ChREBP) induces the expression of hepatic glycolytic and lipogenic genes. The farnesoid X receptor (FXR) is a nuclear bile acid receptor controlling bile acid, lipid, and glucose homeostasis. FXR negatively regulates hepatic glycolysis and lipogenesis in mouse liver. The aim of this study was to determine whether FXR regulates the transcriptional activity of ChREBP in human hepatocytes and to unravel the...

  12. Prenatal cocaine reduces AMPA receptor synaptic expression through hyperphosphorylation of the synaptic anchoring protein GRIP

    OpenAIRE

    Bakshi, Kalindi; Gennaro, Serena; Chan, Christopher Y.; Kosciuk, Mary; Liu, Jingjing; Stucky, Andres; Trenkner, Ekkehart; FRIEDMAN, EITAN; Nagele, Robert G; Wang, Hoau-Yan

    2009-01-01

    Prenatal cocaine exposure produces sustained neurobehavioral and brain synaptic changes closely resembling those of animals with defective alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid-type glutamatergic receptors (AMPARs). We hypothesized that prenatal cocaine exposure attenuates AMPAR signaling by interfering with AMPAR synaptic targeting. AMPAR function is governed by receptor cycling on and off the synaptic membrane through its interaction with GRIP, a PDZ domain protein that i...

  13. Trace amines: Identification of a family of mammalian G protein-coupled receptors

    OpenAIRE

    Borowsky, Beth; Adham, Nika; Jones, Kenneth A.; Raddatz, Rita; Artymyshyn, Roman; Ogozalek, Kristine L.; Durkin, Margaret M.; Lakhlani, Parul P.; Bonini, James A.; Pathirana, Sudam; Boyle, Noel; Pu, Xiaosui; Kouranova, Evguenia; Lichtblau, Harvey; Ochoa, F. Yulina

    2001-01-01

    Tyramine, β-phenylethylamine, tryptamine, and octopamine are biogenic amines present in trace levels in mammalian nervous systems. Although some “trace amines” have clearly defined roles as neurotransmitters in invertebrates, the extent to which they function as true neurotransmitters in vertebrates has remained speculative. Using a degenerate PCR approach, we have identified 15 G protein-coupled receptors (GPCR) from human and rodent tissues. Together with the orphan receptor PNR, these rece...

  14. The Origins of Diversity and Specificity in G Protein-Coupled Receptor Signaling

    OpenAIRE

    Maudsley, Stuart; Martin, Bronwen; Luttrell, Louis M

    2005-01-01

    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 appreciat...

  15. Interactions between Human Liver Fatty Acid Binding Protein and Peroxisome Proliferator Activated Receptor Selective Drugs

    OpenAIRE

    Tony Velkov

    2013-01-01

    Fatty acid binding proteins (FABPs) act as intracellular shuttles for fatty acids as well as lipophilic xenobiotics to the nucleus, where these ligands are released to a group of nuclear receptors called the peroxisome proliferator activated receptors (PPARs). PPAR mediated gene activation is ultimately involved in maintenance of cellular homeostasis through the transcriptional regulation of metabolic enzymes and transporters that target the activating ligand. Here we show that liver- (L-) FA...

  16. Jun Dimerization Protein 2 Functions as a Progesterone Receptor N-Terminal Domain Coactivator

    OpenAIRE

    Wardell, Suzanne E.; Boonyaratanakornkit, Viroj; Adelman, James S.; Aronheim, Ami; Edwards, Dean P.

    2002-01-01

    The progesterone receptor (PR) contains two transcription activation function (AF) domains, constitutive AF-1 in the N terminus and AF-2 in the C terminus. AF-2 activity is mediated by a hormone-dependent interaction with a family of steroid receptor coactivators (SRCs). SRC-1 can also stimulate AF-1 activity through a secondary domain that interacts simultaneously with the primary AF-2 interaction site. Other protein interactions and mechanisms that mediate AF-1 activity are not well defined...

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

    OpenAIRE

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

    2012-01-01

    Abstract Background G-protein-coupled estrogen receptor (GPER/GPR30) was reported to bind 17β-estradiol (E2), tamoxifen, and ICI 182,780 (fulvestrant) and promotes activation of epidermal growth factor receptor (EGFR)-mediated signaling in breast, endometrial and thyroid cancer cells. Although lung adenocarcinomas express estrogen receptors α and β (ERα and ERβ), the expression of GPER in lung cancer has not been investigated. The purpose of this study was to examine the expression of GPER in...

  18. Towards predictive docking at aminergic G-protein coupled receptors

    Czech Academy of Sciences Publication Activity Database

    Jakubík, Jan; El-Fakahany, E. E.; Doležal, Vladimír

    2015-01-01

    Roč. 21, č. 11 (2015), s. 284. ISSN 1610-2940 R&D Projects: GA ČR(CZ) GBP304/12/G069; GA ČR(CZ) GA14-05696S; GA ČR(CZ) GA305/09/0681 Institutional support: RVO:67985823 Keywords : induced-fit docking * pose scoring * molecular dynamics * ligand-receptor interaction Subject RIV: FH - Neurology Impact factor: 1.736, year: 2014

  19. The low density lipoprotein receptor-related protein (LRP) 1 and its function in lung diseases.

    Science.gov (United States)

    Wujak, L; Markart, P; Wygrecka, M

    2016-07-01

    The low density lipoprotein receptor-related protein (LRP) 1 is a ubiquitously expressed, versatile cell surface transmembrane receptor involved in embryonic development and adult tissue homeostasis. LRP1 binds and endocytoses a broad spectrum of over 40 ligands identified thus far, including lipoproteins, extracellular matrix proteins, proteases and protease/inhibitor complexes and growth factors. Interactions with other membrane receptors and intracellular adaptors/scaffolding proteins allow LRP1 to modulate cell migration, survival, proliferation and (trans) differentiation. Because LRP1 displays a wide-range of interactions and activities, its expression and function is temporally and spatially tightly controlled. It is not, therefore, surprising that deregulation of LRP1 production and/or activity is observed in several diseases. In this review, we will systematically examine the evidence for the role of LRP1 in human pathologies placing special emphasis on LRP1-mediated pathogenesis of the lung. PMID:26926950

  20. Cannabinoid receptor 2 expression modulates Gβ(1)γ(2) protein interaction with the activator of G protein signalling 2/dynein light chain protein Tctex-1.

    Science.gov (United States)

    Nagler, Marina; Palkowitsch, Lysann; Rading, Sebastian; Moepps, Barbara; Karsak, Meliha

    2016-01-01

    The activator of G protein signalling AGS2 (Tctex-1) forms protein complexes with Gβγ, and controls cell proliferation by regulating cell cycle progression. A direct interaction of Tctex-1 with various G protein-coupled receptors has been reported. Since the carboxyl terminal portion of CB2 carries a putative Tctex-1 binding motif, we investigated the potential interplay of CB2 and Tctex-1 in the absence and presence of Gβγ. The supposed interaction of cannabinoid receptor CB2 with Tctex-1 and the influence of CB2 on the formation of Tctex-1-Gβγ-complexes were studied by co- and/or immunoprecipitation experiments in transiently transfected HEK293 cells. The analysis on Tctex-1 protein was performed in the absence and presence of the ligands JWH 133, 2-AG, and AM 630, the protein biosynthesis inhibitor cycloheximide or the protein degradation blockers MG132, NH4Cl/leupeptin or bafilomycin. Our results show that CB2 neither directly nor indirectly via Gβγ interacts with Tctex-1, but competes with Tctex-1 in binding to Gβγ. The Tctex-1-Gβγ protein interaction was disrupted by CB2 receptor expression resulting in a release of Tctex-1 from the complex, and its degradation by the proteasome and partly by lysosomes. The decrease in Tctex-1 protein levels is induced by CB2 expression "dose-dependently" and is independent of stimulation by agonist or blocking by an inverse agonist treatment. The results suggest that CB2 receptor expression independent of its activation by agonists is sufficient to competitively disrupt Gβγ-Tctex-1 complexes, and to initiate Tctex-1 degradation. These findings implicate that CB2 receptor expression modifies the stability of intracellular protein complexes by a non-canonical pathway. PMID:26410677

  1. Novel cAMP binding protein-BP (CREBBP) mutation in a girl with Rubinstein-Taybi syndrome, GH deficiency, Arnold Chiari malformation and pituitary hypoplasia

    OpenAIRE

    Marzuillo, Pierluigi; Grandone, Anna; Coppola, Ruggero; Cozzolino, Domenico; Festa, Adalgisa; Messa, Federica; Luongo, Caterina; del Giudice, Emanuele Miraglia; Perrone, Laura

    2013-01-01

    Background Rubinstein-Taybi syndrome (RTS) is a rare autosomal dominant disorder (prevalence 1:125,000) characterised by broad thumbs and halluces, facial dysmorphism, psychomotor development delay, skeletal defects, abnormalities in the posterior fossa and short stature. The known genetic causes are point mutations or deletions of the cAMP-response element binding protein-BP (CREBBP) (50-60% of the cases) and of the homologous gene E1A-binding protein (EP300) (5%). Case presentation We descr...

  2. Novel bioluminescent binding assays for interaction studies of protein/peptide hormones with their receptors.

    Science.gov (United States)

    Liu, Ya-Li; Guo, Zhan-Yun

    2016-05-01

    Protein/peptide hormones are the largest group of endogenous signaling molecules and exert various biological functions by binding to specific cell membrane receptors. To study the interactions between these hormones and their receptors, quantitative ligand-receptor binding assays have been widely used for decades. However, the assays conventionally relied on the use of radioligands, which have some major drawbacks and can only be used in laboratories with a radioactive material license. We recently developed novel bioluminescent binding assays for several protein/peptide hormones using the brightest bioluminescent reporter known to date, nanoluciferase (NanoLuc). The NanoLuc reporter can be either chemically conjugated to an appropriate position, or genetically fused at one terminus, of protein/peptide hormones. Compared to conventional radioligands, these bioluminescent ligands have higher sensitivity, better safety, and longer shelf lives, and thus, represent a novel class of non-radioactive tracers for quantitative receptor binding assays. In the present review, we provide some general considerations and specific examples for setting up the bioluminescent binding assays. Such techniques can be applied to other protein/peptide hormones in future to facilitate their interaction studies with their receptors. PMID:27020777

  3. Predicting the Coupling Specificity of G-protein Coupled Receptors to G-proteins by Support Vector Machines

    Institute of Scientific and Technical Information of China (English)

    Cui-Ping Guan; Zhen-Ran Jiang; Yan-Hong Zhou

    2005-01-01

    G-protein coupled receptors (GPCRs) represent one of the most important classes of drug targets for pharmaceutical industry and play important roles in cellular signal transduction. Predicting the coupling specificity of GPCRs to G-proteins is vital for further understanding the mechanism of signal transduction and the function of the receptors within a cell, which can provide new clues for pharmaceutical research and development. In this study, the features of amino acid compositions and physiochemical properties of the full-length GPCR sequences have been analyzed and extracted. Based on these features, classifiers have been developed to predict the coupling specificity of GPCRs to G-proteins using support vector machines. The testing results show that this method could obtain better prediction accuracy.

  4. Characterization of rabbit ileal receptors for Clostridium difficile toxin A. Evidence for a receptor-coupled G protein

    International Nuclear Information System (INIS)

    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

  5. 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.

  6. 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. PMID:27292106

  7. 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.

  8. Polyamine biosynthesis inhibitors alter protein-protein interactions involving estrogen receptor in MCF-7 breast cancer cells.

    Science.gov (United States)

    Thomas, T; Shah, N; Klinge, C M; Faaland, C A; Adihkarakunnathu, S; Gallo, M A; Thomas, T J

    1999-04-01

    We investigated the effects of polyamine biosynthesis inhibition on the estrogenic signaling pathway of MCF-7 breast cancer cells using a protein-protein interaction system. Estrogen receptor (ER) linked to glutathione-S-transferase (GST) was used to examine the effects of two polyamine biosynthesis inhibitors, difluoromethylornithine (DFMO) and CGP 48664. ER was specifically associated with a 45 kDa protein in control cells. In cells treated with estradiol, nine proteins were associated with ER. Cells treated with polyamine biosynthesis inhibitors in the absence of estradiol retained the binding of their ER with a 45 kDa protein and the ER also showed low-affinity interactions with a number of cellular proteins; however, these associations were decreased by the presence of estradiol and the inhibitors. When samples from the estradiol+DFMO treatment group were incubated with spermidine prior to GST-ER pull down assay, an increased association of several proteins with ER was detected. The intensity of the ER-associated 45 kDa protein increased by 10-fold in the presence of 1000 microM spermidine. These results indicate a specific role for spermidine in ER association of proteins. Western blot analysis of samples eluted from GST-ER showed the presence of chicken ovalbumin upstream promoter-transcription factor, an orphan nuclear receptor, and the endogenous full-length ER. These results show that multiple proteins associate with ER and that the binding of some of these proteins is highly sensitive to intracellular polyamine concentrations. Overall, our results indicate the importance of the polyamine pathway in the gene regulatory function of estradiol in breast cancer cells. PMID:10194516

  9. A comparative approach expands the protein-protein interaction node of the immune receptor XA21 in wheat and rice

    OpenAIRE

    Yang, Baoju; Ruan, Randy; Cantu, Dario; Wang, Xiaodong; Ji, Wanquan; Ronald, Pamela C; Dubcovsky, Jorge

    2013-01-01

    The rice (Oryza sativa) OsXA21 receptor kinase is a well-studied immune receptor that initiates a signal transduction pathway leading to resistance to Xanthomonas oryzae pv. oryzae. Two homologs of OsXA21 were identified in wheat (Triticum aestivum): TaXA21-like1 located in a syntenic region with OsXA21, and TaXA21-like2 located in a non-syntenic region. Proteins encoded by these two wheat genes interact with four wheat orthologs of known OsXA21 interactors. In this study, we screened a wheat...

  10. β2 Adrenergic Receptor Fluorescent Protein Fusions Traffic to the Plasma Membrane and Retain Functionality

    Science.gov (United States)

    Bubnell, Jaclyn; Pfister, Patrick; Sapar, Maria L.; Rogers, Matthew E.; Feinstein, Paul

    2013-01-01

    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. PMID:24086401

  11. Effects of dopamine 1 receptor agonists on the content of cAMP of mesenteric and pulmonary arteries in rat%DA1受体激动剂对大鼠离体肺动脉和肠动脉cAMP生成量的影响

    Institute of Scientific and Technical Information of China (English)

    朱琳; 刘爱华; 赵荣瑞

    2001-01-01

    Aim:To evaluate and compare the effects of DA1 receptor agonist, fenoldopam, on rat pulmonary and mesenteric arteries.Methods:Using radioimmunoassay. The effects of fenoldopam on the cAMP generating system in rat mesenteric and pulmonary arteries were determined. Result:The selective DA1-receptors agonist, fenoldopam, induced a dose-related increases cAMP formation in mesenteric and pulmonary arteries; however, the magnitude of increase in the mesenteric artery was remarkably greater than that in the pulmonary artery. The selective DA1-receptors antagonist, SCH23390, blocked fenoldopam-induced cAMP production, while the selective DA2-receptors antagonist, domperidone, was without effect on the increase of cAMP elicited by fenoldopam. Conclusion:The results showed the existence of DA1-receptors associated with stimulation of AC activity in rat mesenteric and pulmonary arteries. However, there are much fewer receptor sites in the pulmonary artery than in mesenteric artery, suggesting less physioloical importace of such receptors in the pulmonary artery than that in the mesenteric artery.%目的:对比分析多巴胺1(DA1)受体激动剂非诺多泮(FODA)对大鼠离体肺动脉和肠动脉cAMP含量的影响。方法:采用放射免疫测定法,测定FODA对肺动脉和肠系膜动脉cAMP生成量的影响以及DA受体拮抗剂对FODA诱发肠动脉、肺动脉血管cAMP变化的影响。结果:非诺多泮可剂量依赖性增加肠、肺动脉cAMP的生成量,肠动脉cAMP的生成量显著高于肺动脉cAMP的生成量。选择性多巴胺1(DA1)受体阻断剂SCH23390能够阻断非诺多泮所引起的肺动脉和肠动脉cAMP生成量增加,多巴胺2(DA2)受体阻断剂Domperidone则不影响非诺多泮的反应。结论:大鼠肺动脉和肠动脉均存在有刺激腺苷酸环化酶(AC)活性的DA1受体,但肺动脉DA1受体的位点数明显少于肠动脉DA1受体位点数,提示肺动脉DA1受体的生理反应弱于肠动脉。

  12. Gc protein-derived macrophage-activating factor (GcMAF) stimulates cAMP formation in human mononuclear cells and inhibits angiogenesis in chick embryo chorionallantoic membrane assay.

    Science.gov (United States)

    Pacini, Stefania; Morucci, Gabriele; Punzi, Tiziana; Gulisano, Massimo; Ruggiero, Marco

    2011-04-01

    The effects of Gc protein-derived macrophage-activating factor (GcMAF) have been studied in cancer and other conditions where angiogenesis is deregulated. In this study, we demonstrate for the first time that the mitogenic response of human peripheral blood mononuclear cells (PBMCs) to GcMAF was associated with 3'-5'-cyclic adenosine monophosphate (cAMP) formation. The effect was dose dependent, and maximal stimulation was achieved using 0.1 ng/ml. Heparin inhibited the stimulatory effect of GcMAF on PBMCs. In addition, we demonstrate that GcMAF (1 ng/ml) inhibited prostaglandin E(1)- and human breast cancer cell-stimulated angiogenesis in chick embryo chorionallantoic membrane (CAM) assay. Finally, we tested different GcMAF preparations on CAM, and the assay proved to be a reliable, reproducible and inexpensive method to determine the relative potencies of different preparations and their stability; we observed that storage at room temperature for 15 days decreased GcMAF potency by about 50%. These data could prove useful for upcoming clinical trials on GcMAF. PMID:21170647

  13. Garcinol potentiates TRAIL-induced apoptosis through modulation of death receptors and antiapoptotic proteins.

    Science.gov (United States)

    Prasad, Sahdeo; Ravindran, Jayaraj; Sung, Bokyung; Pandey, Manoj K; Aggarwal, Bharat B

    2010-04-01

    Whether garcinol, the active component of Garcinia indica, can modulate the sensitivity of cancer cells to TRAIL, a cytokine currently in phase II clinical trial, was investigated. We found that garcinol potentiated TRAIL-induced apoptosis of cancer cells as indicated by intracellular esterase activity, DNA strand breaks, accumulation of the membrane phospholipid phosphatidylserine, mitochondrial activity, and activation of caspase-8, -9, and -3. We found that garcinol, independent of the cell type, induced both of the TRAIL receptors, death receptor 4 (DR4) and DR5. Garcinol neither induced the receptors on normal cells nor sensitized them to TRAIL. Deletion of DR5 or DR4 by small interfering RNA significantly reduced the apoptosis induced by TRAIL and garcinol. In addition, garcinol downregulated various cell survival proteins including survivin, bcl-2, XIAP, and cFLIP, and induced bid cleavage, bax, and cytochrome c release. Induction of death receptors by garcinol was found to be independent of modulation of CCAAT/enhancer-binding protein-homologous protein, p53, bax, extracellular signal-regulated kinase, or c-Jun-NH(2)-kinase. The effect of garcinol was mediated through the generation of reactive oxygen species, in as much as induction of both death receptors, modulation of antiapoptotic and proapoptotic proteins, and potentiation of TRAIL-induced apoptosis were abolished by N-acetyl cysteine and glutathione. Interestingly, garcinol also converted TRAIL-resistant cells into TRAIL-sensitive cells. Overall, our results indicate that garcinol can potentiate TRAIL-induced apoptosis through upregulation of death receptors and downregulation of antiapoptotic proteins. Mol Cancer Ther; 9(4); 856-68. (c)2010 AACR. PMID:20371723

  14. Human eosinophil major basic protein is an endogenous allosteric antagonist at the inhibitory muscarinic M2 receptor.

    OpenAIRE

    Jacoby, D. B.; Gleich, G J; Fryer, A. D.

    1993-01-01

    The effect of human eosinophil major basic protein (MBP) as well as other eosinophil proteins, on binding of [3H]N-methyl-scopolamine ([3H]NMS: 1 x 10(-10) M) to muscarinic M2 receptors in heart membranes and M3 receptors in submandibular gland membranes was studied. MBP inhibited specific binding of [3H]NMS to M2 receptors but not to M3 receptors. MBP also inhibited atropine-induced dissociation of [3H]NMS-receptor complexes in a dose-dependent fashion, demonstrating that the interaction of ...

  15. Structural heterogeneity of membrane receptors and GTP-binding proteins and its functional consequences for signal transduction

    OpenAIRE

    Boege, Fritz; Neumann, Eberhard; Helmreich, Ernst J. M.

    1991-01-01

    Recent information obtained, mainly by recombinant cDNA technology, on structural heterogeneity of hormone and transmitter receptors, of GTP-binding proteins (G-proteins) and, especially, of G-protein-linked receptors is reviewed and the implications of structural heterogeneity for diversity of hormone and transmitter actions is discussed. For the future, three-dimensional structural analysis of membrane proteins participating in signal transmission and transduction pathways is needed in orde...

  16. "cAMP sponge": a buffer for cyclic adenosine 3', 5'-monophosphate.

    Directory of Open Access Journals (Sweden)

    Konstantinos Lefkimmiatis

    Full Text Available BACKGROUND: While intracellular buffers are widely used to study calcium signaling, no such tool exists for the other major second messenger, cyclic AMP (cAMP. METHODS/PRINCIPAL FINDINGS: Here we describe a genetically encoded buffer for cAMP based on the high-affinity cAMP-binding carboxy-terminus of the regulatory subunit RIbeta of protein kinase A (PKA. Addition of targeting sequences permitted localization of this fragment to the extra-nuclear compartment, while tagging with mCherry allowed quantification of its expression at the single cell level. This construct (named "cAMP sponge" was shown to selectively bind cAMP in vitro. Its expression significantly suppressed agonist-induced cAMP signals and the downstream activation of PKA within the cytosol as measured by FRET-based sensors in single living cells. Point mutations in the cAMP-binding domains of the construct rendered the chimera unable to bind cAMP in vitro or in situ. Cyclic AMP sponge was fruitfully applied to examine feedback regulation of gap junction-mediated transfer of cAMP in epithelial cell couplets. CONCLUSIONS: This newest member of the cAMP toolbox has the potential to reveal unique biological functions of cAMP, including insight into the functional significance of compartmentalized signaling events.

  17. 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…

  18. 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

    discovery is described, and proof-of-concept data from a pilot screen with a CXCR4 assay are presented. This chemokine receptor is a highly relevant drug target which plays an important role in the pathogenesis of inflammatory disease and also has been shown to be a co-receptor for entry of HIV into cells...

  19. Nicotinic acetylcholine receptor α7 subunits with a C2 cytoplasmic loop yellow fluorescent protein insertion form functional receptors

    Institute of Scientific and Technical Information of China (English)

    Teresa A MURRAY; Qiang LIU; Paul WHITEAKER; Jie WU; Ronald J LUKAS

    2009-01-01

    Aim: Several nicotinic acetylcholine receptor (nAChR) subunits have been engineered as fluorescent protein (FP) fusions and exploited to illuminate features of nAChRs. The aim of this work was to create a FP fusion in the nAChR a.7 subunit without compromising formation of functional receptors.Methods: A gene construct was generated to introduce yellow fluorescent protein (YFP), in frame, into the otherwise unaltered, large, second cytoplamsic loop between the third and fourth transmembrane domains of the mouse nAChR al sub-unit (a7Y). SH-EP1 cells were transfected with mouse nAChR wild type a.7 subunits (a.7) or with a7Y subunits, alone or with the chaperone protein, hRJC-3. Receptor function was assessed using whole-cell current recording. Receptor expression was measured with 125I-labeled a-bungarotoxin (I-Bgt) binding, laser scanning confocal microscopy, and total internal reflectance fluorescence (TIRF) microscopy.Results: Whole-cell currents revealed that a7Y nAChRs and al nAChRs were functional with comparable EC50 values for the a7 nAChR-selective agonist, choline, and IC50 values for the a.7 nAChR-selective antagonist, methyllycaconitine. I-Bgt binding was detected only after co-expression with hRIC-3. Confocal microscopy revealed that a7Y had primarily intracel-lular rather than surface expression. TIRF microscopy confirmed that little a7Y localized to the plasma membrane, typical of a7 nAChRs.Conclusion: nAChRs composed as homooligomers of a7Y subunits containing cytoplasmic loop YFP have functional, ligand binding, and trafficking characteristics similar to those of a.7 nAChRs. a7Y nAChRs may be used to elucidate properties of a.7 nAChRs and to identify and develop novel probes for these receptors, perhaps in high-throughput fashion.

  20. Documentation of angiotensin II receptors in glomerular epithelial cells

    Science.gov (United States)

    Sharma, M.; Sharma, R.; Greene, A. S.; McCarthy, E. T.; Savin, V. J.; Cowley, A. W. (Principal Investigator)

    1998-01-01

    Angiotensin II decreases glomerular filtration rate, renal plasma flow, and glomerular capillary hydraulic conductivity. Although angiotensin II receptors have been demonstrated in mesangial cells and proximal tubule cells, the presence of angiotensin II receptors in glomerular epithelial cells has not previously been shown. Previously, we have reported that angiotensin II caused an accumulation of cAMP and a reorganization of the actin cytoskeleton in cultured glomerular epithelial cells. Current studies were conducted to verify the presence of angiotensin II receptors by immunological and non-peptide receptor ligand binding techniques and to ascertain the activation of intracellular signal transduction in glomerular epithelial cells in response to angiotensin II. Confluent monolayer cultures of glomerular epithelial cells were incubated with angiotensin II, with or without losartan and/or PD-123,319 in the medium. Membrane vesicle preparations were obtained by homogenization of washed cells followed by centrifugation. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of membrane proteins followed by multiscreen immunoblotting was used to determine the presence of angiotensin II receptor type 1 (AT1) or type 2 (AT2). Angiotensin II-mediated signal transduction in glomerular epithelial cells was studied by measuring the levels of cAMP, using radioimmunoassay. Results obtained in these experiments showed the presence of both AT1 and AT2 receptor types in glomerular epithelial cells. Angiotensin II was found to cause an accumulation of cAMP in glomerular epithelial cells, which could be prevented only by simultaneous use of losartan and PD-123,319, antagonists for AT1 and AT2, respectively. The presence of both AT1 and AT2 receptors and an increase in cAMP indicate that glomerular epithelial cells respond to angiotensin II in a manner distinct from that of mesangial cells or proximal tubular epithelial cells. Our results suggest that glomerular epithelial

  1. Salt Bridges Overlapping the Gonadotropin-Releasing Hormone Receptor Agonist Binding Site Reveal a Coincidence Detector for G Protein-Coupled Receptor Activation

    OpenAIRE

    Janovick, Jo Ann; Pogozheva, Irina D.; Mosberg, Henry I.; Conn, P. Michael

    2011-01-01

    G protein-coupled receptors (GPCRs) play central roles in most physiological functions, and mutations in them cause heritable diseases. Whereas crystal structures provide details about the structure of GPCRs, there is little information that identifies structural features that permit receptors to pass the cellular quality control system or are involved in transition from the ground state to the ligand-activated state. The gonadotropin-releasing hormone receptor (GnRHR), because of its small s...

  2. Identification of G protein-coupled receptors for Drosophila PRXamide peptides, CCAP, corazonin, and AKH supports a theory of ligand-receptor coevolution

    OpenAIRE

    Park, Yoonseong; KIM, YOUNG-JOON; Adams, Michael E.

    2002-01-01

    G-protein coupled receptors (GPCRs) are ancient, ubiquitous sensors vital to environmental and physiological signaling throughout organismal life. With the publication of the Drosophila genome, numerous “orphan” GPCRs have become available for functional analysis. Here we characterize two groups of GPCRs predicted as receptors for peptides with a C-terminal amino acid sequence motif consisting of −PRXamide (PRXa). Assuming ligand-receptor coevolution, two alternative hypotheses were construct...

  3. 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.

  4. The G protein-coupled receptors in the pufferfish Takifugu rubripes

    OpenAIRE

    Sundar Durai; Kumar Sonu; Sarkar Anita

    2011-01-01

    Abstract Background Guanine protein-coupled receptors (GPCRs) constitute a eukaryotic transmembrane protein family and function as “molecular switches” in the second messenger cascades and are found in all organisms between yeast and humans. They form the single, biggest drug-target family due to their versatility of action and their role in several physiological functions, being active players in detecting the presence of light, a variety of smells and tastes, amino acids, nucleotides, lipid...

  5. Selective Accumulation of Raft-Associated Membrane Protein Lat in T Cell Receptor Signaling Assemblies

    OpenAIRE

    Harder, Thomas; Kuhn, Marina

    2000-01-01

    Activation of T cell antigen receptor (TCR) induces tyrosine phosphorylations that mediate the assembly of signaling protein complexes. Moreover, cholesterol-sphingolipid raft membrane domains have been implicated to play a role in TCR signal transduction. Here, we studied the assembly of TCR with signal transduction proteins and raft markers in plasma membrane subdomains of Jurkat T leukemic cells. We employed a novel method to immunoisolate plasma membrane subfragments that were highly conc...

  6. The cyclic-AMP receptor protein (CRP) regulon in Aggregatibacter actinomycetemcomitans includes leukotoxin

    OpenAIRE

    Feuerbacher, Leigh A.; Burgum, Alex; Kolodrubetz, David

    2011-01-01

    The cyclic-AMP receptor protein (CRP) acts as a global regulatory protein among bacteria. Here, the CRP regulon has been defined in Aggregatibacter actinomycetemcomitans using microarray analysis of A. actinomycetemcomitans strain JP2 wild type cells compared to an isogenic crp deletion mutant. Genes whose expression levels changed at least 2-fold with p ≤ 0.05 were considered significant. Of the 300 genes identified as being CRP-regulated, 139 were CRP-activated, including leukotoxin, with t...

  7. Molecular characterization of receptor binding proteins and immunogens of virulent Treponema pallidum

    OpenAIRE

    1980-01-01

    Receptor binding proteins of Treponema pallidum were identified by incubation of [35S]methionine-labeled, soluble T. pallidum preparations with formaldehyde-fixed HEp-2 cells. Three major treponemal proteins (bands 1--3) that avidly bound to the eucaryotic cell surface were detected by sodium dodecylsulfate-polyacrylamide gel electrophoresis and fluorography. Brief trypsin treatment of HEp-2 cells before formaldehyde fixation reduced the extent of the interaction of these treponemal macromole...

  8. Computing Highly Correlated Positions Using Mutual Information and Graph Theory for G Protein-Coupled Receptors

    OpenAIRE

    Fatakia, Sarosh N.; Costanzi, Stefano; Chow, Carson C.

    2009-01-01

    G protein-coupled receptors (GPCRs) are a superfamily of seven transmembrane-spanning proteins involved in a wide array of physiological functions and are the most common targets of pharmaceuticals. This study aims to identify a cohort or clique of positions that share high mutual information. Using a multiple sequence alignment of the transmembrane (TM) domains, we calculated the mutual information between all inter-TM pairs of aligned positions and ranked the pairs by mutual information. A ...

  9. Domains involved in the specificity of G protein activation in phospholipase C-coupled metabotropic glutamate receptors.

    OpenAIRE

    Pin, J P; Joly, C; Heinemann, S F; Bockaert, J.

    1994-01-01

    G protein-coupled glutamate receptors (mGluR) have recently been characterized. These receptors have seven putative transmembrane domains, but display no sequence homology with the large family of G protein-coupled receptors. They constitute therefore a new family of receptors. Whereas mGluR1 and mGluR5 activate phospholipase C (PLC), mGluR2, mGluR3, mGluR4 and mGluR6 inhibit adenylyl cyclase (AC) activity. The third putative intracellular loop, which determines the G protein specificity in m...

  10. 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.

  11. A comparative study of protein patterns of human estrogen receptor

    Czech Academy of Sciences Publication Activity Database

    Flodrová, Dana; Toporová, L.; Macejová, D.; Laštovičková, Markéta; Brtko, J.; Bobálová, Janette

    2016-01-01

    Roč. 35, - (2016), s. 387-392. ISSN 0231-5882 R&D Projects: GA AV ČR SAV-15-01 Institutional support: RVO:68081715 Keywords : cell line * breast cancer * protein * mass spectrometry Subject RIV: CB - Analytical Chemistry, Separation Impact factor: 1.173, year: 2014

  12. Imaging G protein-coupled receptors while quantifying their ligand-binding free-energy landscape.

    Science.gov (United States)

    Alsteens, David; Pfreundschuh, Moritz; Zhang, Cheng; Spoerri, Patrizia M; Coughlin, Shaun R; Kobilka, Brian K; Müller, Daniel J

    2015-09-01

    Imaging native membrane receptors and testing how they interact with ligands is of fundamental interest in the life sciences but has proven remarkably difficult to accomplish. Here, we introduce an approach that uses force-distance curve-based atomic force microscopy to simultaneously image single native G protein-coupled receptors in membranes and quantify their dynamic binding strength to native and synthetic ligands. We measured kinetic and thermodynamic parameters for individual protease-activated receptor-1 (PAR1) molecules in the absence and presence of antagonists, and these measurements enabled us to describe PAR1's ligand-binding free-energy landscape with high accuracy. Our nanoscopic method opens an avenue to directly image and characterize ligand binding of native membrane receptors. PMID:26167642

  13. Grp78 is involved in retention of mutant low density lipoprotein receptor protein in the endoplasmic reticulum

    DEFF Research Database (Denmark)

    Jørgensen, M M; Jensen, Ole Nørregaard; Holst, H U;

    2000-01-01

    The low density lipoprotein (LDL) receptor is responsible for removing the majority of the LDL cholesterol from the plasma. Mutations in the LDL receptor gene cause the disease familial hypercholesterolemia (FH). Approximately 50% of the mutations in the LDL receptor gene in patients with FH lead...... to receptor proteins that are retained in the endoplasmic reticulum (ER). Misfolding of mutant LDL receptors is a probable cause of this ER retention, resulting in no functional LDL receptors at the cell surface. However, the specific factors and mechanisms responsible for retention of mutant LDL...

  14. Low density lipoprotein receptor-related protein-2/megalin is expressed in oligodendrocytes in the mouse spinal cord white matter

    DEFF Research Database (Denmark)

    Wicher, Grzegorz; Larsson, Mårten; Svenningsen, Åsa Fex; Gyllencreutz, Erika; Rask, Lars; Aldskogius, Håkan

    2006-01-01

    Lipoprotein receptor-related protein-2 (LRP2)/megalin is a member of the low density lipoprotein receptor (LDLR) family, and is essential in absorptive epithelia for endocytosis of lipoproteins, low molecular weight proteins, cholesterol and vitamins, as well as in cellular signaling. Previous st...

  15. Ku proteins function as corepressors to regulate farnesoid X receptor-mediated gene expression

    Energy Technology Data Exchange (ETDEWEB)

    Ohno, Masae; Kunimoto, Masaaki; Nishizuka, Makoto; Osada, Shigehiro [Department of Molecular Biology, Graduate School of Pharmaceutical Sciences, Nagoya City University, 3-1 Tanabe-dori, Mizuho-ku, Nagoya, Aichi 467-8603 (Japan); Imagawa, Masayoshi, E-mail: imagawa@phar.nagoya-cu.ac.jp [Department of Molecular Biology, Graduate School of Pharmaceutical Sciences, Nagoya City University, 3-1 Tanabe-dori, Mizuho-ku, Nagoya, Aichi 467-8603 (Japan)

    2009-12-18

    The farnesoid X receptor (FXR; NR1H4) is a member of the nuclear receptor superfamily and regulates the expression of genes involved in enterohepatic circulation and the metabolism of bile acids. Based on functional analyses, nuclear receptors are divided into regions A-F. To explore the cofactors interacting with FXR, we performed a pull-down assay using GST-fused to the N-terminal A/B region and the C region, which are required for the ligand-independent transactivation and DNA-binding, respectively, of FXR, and nuclear extracts from HeLa cells. We identified DNA-dependent protein kinase catalytic subunit (DNA-PKcs), Ku80, and Ku70 as FXR associated factors. These proteins are known to have an important role in DNA repair, recombination, and transcription. DNA-PKcs mainly interacted with the A/B region of FXR, whereas the Ku proteins interacted with the C region and with the D region (hinge region). Chromatin immunoprecipitation assays revealed that the Ku proteins associated with FXR on the bile salt export pump (BSEP) promoter. Furthermore, we demonstrated that ectopic expression of the Ku proteins decreased the promoter activity and expression of BSEP gene mediated by FXR. These results suggest that the Ku proteins function as corepressors for FXR.

  16. Ku proteins function as corepressors to regulate farnesoid X receptor-mediated gene expression

    International Nuclear Information System (INIS)

    The farnesoid X receptor (FXR; NR1H4) is a member of the nuclear receptor superfamily and regulates the expression of genes involved in enterohepatic circulation and the metabolism of bile acids. Based on functional analyses, nuclear receptors are divided into regions A-F. To explore the cofactors interacting with FXR, we performed a pull-down assay using GST-fused to the N-terminal A/B region and the C region, which are required for the ligand-independent transactivation and DNA-binding, respectively, of FXR, and nuclear extracts from HeLa cells. We identified DNA-dependent protein kinase catalytic subunit (DNA-PKcs), Ku80, and Ku70 as FXR associated factors. These proteins are known to have an important role in DNA repair, recombination, and transcription. DNA-PKcs mainly interacted with the A/B region of FXR, whereas the Ku proteins interacted with the C region and with the D region (hinge region). Chromatin immunoprecipitation assays revealed that the Ku proteins associated with FXR on the bile salt export pump (BSEP) promoter. Furthermore, we demonstrated that ectopic expression of the Ku proteins decreased the promoter activity and expression of BSEP gene mediated by FXR. These results suggest that the Ku proteins function as corepressors for FXR.

  17. 0610009K11Rik, a testis-specific and germ cell nuclear receptor-interacting protein

    International Nuclear Information System (INIS)

    Using an in silico approach, a putative nuclear receptor-interacting protein 0610009K11Rik was identified in mouse testis. We named this gene testis-specific nuclear receptor-interacting protein-1 (Tnrip-1). Tnrip-1 was predominantly expressed in the testis of adult mouse tissues. Expression of Tnrip-1 in the testis was regulated during postnatal development, with robust expression in 14-day-old or older testes. In situ hybridization analyses showed that Tnrip-1 is highly expressed in pachytene spermatocytes and spermatids. Consistent with its mRNA expression, Tnrip-1 protein was detected in adult mouse testes. Immunohistochemical studies showed that Tnrip-1 is a nuclear protein and mainly expressed in pachytene spermatocytes and round spermatids. Moreover, co-immunoprecipitation analyses showed that endogenous Tnrip-1 protein can interact with germ cell nuclear receptor (GCNF) in adult mouse testes. Our results suggest that Tnrip-1 is a testis-specific and GCNF-interacting protein which may be involved in the modulation of GCNF-mediated gene transcription in spermatogenic cells within the testis

  18. A new crystal structure fragment-based pharmacophore method for G protein-coupled receptors

    DEFF Research Database (Denmark)

    Fidom, Kimberley; Isberg, Vignir; Hauser, Alexander Sebastian; Mordalski, Stefan; Lehto, Thomas; Bojarski, Andrzej J; Gloriam, David E

    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 and...... ligands are known. 47% of the class A G protein-coupled receptors can be targeted with at least four-element pharmacophores. The fragment libraries can also be used to grow known ligands or for rotamer refinement of homology models. Researchers can download the complete fragment library or a subset...... 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 for...

  19. 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.

  20. Identification of the first surrogate agonists for the G protein-coupled receptor GPR132

    DEFF Research Database (Denmark)

    Shehata, Mohamed A.; Christensen, Hanna Belcik; Isberg, Vignir;

    2015-01-01

    GPR132 is an orphan class A G protein-coupled receptor. It has been proposed to be activated by protons and to regulate apoptosis, atherosclerosis and inflammation, but these results are still preliminary. In the current work, we designed and screened a focused compound library using a β-arrestin......GPR132 is an orphan class A G protein-coupled receptor. It has been proposed to be activated by protons and to regulate apoptosis, atherosclerosis and inflammation, but these results are still preliminary. In the current work, we designed and screened a focused compound library using a β......-arrestin recruitment assay, and thereby identified the first disclosed surrogate GPR132 agonist 1 with a potency of 3.4 μM. This constitutes the first available pharmacological tool for the in vitro characterization of the orphan receptor GPR132. The testing of 32 analogs furthermore identified a number of compounds...

  1. Structural basis for modulation of a G-protein-coupled receptor by allosteric drugs

    Science.gov (United States)

    Dror, Ron O.; Green, Hillary F.; Valant, Celine; Borhani, David W.; Valcourt, James R.; Pan, Albert C.; Arlow, Daniel H.; Canals, Meritxell; Lane, J. Robert; Rahmani, Raphaël; Baell, Jonathan B.; Sexton, Patrick M.; Christopoulos, Arthur; Shaw, David E.

    2013-11-01

    The design of G-protein-coupled receptor (GPCR) allosteric modulators, an active area of modern pharmaceutical research, has proved challenging because neither the binding modes nor the molecular mechanisms of such drugs are known. Here we determine binding sites, bound conformations and specific drug-receptor interactions for several allosteric modulators of the M2 muscarinic acetylcholine receptor (M2 receptor), a prototypical family A GPCR, using atomic-level simulations in which the modulators spontaneously associate with the receptor. Despite substantial structural diversity, all modulators form cation-π interactions with clusters of aromatic residues in the receptor extracellular vestibule, approximately 15Å from the classical, `orthosteric' ligand-binding site. We validate the observed modulator binding modes through radioligand binding experiments on receptor mutants designed, on the basis of our simulations, either to increase or to decrease modulator affinity. Simulations also revealed mechanisms that contribute to positive and negative allosteric modulation of classical ligand binding, including coupled conformational changes of the two binding sites and electrostatic interactions between ligands in these sites. These observations enabled the design of chemical modifications that substantially alter a modulator's allosteric effects. Our findings thus provide a structural basis for the rational design of allosteric modulators targeting muscarinic and possibly other GPCRs.

  2. Purification and partial characterization of a receptor protein for mouse interferon γ

    International Nuclear Information System (INIS)

    A receptor protein for mouse interferon γ has been purified from solubilized plasma membranes of the mouse monomyelocytic cell line WEHI-3. Sequential wheat germ agglutinin and ligand affinity chromatography of membranes extracted with octyl β-D-glucopyranoside resulted in at least a 680-fold purification of the receptor, as measured by precipitating it in association with liposomes composed of phosphatidylcholine. The purified receptor bound 125I-labeled recombinant mouse interferon γ (rMuIFN-γ) with a Kd of 10 nM, a value comparable to that obtained with isolated membranes, PAGE analysis of radiolabeled (with either 35S or 125I) receptor preparations consistently revealed a major band of 95 kDa. This species was degraged with time to smaller fragments, GR-20, a monoclonal antibody against the receptor, completely inhibited specific binding of 125I-labeled rMuIFN-γ to WEHI-3 cells, blocked the induction of priming by rMuIFN-γ of macrophage-mediated tumor cell killing, removed binding activity for 125I-labeled rMuIFN-γ from solubilized membranes, and immunoprecipitated a single 95-kDa protein from the extract of surface labeled (125I) WEHI-3 cells. Cross-linking of 125I-labeled rMuIFN-γ to its receptor yielded a complex of 125 ± 5 kDa, consistent with the binding of the dimeric form of mouse interferon γ (32 kDa) to a membrane protein of 95 kDa. These data suggest that the receptor for mouse interferon γ is a glycoprotein of 95 kDa

  3. The Membrane Receptor for Plasma Retinol Binding Protein, a New Type of Cell-Surface Receptor

    OpenAIRE

    Sun, Hui; KAWAGUCHI, RIKI

    2011-01-01

    Vitamin A is essential for diverse aspects of life ranging from embryogenesis to the proper functioning of most adult organs. Its derivatives (retinoid) have potent biological activities such as regulating cell growth and differentiation. Plasma retinol binding protein (RBP) is the specific vitamin A carrier protein in the blood that binds to vitamin A with high affinity and delivers it to target organs. A large amount of evidence has accumulated over the past decades supporting the existence...

  4. Orphan G protein receptor GPR55 as an emerging target in cancer therapy and management

    International Nuclear Information System (INIS)

    G protein-coupled receptors (GPCRs) modulate a vast array of cellular processes. The current review gives an overview of the general characteristics of GPCRs and their role in physiological conditions. In addition, it describes the current knowledge of the physiological and pathophysiological functions of GPR55, an orphan GPCR, and how it can be exploited as a therapeutic target to combat various cancers

  5. Differential effect of NMDA and AMPA receptor blockade on protein synthesis in the rat infarct borderzone

    DEFF Research Database (Denmark)

    Christensen, Thomas; Bruhn, T; Frank, L; Diemer, Nils Henrik

    We investigated whether the known neuroprotective effects of two selective glutamate receptor antagonists, the NMDA antagonist MK-801 and the AMPA antagonist NBQX, are reflected in the regional cerebral protein synthesis rates (CPSR) in rats with middle cerebral artery occlusion (MCAO). Rats trea...

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

    DEFF Research Database (Denmark)

    Liebscher, Ines; Ackley, Brian; Araç, Demet;

    2014-01-01

    The class of adhesion G protein-coupled receptors (aGPCRs), with 33 human homologs, is the second largest family of GPCRs. In addition to a seven-transmembrane α-helix-a structural feature of all GPCRs-the class of aGPCRs is characterized by the presence of a large N-terminal extracellular region...

  7. International Union of Basic and Clinical Pharmacology. XCIV. Adhesion G protein-coupled receptors

    DEFF Research Database (Denmark)

    Hamann, Jörg; Aust, Gabriela; Araç, Demet;

    2015-01-01

    The Adhesion family forms a large branch of the pharmacologically important superfamily of G protein-coupled receptors (GPCRs). As Adhesion GPCRs increasingly receive attention from a wide spectrum of biomedical fields, the Adhesion GPCR Consortium, together with the International Union of Basic ...

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

    DEFF Research Database (Denmark)

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

    2012-01-01

    Human and animal studies suggest that estrogens are involved in the processing of nociceptive sensory information and analgesic responses in the central nervous system. Rapid pronociceptive estrogenic effects have been reported, some of which likely involve G protein-coupled estrogen receptor (GPER...

  9. Nuclear translocation of EGF receptor regulated by Epstein-Barr virus encoded latent membrane protein 1

    Institute of Scientific and Technical Information of China (English)

    TAO; Yongguang; SONG; Xin; TAN; Yunnian; LIN; Xiaofeng; ZH

    2004-01-01

    Epstein-Barr virus (EBV) encoded latent membrane protein 1 (LMP1) is considered to be the major oncogenic protein of EBV encoded proteins, and also it has always been the core of the oncogenic mechanism of EBV. Traditional receptor theory demonstrates that cell surface receptors exert biological functions on the membrane, which neither enter into the nucleus nor directly affect the transcription of the target genes. But, advanced studies on nuclear translocation of the epidermal growth factor receptor (EGFR) family have greatly developed our knowledge of the biological function of cell surface receptors. In this study, we used Tet-on LMP1 HNE2 cell line as a cell model, which is a dual-stable LMP1 integrated NPC cell line and the expression of LMP1 in which could be regulated by Tet system. We found that LMP1 could regulate the nuclear translocation of EGFR in a dose-dependent manner from both quantitative and qualitative levels through the Western blot analysis and the immunofluorescent analysis with a laser scanning confocal microscope. We further demonstrated that the nuclear localization sequence of EGFR played some roles in the location of the protein within the nucleus under LMP1 regulation, and the nuclear accumulation of EGFR regulated by LMP1 was in a ligand-independent manner. These findings provide a novel view that the regulation of LMP1 on the nuclear translocation of EGFR is critical for the process of nasopharyngeal carcinoma.

  10. Problem-Solving Test: Vitellogenin and Vitellogenin-Receptor Recognition: An Example of Protein Interaction

    Science.gov (United States)

    Hernandez-Cortes, Patricia

    2012-01-01

    Vitellogenin (Vtg) is a lipid transfer protein that carries yolk to the ovary. The vitellogenin receptor (VtgR) mediates the uptake of Vtg into the oocyte of oviparous animals; its structure includes eight ligand-binding repeats (LBR). The binding site of VtgR and Vtg and the location of the interaction within the molecules are at these LBR.…

  11. Identification of the first surrogate agonists for the G protein-coupled receptor GPR132

    DEFF Research Database (Denmark)

    Shehata, Mohamed A.; Christensen, Hanna Belcik; Isberg, Vignir; Pedersen, Daniel Sejer; Bender, Andreas; Bräuner-Osborne, Hans; Gloriam, David E.

    2015-01-01

    GPR132 is an orphan class A G protein-coupled receptor. It has been proposed to be activated by protons and to regulate apoptosis, atherosclerosis and inflammation, but these results are still preliminary. In the current work, we designed and screened a focused compound library using a β...

  12. 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

    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...

  13. Beta-arrestin-mediated activation of MAPK by inverse agonists reveals distinct active conformations for G protein-coupled receptors.

    OpenAIRE

    Azzi, Mounia; Pascale G. Charest; Angers, Stéphane; Rousseau, Guy; Kohout, Trudy; Bouvier, Michel; Piñeyro, Graciela

    2003-01-01

    It is becoming increasingly clear that signaling via G protein-coupled receptors is a diverse phenomenon involving receptor interaction with a variety of signaling partners. Despite this diversity, receptor ligands are commonly classified only according to their ability to modify G protein-dependent signaling. Here we show that beta2AR ligands like ICI118551 and propranolol, which are inverse agonists for Gs-stimulated adenylyl cyclase, induce partial agonist responses for the mitogen-activat...

  14. Intracellular adenosine 3',5'-phosphate formation is essential for down-regulation of surface adenosine 3',5'-phosphate receptors in Dictyostelium

    OpenAIRE

    Van Haastert, Peter J. M.

    1994-01-01

    Dictyostelium discoideum cells contain cell surface cyclic AMP (cAMP) receptors that bind cAMP as a first messenger and intracellular cAMP receptors that bind cAMP as a second messenger. Prolonged incubation of Dictyostelium cells with cAMP induces a sequential process of phosphorylation, sequestration and down-regulation of the surface receptors. The role of intracellular cAMP in down-regulation of surface receptors was investigated. Down-regulation of receptors does not occur under conditio...

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

    OpenAIRE

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

    2011-01-01

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

  16. Prunetin signals via G-protein-coupled receptor, GPR30(GPER1): Stimulation of adenylyl cyclase and cAMP-mediated activation of MAPK signaling induces Runx2 expression in osteoblasts to promote bone regeneration.

    Science.gov (United States)

    Khan, Kainat; Pal, Subhashis; Yadav, Manisha; Maurya, Rakesh; Trivedi, Arun Kumar; Sanyal, Sabyasachi; Chattopadhyay, Naibedya

    2015-12-01

    Prunetin is found in red clover and fruit of Prunus avium (red cherry). The effect of prunetin on osteoblast function, its mode of action and bone regeneration in vivo were investigated. Cultures of primary osteoblasts, osteoblastic cell line and HEK293T cells were used for various in vitro studies. Adult female rats received drill-hole injury at the femur diaphysis to assess the bone regenerative effect of prunetin. Prunetin at 10nM significantly (a) increased proliferation and differentiation of primary cultures of osteoblasts harvested from rats and (b) promoted formation of mineralized nodules by bone marrow stromal/osteoprogenitor cells. At this concentration, prunetin did not activate any of the two nuclear estrogen receptors (α and β). However, prunetin triggered signaling via a G-protein-coupled receptor, GPR30/GPER1, and enhanced cAMP levels in osteoblasts. G15, a selective GPR30 antagonist, abolished prunetin-induced increases in osteoblast proliferation, differentiation and intracellular cAMP. In osteoblasts, prunetin up-regulated runt-related transcription factor 2 (Runx2) protein through cAMP-dependent Erk/MAP kinase activation that ultimately resulted in the up-regulation of GPR30. Administration of prunetin at 0.25mg/kg given to rats stimulated bone regeneration at the site of drill hole and up-regulated Runx2 expression in the fractured callus and the effect was comparable to human parathyroid hormone, the only clinically used osteogenic therapy. We conclude that prunetin promotes osteoinduction in vivo and the mechanism is defined by signaling through GPR30 resulting in the up-regulation of the key osteogenic gene Runx2 that in turn up-regulates GPR30. PMID:26345541

  17. The role of ECL2 in CGRP receptor activation: a combined modelling and experimental approach

    Science.gov (United States)

    Woolley, Michael. J.; Watkins, Harriet A.; Taddese, Bruck; Karakullukcu, Z. Gamze; Barwell, James; Smith, Kevin J.; Hay, Debbie L.; Poyner, David R.; Reynolds, Christopher A.; Conner, Alex C.

    2013-01-01

    The calcitonin gene-related peptide (CGRP) receptor is a complex of a calcitonin receptor-like receptor (CLR), which is a family B G-protein-coupled receptor (GPCR) and receptor activity modifying protein 1. The role of the second extracellular loop (ECL2) of CLR in binding CGRP and coupling to Gs was investigated using a combination of mutagenesis and modelling. An alanine scan of residues 271–294 of CLR showed that the ability of CGRP to produce cAMP was impaired by point mutations at 13 residues; most of these also impaired the response to adrenomedullin (AM). These data were used to select probable ECL2-modelled conformations that are involved in agonist binding, allowing the identification of the likely contacts between the peptide and receptor. The implications of the most likely structures for receptor activation are discussed. PMID:24047872

  18. 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. PMID:27330080

  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; Bendtsen, L; Sand, O; Sottrup-Jensen, Lars

    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 that...

  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; Bendtsen, Lone; Sand, Ole; Sottrup-Jensen, Lars

    , 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...

  1. Multimodular biosensors reveal a novel platform for activation of G proteins by growth factor receptors

    Science.gov (United States)

    Midde, Krishna K.; Aznar, Nicolas; Laederich, Melanie B.; Ma, Gary S.; Kunkel, Maya T.; Newton, Alexandra C.; Ghosh, Pradipta

    2015-01-01

    Environmental cues are transmitted to the interior of the cell via a complex network of signaling hubs. Receptor tyrosine kinases (RTKs) and trimeric G proteins are two such major signaling hubs in eukaryotes. Conventionally, canonical signal transduction via trimeric G proteins is thought to be triggered exclusively by G protein-coupled receptors. Here we used molecular engineering to develop modular fluorescent biosensors that exploit the remarkable specificity of bimolecular recognition, i.e., of both G proteins and RTKs, and reveal the workings of a novel platform for activation of G proteins by RTKs in single living cells. Comprised of the unique modular makeup of guanidine exchange factor Gα-interacting vesicle-associated protein (GIV)/girdin, a guanidine exchange factor that links G proteins to a variety of RTKs, these biosensors provide direct evidence that RTK–GIV–Gαi ternary complexes are formed in living cells and that Gαi is transactivated within minutes after growth factor stimulation at the plasma membrane. Thus, GIV-derived biosensors provide a versatile strategy for visualizing, monitoring, and manipulating the dynamic association of Gαi with RTKs for noncanonical transactivation of G proteins in cells and illuminate a fundamental signaling event regulated by GIV during diverse cellular processes and pathophysiologic states. PMID:25713130

  2. Structure-based rational design of a Toll-like receptor 4 (TLR4 decoy receptor with high binding affinity for a target protein.

    Directory of Open Access Journals (Sweden)

    Jieun Han

    Full Text Available Repeat proteins are increasingly attracting much attention as alternative scaffolds to immunoglobulin antibodies due to their unique structural features. Nonetheless, engineering interaction interface and understanding molecular basis for affinity maturation of repeat proteins still remain a challenge. Here, we present a structure-based rational design of a repeat protein with high binding affinity for a target protein. As a model repeat protein, a Toll-like receptor4 (TLR4 decoy receptor composed of leucine-rich repeat (LRR modules was used, and its interaction interface was rationally engineered to increase the binding affinity for myeloid differentiation protein 2 (MD2. Based on the complex crystal structure of the decoy receptor with MD2, we first designed single amino acid substitutions in the decoy receptor, and obtained three variants showing a binding affinity (K(D one-order of magnitude higher than the wild-type decoy receptor. The interacting modes and contributions of individual residues were elucidated by analyzing the crystal structures of the single variants. To further increase the binding affinity, single positive mutations were combined, and two double mutants were shown to have about 3000- and 565-fold higher binding affinities than the wild-type decoy receptor. Molecular dynamics simulations and energetic analysis indicate that an additive effect by two mutations occurring at nearby modules was the major contributor to the remarkable increase in the binding affinities.

  3. Identification of a DNA binding protein cooperating with estrogen receptor as RIZ (retinoblastoma interacting zinc finger protein).

    Science.gov (United States)

    Medici, N; Abbondanza, C; Nigro, V; Rossi, V; Piluso, G; Belsito, A; Gallo, L; Roscigno, A; Bontempo, P; Puca, A A; Molinari, A M; Moncharmont, B; Puca, G A

    1999-11-01

    Double-stranded DNA fragments were selected from a random pool by repeated cycles of estrogen receptor-specific immunoprecipitation in the presence of a nuclear extract and PCR amplification (cyclic amplification and selection of target, CAST, for multiple elements). Fragments were cloned and sequence analysis indicated the 5-nucleotide word TTGGC was the most recurrent sequence unrelated to the known estrogen responsive element. Screening a HeLa cell expression library with a probe designed with multiple repeats of this sequence resulted in the identification of a 1700-aa protein showing a complete homology with the product of the human retinoblastoma-interacting zinc-finger gene RIZ. In transfection experiments, RIZ protein was able to bestow estrogen inducibility to a promoter containing an incomplete estrogen responsive element and a TTGGC motif. RIZ protein present in MCF-7 cell nuclear extract retarded the TTGGC-containing probe in an EMSA. Estrogen receptor was co-immunoprecipitated from MCF-7 cell extract by antibodies to RIZ protein and vice versa, thus indicating an existing interaction between these two proteins. PMID:10544042

  4. G protein-coupled receptor 30 is an estrogen receptor in the plasma membrane

    International Nuclear Information System (INIS)

    Recently, GPR30 was reported to be a novel estrogen receptor; however, its intracellular localization has remained controversial. To investigate the intracellular localization of GPR30 in vivo, we produced four kinds of polyclonal antibodies for distinct epitopes on GPR30. Immunocytochemical observations using anti-GPR30 antibody and anti-FLAG antibody show that FLAG-GPR30 localizes to the plasma membrane 24 h after transfection. Treatment with estrogen (17β-estradiol or E2) causes an elevation in the intracellular Ca2+ concentration ([Ca2+]i) within 10 s in HeLa cells expressing FLAG-GPR30. In addition, E2 induces the translocation of GPR30 from the plasma membrane to the cytoplasm by 1 h after stimulation. Immunohistochemical analysis shows that GPR30 exists on the cell surface of CA2 pyramidal neuronal cells. The images on transmission electron microscopy show that GPR30 is localized to a particular region associated with the plasma membranes of the pyramidal cells. These data indicate that GPR30, a transmembrane receptor for estrogen, is localized to the plasma membrane of CA2 pyramidal neuronal cells of the hippocampus in rat brain

  5. Stereoselective synthesis, biological evaluation, and modeling of novel bile acid-derived G-protein coupled bile acid receptor 1 (GP-BAR1, TGR5) agonists.

    Science.gov (United States)

    Yu, Donna D; Sousa, Kyle M; Mattern, Daniell L; Wagner, Jeffrey; Fu, Xianghui; Vaidehi, Nagarajan; Forman, Barry M; Huang, Wendong

    2015-04-01

    GP-BAR1 (also known as TGR5), a novel G-protein coupled receptor regulating various non-genomic functions via bile acid signaling, has emerged as a promising target for metabolic disorders, including obesity and type II diabetes. However, given that many bile acids (BAs) are poorly tolerated for systemic therapeutic use, there is significant need to develop GP-BAR1 agonists with improved potency and specificity and there also is significant impetus to develop a stereoselective synthetic methodology for GP-BAR1 agonists. Here, we report the development of highly stereo-controlled strategies to investigate a series of naturally occurring bile acid derivatives with markedly enhanced GP-BAR1 activity. These novel GP-BAR1 agonists are evaluated in vitro using luciferase-based reporter and cAMP assays to elucidate their biological properties. In vivo studies revealed that the GP-BAR1 agonist 23(S)-m-LCA increased intestinal GLP-1 transcripts by 26-fold. Additionally, computational modeling studies of selected ligands that exhibit enhanced potency and specificity for GP-BAR1 provide information on potential binding sites for these ligands in GP-BAR1. PMID:25735208

  6. Differential Requirement of the Extracellular Domain in Activation of Class B G Protein-coupled Receptors.

    Science.gov (United States)

    Zhao, Li-Hua; Yin, Yanting; Yang, Dehua; Liu, Bo; Hou, Li; Wang, Xiaoxi; Pal, Kuntal; Jiang, Yi; Feng, Yang; Cai, Xiaoqing; Dai, Antao; Liu, Mingyao; Wang, Ming-Wei; Melcher, Karsten; Xu, H Eric

    2016-07-15

    G protein-coupled receptors (GPCRs) from the secretin-like (class B) family are key players in hormonal homeostasis and are important drug targets for the treatment of metabolic disorders and neuronal diseases. They consist of a large N-terminal extracellular domain (ECD) and a transmembrane domain (TMD) with the GPCR signature of seven transmembrane helices. Class B GPCRs are activated by peptide hormones with their C termini bound to the receptor ECD and their N termini bound to the TMD. It is thought that the ECD functions as an affinity trap to bind and localize the hormone to the receptor. This in turn would allow the hormone N terminus to insert into the TMD and induce conformational changes of the TMD to activate downstream signaling. In contrast to this prevailing model, we demonstrate that human class B GPCRs vary widely in their requirement of the ECD for activation. In one group, represented by corticotrophin-releasing factor receptor 1 (CRF1R), parathyroid hormone receptor (PTH1R), and pituitary adenylate cyclase activating polypeptide type 1 receptor (PAC1R), the ECD requirement for high affinity hormone binding can be bypassed by induced proximity and mass action effects, whereas in the other group, represented by glucagon receptor (GCGR) and glucagon-like peptide-1 receptor (GLP-1R), the ECD is required for signaling even when the hormone is covalently linked to the TMD. Furthermore, the activation of GLP-1R by small molecules that interact with the intracellular side of the receptor is dependent on the presence of its ECD, suggesting a direct role of the ECD in GLP-1R activation. PMID:27226600

  7. Binding studies using Pichia pastoris expressed human aryl hydrocarbon receptor and aryl hydrocarbon receptor nuclear translocator proteins.

    Science.gov (United States)

    Zheng, Yujuan; Xie, Jinghang; Huang, Xin; Dong, Jin; Park, Miki S; Chan, William K

    2016-06-01

    The aryl hydrocarbon receptor (AHR) is a transcription factor which activates gene transcription by binding to its corresponding enhancer as the heterodimer, which is consisted of AHR and the aryl hydrocarbon receptor nuclear translocator (ARNT). Human AHR can be rather difficult to study, when compared among the AHR of other species, since it is relatively unstable and less sensitive to some ligands in vitro. Overexpression of human AHR has been limited to the baculovirus expression, which is costly and tedious due to the need of repetitive baculovirus production. Here we explored whether we could generate abundant amounts of human AHR and ARNT in a better overexpression system for functional study. We observed that human AHR and ARNT can be expressed in Pichia pastoris with yields that are comparable to the baculovirus system only if their cDNAs are optimized for Pichia expression. Fusion with a c-myc tag at their C-termini seems to increase the expression yield. These Pichia expressed proteins can effectively heterodimerize and form the ternary AHR/ARNT/enhancer complex in the presence of β-naphthoflavone or kynurenine. Limited proteolysis using thermolysin can be used to study the heterodimerization of these human AHR and ARNT proteins. PMID:26923060

  8. G-protein coupled receptor BAI3 promotes myoblast fusion in vertebrates.

    Science.gov (United States)

    Hamoud, Noumeira; Tran, Viviane; Croteau, Louis-Philippe; Kania, Artur; Côté, Jean-François

    2014-03-11

    Muscle fibers form as a result of myoblast fusion, yet the cell surface receptors regulating this process are unknown in vertebrates. In Drosophila, myoblast fusion involves the activation of the Rac pathway by the guanine nucleotide exchange factor Myoblast City and its scaffolding protein ELMO, downstream of cell-surface cell-adhesion receptors. We previously showed that the mammalian ortholog of Myoblast City, DOCK1, functions in an evolutionarily conserved manner to promote myoblast fusion in mice. In search for regulators of myoblast fusion, we identified the G-protein coupled receptor brain-specific angiogenesis inhibitor (BAI3) as a cell surface protein that interacts with ELMO. In cultured cells, BAI3 or ELMO1/2 loss of function severely impaired myoblast fusion without affecting differentiation and cannot be rescued by reexpression of BAI3 mutants deficient in ELMO binding. The related BAI protein family member, BAI1, is functionally distinct from BAI3, because it cannot rescue the myoblast fusion defects caused by the loss of BAI3 function. Finally, embryonic muscle precursor expression of a BAI3 mutant unable to bind ELMO was sufficient to block myoblast fusion in vivo. Collectively, our findings provide a role for BAI3 in the relay of extracellular fusion signals to their intracellular effectors, identifying it as an essential transmembrane protein for embryonic vertebrate myoblast fusion. PMID:24567399

  9. GNL3L Inhibits Estrogen Receptor-Related Protein Activities by Competing for Coactivator Binding

    OpenAIRE

    Yasumoto, Hiroaki; Meng, Lingjun; Lin, Tao; Zhu, Qubo; Tsai, Robert Y.L.

    2007-01-01

    Guanine-nucleotide binding protein 3-like (GNL3L) is the closest homologue of a stem cell-enriched factor nucleostemin in vertebrates. They share the same yeast orthologue, Grn1p, but only GNL3L can rescue the growth-deficient phenotype in Grn1p-null yeasts. To determine the unique function of GNL3L, we identified estrogen receptor-related protein-γ (ERRγ) as a GNL3L-specific binding protein. GNL3L and ERRγ are coexpressed in the eye, kidney and muscle, and co-reside in the nucleoplasm. The i...

  10. Demonstration of functional low-density lipoprotein receptors by protein blotting in fibroblasts from a subject with homozygous receptor-negative familial hypercholesterolemia

    International Nuclear Information System (INIS)

    We report the detection of low-density lipoprotein (LDL) receptors by the technique of receptor blotting in fibroblasts from a patient with homozygous familial hypercholesterolemia (FHC) previously classified as ''receptor negative.'' Solubilized receptors were electrophoresed, transferred to nitrocellulose paper, treated with LDL followed by radiolabeled antibody to LDL, and visualized by autoradiography. GM 2000 FHC fibroblasts revealed LDL receptors with an apparent molecular weight of approximately 140,000, the same as in normal cells. LDL receptor activity by blotting in GM 2000 cells was greatly diminished in comparison with normal cells, but was calcium dependent. Receptor activity was also detectable by conventional monolayer binding and degradation assays. Thus, GM 2000 cells have profoundly diminished LDL receptor activity, but retain the genetic capacity to make LDL receptor material of normal molecular weight that is capable of binding LDL. Previous studies have demonstrated the presence of trace amounts of immunoreactive LDL receptor protein in fibroblasts from some receptor-negative FHC homozygotes. These studies are extended by demonstrating the ability of this material to bind LDL

  11. 36 CFR 13.1208 - Lake Camp.

    Science.gov (United States)

    2010-07-01

    ... 36 Parks, Forests, and Public Property 1 2010-07-01 2010-07-01 false Lake Camp. 13.1208 Section 13... § 13.1208 Lake Camp. Leaving a boat, trailer, or vehicle unattended for more than 72 hours at the facilities associated with the Lake Camp launching ramp is prohibited without authorization from...

  12. Extension Sustainability Camp: Design, Implementation, and Evaluation

    Science.gov (United States)

    Brain, Roslynn; Upton, Sally; Tingey, Brett

    2015-01-01

    Sustainability Camps provide an opportunity for Extension educators to be in the forefront of sustainability outreach and to meet the growing demand for sustainability education. This article shares development, implementation, and evaluation of an Extension Sustainability Camp for youth, grades 4-6. Camp impact was measured via daily pre-and…

  13. Amyloid β-protein differentially affects NMDA receptor- and GABAA receptor-mediated currents in rat hippocampal CA1 neurons

    Institute of Scientific and Technical Information of China (English)

    Junfang Zhang; Lei Hou; Xiuping Gao; Fen Guo; Wei Jing; Jinshun Qi; Jiantian Qiao

    2009-01-01

    Although the aggregated amyloid β-protein (Aβ) in senile plaques is one of the key neuropathological features of Alzheimer's disease (AD), soluble forms of Aβ also interfere with synaptic plasticity at the early stage of AD. The suppressive action of acute application of Aβ on hippocampal long-term potentiation (LTP) has been reported widely, whereas the mechanism underlying the effects of Aβ is still mostly unknown. The present study, using the whole-cell patch clamp technique, investigated the effects of Aβ fragments (Aβ25-35 and Aβ31-35) on the LTP induction-related postsynaptic ligand-gated channel currents in isolated hippocampal CA1 neurons. The results showed a rapid but opposite action of both peptides on excitatory and inhibitory receptor currents. Glutamate application-induced currents were suppressed by A β25-35 in a dose-dependent manner, and further N-methyl-I>aspartate (NMDA) receptor-mediated currents were selec-tively inhibited. In contrast, pretreatment with Aβ fragments potentiated γ-aminobutyric acid (GABA)-induced whole-cell currents. As a control, Aβ35-31 the reversed sequence of Aβ35-31 showed no effect on the currents induced by glutamate, NMDA or GABA. These results may partly explain the impaired effects of Aβ on hippocampal LTP, and suggest that the functional down-regulation of N M DA receptors and up-regulation of GABAA receptors may play an important role in remodeling the hippocampal synaptic plasticity in early AD.

  14. 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.

  15. Applications of Research to Camping.

    Science.gov (United States)

    Henderson, Karla A.

    1987-01-01

    Considers contributions of basic/theoretical, applied/practical, and marketing research to the field of camping. Outlines research concerns: application of qualitative methods, practical application of marketing research, effective research dissemination, and focus on longitudinal studies using larger samples. Affirms role of research to document…

  16. Lyme Disease Comes to Camp.

    Science.gov (United States)

    Peterson, Michael

    1989-01-01

    Describes one summer camp's plan for dealing with Lyme disease. Describes the disease and the deer tick. Recommends avoiding tick exposure through clothing, frequent examination, showers, and avoiding high grass and brushy areas, and using chemical insect repellents and chemicals to kill ticks in deer mouse nests. (DHP)

  17. AVTC Hosts TechnoCamp

    Science.gov (United States)

    Miner, Brenda

    2006-01-01

    The Area Vo-Tech Center (AVTC) in Russellville, Arkansas, recently hosted its first TechnoCamp to encourage enrollment based on the aptitude and interest level of the students enrolling in the various programs. The center currently offers student enrollment in auto technology, computer engineering, cosmetology, construction technology, drafting…

  18. G protein-coupled receptor 39 deficiency is associated with pancreatic islet dysfunction

    DEFF Research Database (Denmark)

    Holst, Birgitte; Egerod, Kristoffer L; Jin, Chunyu;

    2009-01-01

    G protein-coupled receptor (GPR)-39 is a seven-transmembrane receptor expressed mainly in endocrine and metabolic tissues that acts as a Zn(++) sensor signaling mainly through the G(q) and G(12/13) pathways. The expression of GPR39 is regulated by hepatocyte nuclear factor (HNF)-1alpha and HNF-4a...... insulin in response to glucose stimulation than islets from wild-type littermates. It is concluded that GPR39 is involved in the control of endocrine pancreatic function, and it is suggested that this receptor could be a novel potential target for the treatment of diabetes.......G protein-coupled receptor (GPR)-39 is a seven-transmembrane receptor expressed mainly in endocrine and metabolic tissues that acts as a Zn(++) sensor signaling mainly through the G(q) and G(12/13) pathways. The expression of GPR39 is regulated by hepatocyte nuclear factor (HNF)-1alpha and HNF-4......alpha, and in the present study, we addressed the importance of GPR39 for glucose homeostasis and pancreatic islets function. The expression and localization of GPR39 were characterized in the endocrine pancreas and pancreatic cell lines. Gpr39(-/-) mice were studied in vivo, especially in respect of...

  19. Alteration in contractile G-protein coupled receptor expression by moist snuff and nicotine in rat cerebral arteries

    DEFF Research Database (Denmark)

    Sandhu, Hardip; Xu, Cang-Bao; Edvinsson, Lars

    2011-01-01

    vasocontractile G-protein coupled receptors (GPCR), such as endothelin ET(B), serotonin 5-HT(1B), and thromboxane A(2) TP receptors, in rat cerebral arteries. Studies show that these vasocontractile GPCR show alterations by lipid-soluble cigarette smoke particles via activation of mitogen-activated protein...... 5-carboxamidotryptamine, and TP receptor agonist U46619 were investigated by a sensitive myograph. The expression of ET(B), 5-HT(1B), and TP receptors was studied at mRNA and protein levels using quantitative real-time PCR and immunohistochemistry, respectively. Organ culture with WSS or DSS (25ng...... kept at plasma level of snus users (25ng nicotine/ml). A high dose (250ng nicotine/ml) was also included due to the previous results showing alteration in the GPCR expression by nicotine at this concentration. Contractile responses to the ET(B) receptor agonist sarafotoxin 6c, 5-HT(1B) receptor agonist...

  20. Influence of berberine on protein tyrosine kinase of erythrocyte insulin receptors from type 2 diabetes mellitus

    Institute of Scientific and Technical Information of China (English)

    Xianglei Deng; Xinrong Li; Chenggong Tian

    2005-01-01

    Objective: Bererine has been used to treat type 2 diabetes mellitus in Chinese traditional medicine because of its hypoglycemic effect. In this report, we compared the intrinsic tyrosine kinase activities of erythrocyte insulin receptors from type 2 diabetes mellitus with or without stimulation by berberine in vitro. Methods: Preparations containing insulin receptors were obtained from soluble human erythrocytes, and the insulin receptors were partially purified by affinity chromatography. The tyrosine kinase activity was measured by the exogenous substrate phosphorylation. Results: Both the membrane tyrosine kinase activity and the purified receptor tyrosine kinase activity from diabetics decreased significantly compared with those of normal individuals (reduced by 67.4 % and 47.2 %, respectively).After incubation with berberine, there is a statistical difference in the activity of membrane tyrosine kinase for diabetic patients (a 150% increase). Bererine had no effect on the tyrosine kinase activity of purified insulin receptors. Conclusion: We concluded from these results that berberine was able to improve the insulin sensitivity by increasing the protein tyrosine kinase activity of membrane-bound insulin receptors from type 2 diabetes mellitus.

  1. The amphioxus (Branchiostoma floridae genome contains a highly diversified set of G protein-coupled receptors

    Directory of Open Access Journals (Sweden)

    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.

  2. Protein-tyrosine-phosphatase SHPTP2 couples platelet-derived growth factor receptor beta to Ras.

    OpenAIRE

    Bennett, A.M.; Tang, T. L.; SUGIMOTO, S; Walsh, C T; Neel, B G

    1994-01-01

    Protein-tyrosine-phosphatase SHPTP2 (Syp/PTP-1D/PTP2C) is the homologue of the Drosophila corkscrew (csw) gene product, which transmits positive signals from receptor tyrosine kinases. Likewise, SHPTP2 has been implicated in positive signaling from platelet-derived growth factor receptor beta (PDGFR). Upon PDGF stimulation, SHPTP2 binds to the PDGFR and becomes tyrosine-phosphorylated. We have identified tyrosine-542 (pY542TNI) as the major in vivo site of SHPTP2 tyrosine phosphorylation. The...

  3. Molecular interactions between chondroitin-dermatan sulfate and growth factors/receptors/matrix proteins.

    Science.gov (United States)

    Mizumoto, Shuji; Yamada, Shuhei; Sugahara, Kazuyuki

    2015-10-01

    Recent functional studies on chondroitin sulfate-dermatan sulfate (CS-DS) demonstrated its indispensable roles in various biological events including brain development and cancer. CS-DS proteoglycans exert their physiological activity through interactions with specific proteins including growth factors, cell surface receptors, and matrix proteins. The characterization of these interactions is essential for regulating the biological functions of CS-DS proteoglycans. Although amino acid sequences on the bioactive proteins required for these interactions have already been elucidated, the specific saccharide sequences involved in the binding of CS-DS to target proteins have not yet been sufficiently identified. In this review, recent findings are described on the interaction between CS-DS and some proteins which are especially involved in the central nervous system and cancer development/metastasis. PMID:26164146

  4. Involvement of calcium-calmodulin-dependent protein kinase II in endothelin receptor expression in rat cerebral arteries

    DEFF Research Database (Denmark)

    Waldsee, Roya; Ahnstedt, Hilda; Eftekhari, Sajedeh;

    2010-01-01

    Experimental cerebral ischemia and organ culture of cerebral arteries result in the enhanced expression of endothelin ET(B) receptors in smooth muscle cells via increased transcription. The present study was designed to evaluate the involvement of calcium-calmodulin-dependent protein kinase (CAMK......(B) receptor agonist) were studied using a sensitive myograph. The mRNA levels of the ET(A) and ET(B) receptors and CAMKII were determined by real-time PCR, and their protein levels were evaluated by immunohistochemistry and Western blot. The mRNA levels of CAMKII and the ET(B) receptor increased during organ...

  5. Functional coupling of a recombinant Human 5-HT5A receptor to G-proteins in HEK-293 cells

    OpenAIRE

    Hurley, P T; McMahon, R. A.; Fanning, P; O'Boyle, K M; Rogers, M; Martin, F

    1998-01-01

    We have cloned, expressed and pharmacologically characterized the Human 5-HT5A receptor.We have shown that ligand activation of the Human 5-HT5A receptor results in functional coupling to G-proteins in HEK-293 cells.Stimulation of the receptor with 5-CT (5-carboxamidotryptamine) resulted in a dose-dependent increase in the % [35S]-GTPγS binding over the basal level. This is the first study to describe such G-protein activation for the Human 5-HT5A receptor in any cell.A dose-dependent inhibit...

  6. Prostaglandin E2 Inhibits NLRP3 Inflammasome Activation through EP4 Receptor and Intracellular Cyclic AMP in Human Macrophages.

    Science.gov (United States)

    Sokolowska, Milena; Chen, Li-Yuan; Liu, Yueqin; Martinez-Anton, Asuncion; Qi, Hai-Yan; Logun, Carolea; Alsaaty, Sara; Park, Yong Hwan; Kastner, Daniel L; Chae, Jae Jin; Shelhamer, James H

    2015-06-01

    PGE2 is a potent lipid mediator involved in maintaining homeostasis but also promotion of acute inflammation or immune suppression in chronic inflammation and cancer. Nucleotide-binding domain, leucine-rich repeat-containing protein (NLR)P3 inflammasome plays an important role in host defense. Uncontrolled activation of the NLRP3 inflammasome, owing to mutations in the NLRP3 gene, causes cryopyrin-associated periodic syndromes. In this study, we showed that NLRP3 inflammasome activation is inhibited by PGE2 in human primary monocyte-derived macrophages. This effect was mediated through PGE2 receptor subtype 4 (EP4) and an increase in intracellular cAMP, independently of protein kinase A or exchange protein directly activated by cAMP. A specific agonist of EP4 mimicked, whereas its antagonist or EP4 knockdown reversed, PGE2-mediated NLRP3 inhibition. PGE2 caused an increase in intracellular cAMP. Blockade of adenylate cyclase by its inhibitor reversed PGE2-mediated NLRP3 inhibition. Increase of intracellular cAMP by an activator of adenylate cyclase or an analog of cAMP, or a blockade of cAMP degradation by phosphodiesterase inhibitor decreased NLRP3 activation. Protein kinase A or exchange protein directly activated by cAMP agonists did not mimic, and their antagonists did not reverse, PGE2-mediated NLRP3 inhibition. Additionally, constitutive IL-1β secretion from LPS-primed PBMCs of cryopyrin-associated periodic fever syndromes patients was substantially reduced by high doses of PGE2. Moreover, blocking cytosolic phospholipase A2α by its inhibitor or small interfering RNA or inhibiting cyclooxygenase 2, resulting in inhibition of endogenous PGE2 production, caused an increase in NLRP3 inflammasome activation. Our results suggest that PGE2 might play a role in maintaining homeostasis during the resolution phase of inflammation and might serve as an autocrine and paracrine regulator. PMID:25917098

  7. Opioid and GABAB receptors differentially couple to an adenylyl cyclase/protein kinase A downstream effector after chronic morphine treatment.

    Directory of Open Access Journals (Sweden)

    Elena Elizabeth Bagley

    2014-06-01

    Full Text Available Opioids are intensely addictive, and cessation of their chronic use is associated with a highly aversive withdrawal syndrome. A cellular hallmark of withdrawal is an opioid sensitive protein kinase A-dependent increase in GABA transporter-1 (GAT-1 currents in periaqueductal gray (PAG neurons. Elevated GAT-1 activity directly increases GABAergic neuronal excitability and synaptic GABA release, which will enhance GABAergic inhibition of PAG output neurons. This reduced activity of PAG output neurons to several brain regions, including the hypothalamus and medulla, contributes to many of the PAG-mediated signs of opioid withdrawal. The GABAB receptor agonist baclofen reduces some of the PAG mediated signs of opioid withdrawal. Like the opioid receptors the GABAB receptor is a Gi/Go coupled G-protein coupled receptor. This suggests it could be modulating GAT-1 activity in PAG neurons through its inhibition of the adenylyl cyclase/protein kinase A pathway. Opioid modulation of the GAT-1 activity can be detected by changes in the reversal potential of opioid membrane currents. We found that when opioids are reducing the GAT-1 cation conductance and increasing the GIRK conductance the opioid agonist reversal potential is much more negative than Ek. Using this approach for GABAB receptors we show that the GABAB receptor agonist, baclofen, does not couple to inhibition of GAT-1 currents during opioid withdrawal. It is possible this differential signaling of the two Gi/Go coupled G-protein coupled receptors is due to the strong compartmentalization of the GABAB receptor that does not favor signaling to the adenylyl cyclase/protein kinase A/GAT-1 pathway. This highlights the importance of studying the effects of G-protein coupled receptors in native tissue with endogenous G-protein coupled receptors and the full complement of relevant proteins and signaling molecules. This study suggests that baclofen reduces opioid withdrawal symptoms through a non-GAT-1

  8. Non-canonical dynamic mechanisms of interaction between the p66Shc protein and Met receptor.

    Science.gov (United States)

    Landry, Mélissa; Pomerleau, Véronique; Saucier, Caroline

    2016-06-01

    Met receptor tyrosine kinase (RTK) is known to bind to the three distinct protein isoforms encoded by the ShcA (Shc) gene. Structure-function studies have unveiled critical roles for p52Shc-dependent signalling pathways in Met-regulated biological functions. The molecular basis of the interaction between the Met and p52Shc proteins is well-defined, but not for the longest protein isoform, p66Shc. In the present study, co-immunoprecipitation assays were performed in human embryonic kidney 293 (HEK293) cells, transiently co-transfected with Met and p66Shc mutants, in order to define the molecular determinants involved in mediating Met-p66Shc interaction. Our results show that p66Shc interacts constitutively with the receptor Met, and the Grb2 (growth factor receptor-bound protein-2) and Gab1 (Grb2-associated binder-1) adaptor proteins. Although its phosphotyrosine-binding domain (PTB) and Src homology 2 (SH2) domains co-ordinate p66Shc binding to non-activated Met receptor, these phosphotyrosine-binding modules, and its collagen homology domain 2 (CH2) region, exert negative constraints. In contrast, p66Shc interaction with the activated Met depends mainly on the integrity of its PTB domain, and to a lesser extent of its SH2 domain. Even though not required for the recruitment of p66Shc, tyrosine phosphorylation of p66Shc by activated Met enhances these interactions by mechanisms not reliant on the integrity of the Met multisubstrate-binding site. In turn, this increases phosphotyrosine-dependent p66Shc-Grb2-Gab1 complex formation away from the receptor, while blocking Grb2 and Gab1 recruitment to activated Met. In conclusion, we identify, for the first time, a novel non-canonical dynamic mode of interaction between Met and the p66 protein isoform of Shc and its effects on rewiring binding effector complexes according to the activation state of the receptor. PMID:27048591

  9. Glutamate Receptor Interacting Protein 1 Regulates CD4(+) CTLA-4 Expression and Transplant Rejection.

    Science.gov (United States)

    Modjeski, K L; Levy, S C; Ture, S K; Field, D J; Shi, G; Ko, K; Zhu, Q; Morrell, C N

    2016-05-01

    PDZ domains are common 80- to 90-amino-acid regions named after the first three proteins discovered to share these domains: postsynaptic density 95, discs large, and zonula occludens. PDZ domain-containing proteins typically interact with the C-terminus of membrane receptors. Glutamate receptor interacting protein 1 (GRIP1), a seven-PDZ domain protein scaffold, regulates glutamate receptor surface expression and trafficking in neurons. We have found that human and mouse T cells also express GRIP1. T cell-specific GRIP1(-/-) mice >11 weeks old had prolonged cardiac allograft survival. Compared with wild-type T cells, in vitro stimulated GRIP1(-/-) T cells had decreased expression of activation markers and increased apoptotic surface marker expression. Surface expression of the strong T cell inhibitory molecule cytotoxic T lymphocyte antigen-4 (CTLA-4) was increased on GRIP1(-/-) T cells from mice >11 weeks old. CTLA-4 increases with T cell stimulation and its surface expression on GRIP1(-/-) T cells remained high after stimulation was removed, indicating a possible internalization defect in GRIP1-deficient T cells. CTLA-4-blocking antibody treatment following heart transplantation led to complete rejection in T cell GRIP1(-/-) mice, indicating that increased CTLA-4 surface expression contributed to the extended graft survival. Our data indicate that GRIP1 regulates T cell activation by regulating CTLA-4 surface expression. PMID:26601915

  10. 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.

  11. 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 R; Warden, Charles D; Wood, David W

    2011-11-15

    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 G(4)S (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

  12. Acetylation of pregnane X receptor protein determines selective function independent of ligand activation

    International Nuclear Information System (INIS)

    Research highlights: → Pregnane X receptor (PXR), a major regulatory protein, is modified by acetylation. → PXR undergoes dynamic deacetylation upon ligand-mediated activation. → SIRT1 partially mediates PXR deacetylation. → 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.

  13. 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.

  14. 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.

  15. A Miniaturized Screen of a Schistosoma mansoni Serotonergic G Protein-Coupled Receptor Identifies Novel Classes of Parasite-Selective Inhibitors.

    Science.gov (United States)

    Chan, John D; McCorvy, John D; Acharya, Sreemoyee; Johns, Malcolm E; Day, Timothy A; Roth, Bryan L; Marchant, Jonathan S

    2016-05-01

    Schistosomiasis is a tropical parasitic disease afflicting ~200 million people worldwide and current therapy depends on a single drug (praziquantel) which exhibits several non-optimal features. These shortcomings underpin the need for next generation anthelmintics, but the process of validating physiologically relevant targets ('target selection') and pharmacologically profiling them is challenging. Remarkably, even though over a quarter of current human therapeutics target rhodopsin-like G protein coupled receptors (GPCRs), no library screen of a flatworm GPCR has yet been reported. Here, we have pharmacologically profiled a schistosome serotonergic GPCR (Sm.5HTR) implicated as a downstream modulator of PZQ efficacy, in a miniaturized screening assay compatible with high content screening. This approach employs a split luciferase based biosensor sensitive to cellular cAMP levels that resolves the proximal kinetics of GPCR modulation in intact cells. Data evidence a divergent pharmacological signature between the parasitic serotonergic receptor and the closest human GPCR homolog (Hs.5HTR7), supporting the feasibility of optimizing parasitic selective pharmacophores. New ligands, and chemical series, with potency and selectivity for Sm.5HTR over Hs.5HTR7 are identified in vitro and validated for in vivo efficacy against schistosomules and adult worms. Sm.5HTR also displayed a property resembling irreversible inactivation, a phenomenon discovered at Hs.5HTR7, which enhances the appeal of this abundantly expressed parasite GPCR as a target for anthelmintic ligand design. Overall, these data underscore the feasibility of profiling flatworm GPCRs in a high throughput screening format competent to resolve different classes of GPCR modulators. Further, these data underscore the promise of Sm.5HTR as a chemotherapeutically vulnerable node for development of next generation anthelmintics. PMID:27187180

  16. A Miniaturized Screen of a Schistosoma mansoni Serotonergic G Protein-Coupled Receptor Identifies Novel Classes of Parasite-Selective Inhibitors

    Science.gov (United States)

    Chan, John D.; McCorvy, John D.; Acharya, Sreemoyee; Day, Timothy A.; Roth, Bryan L.; Marchant, Jonathan S.

    2016-01-01

    Schistosomiasis is a tropical parasitic disease afflicting ~200 million people worldwide and current therapy depends on a single drug (praziquantel) which exhibits several non-optimal features. These shortcomings underpin the need for next generation anthelmintics, but the process of validating physiologically relevant targets (‘target selection’) and pharmacologically profiling them is challenging. Remarkably, even though over a quarter of current human therapeutics target rhodopsin-like G protein coupled receptors (GPCRs), no library screen of a flatworm GPCR has yet been reported. Here, we have pharmacologically profiled a schistosome serotonergic GPCR (Sm.5HTR) implicated as a downstream modulator of PZQ efficacy, in a miniaturized screening assay compatible with high content screening. This approach employs a split luciferase based biosensor sensitive to cellular cAMP levels that resolves the proximal kinetics of GPCR modulation in intact cells. Data evidence a divergent pharmacological signature between the parasitic serotonergic receptor and the closest human GPCR homolog (Hs.5HTR7), supporting the feasibility of optimizing parasitic selective pharmacophores. New ligands, and chemical series, with potency and selectivity for Sm.5HTR over Hs.5HTR7 are identified in vitro and validated for in vivo efficacy against schistosomules and adult worms. Sm.5HTR also displayed a property resembling irreversible inactivation, a phenomenon discovered at Hs.5HTR7, which enhances the appeal of this abundantly expressed parasite GPCR as a target for anthelmintic ligand design. Overall, these data underscore the feasibility of profiling flatworm GPCRs in a high throughput screening format competent to resolve different classes of GPCR modulators. Further, these data underscore the promise of Sm.5HTR as a chemotherapeutically vulnerable node for development of next generation anthelmintics. PMID:27187180

  17. Is juvenile boot camp policy effective

    Directory of Open Access Journals (Sweden)

    Kübra Gültekin

    2012-04-01

    Full Text Available Militaristic boot camps became very popular in the U.S. in the early 1990’s as an alternative to traditional prisons and probation. Less recidivism and less cost were the shibboleths of correctional boot camps. The boot camps are believed to reduce the number of repeat offenders and to lower operational costs. The rehabilitation programs and aftercare activities are thought to bring ongoing changes in inmates’ behaviors. Therefore, boot camps are strongly supported by politicians and the public. Tax dollars are spent to operate the boot camps. However, despite the fact that only two decades have passed since the existence of juvenile boot camps, numerous studies have declared that juvenile boot camp prisons are ineffective in reducing future offenses of inmates, operational costs, and in continually changing the behaviors of young offenders.

  18. Decreased expression of thyroid receptor-associated protein 220 in temporal lobe tissue of patients with refractory epilepsy

    International Nuclear Information System (INIS)

    Purpose: TRAP220 (thyroid hormone receptor-associated protein) functions as a coactivator for nuclear receptors and stimulates transcription by recruiting the TRAP mediator complex to hormone responsive promoter regions. Thus, TRAP220 enhances the function of thyroid/steroid hormone receptors such as thyroid hormone and oestrogen receptors. This study investigated the expression of TRAP220 mRNA and protein level in epileptic brains comparing with human control. Methods: We examined the expression of TRAP220 mRNA and protein levels in temporal lobes from patients with chronic pharmacoresistant epilepsy who have undergone surgery. Results: Expression of TRAP220 mRNA and protein was shown to be decreased significantly in the temporal cortex of the patients with epilepsy. Conclusions: Our work showed that a decrease in TRAP220 mRNA and protein levels may be involved in the pathophysiology of epilepsy and may be associated with impairment of the brain caused by frequent seizures

  19. Endothelial Restoration of Receptor Activity-Modifying Protein 2 Is Sufficient to Rescue Lethality, but Survivors Develop Dilated Cardiomyopathy.

    Science.gov (United States)

    Kechele, Daniel O; Dunworth, William P; Trincot, Claire E; Wetzel-Strong, Sarah E; Li, Manyu; Ma, Hong; Liu, Jiandong; Caron, Kathleen M

    2016-09-01

    RAMPs (receptor activity-modifying proteins) serve as oligomeric modulators for numerous G-protein-coupled receptors, yet elucidating the physiological relevance of these interactions remains complex. Ramp2 null mice are embryonic lethal, with cardiovascular developmental defects similar to those observed in mice null for canonical adrenomedullin/calcitonin receptor-like receptor signaling. We aimed to genetically rescue the Ramp2(-/-) lethality in order to further delineate the spatiotemporal requirements for RAMP2 function during development and thereby enable the elucidation of an expanded repertoire of RAMP2 functions with family B G-protein-coupled receptors in adult homeostasis. Endothelial-specific expression of Ramp2 under the VE-cadherin promoter resulted in the partial rescue of Ramp2(-/-) mice, demonstrating that endothelial expression of Ramp2 is necessary and sufficient for survival. The surviving Ramp2(-/-) Tg animals lived to adulthood and developed spontaneous hypotension and dilated cardiomyopathy, which was not observed in adult mice lacking calcitonin receptor-like receptor. Yet, the hearts of Ramp2(-/-) Tg animals displayed dysregulation of family B G-protein-coupled receptors, including parathyroid hormone and glucagon receptors, as well as their downstream signaling pathways. These data suggest a functional requirement for RAMP2 in the modulation of additional G-protein-coupled receptor pathways in vivo, which is critical for sustained cardiovascular homeostasis. The cardiovascular importance of RAMP2 extends beyond the endothelium and canonical adrenomedullin/calcitonin receptor-like receptor signaling, in which future studies could elucidate novel and pharmacologically tractable pathways for treating cardiovascular diseases. PMID:27402918

  20. Effects of anti-peptide antibody against human M2 muscarinic receptors on the cAMP content in rat ventricles%心肌M2胆碱受体抗体对大鼠心房及心室肌cAMP含量的影响

    Institute of Scientific and Technical Information of China (English)

    朱琳; 李香珍; 冯羡菊; 王文泽; 张琳; 陈爱莲

    2001-01-01

    目的:了解心肌M2胆碱受体抗体(M2-Ab)对大鼠心房和心室肌cAMP含量的影响,并与M2胆碱受体激动剂卡巴可(Carb)的作用进行了对比观察。方法:采用离体生化放射免疫分析法测定M2-Ab及M2胆碱受体激动剂Carb对心肌cAMP含量的影响。结果:①M2-Ab及Carb两者均可剂量依赖性抑制异丙肾上腺素(Isoproterenol,Iso)所刺激的大鼠心房及心室肌cAMP的增加。卡巴可浓度为2 μmol/L,10 μmol/L,50 μmol/L时可分别抑制Iso所刺激的cAMP含量(8.5±1.2)%,(16.2±1.4)%,(29.5±2.1)%,而M2-Ab浓度为50 nmol/L,100 nmol/L,400 nmol/L时,可分别抑制(6.1±0.6)%,(17.3±1.8)%,(31.7±3.1)%(P<0.01)。②Carb(10 μmol/L)及M2-Ab(100 nmol/L)两者可分别抑制基础cAMP(49.2±4.3)%和(64.3±5.1)%。③M受体阻断剂阿托品(Atr)(1.5 μmol/L)不但可阻断Carb对Iso的抑制反应,亦能阻断M2-Ab的这种反应。而相应的抗原性肽段也能阻断M2-Ab的这种反应。结论:M2-Ab抑制Iso引起的心室肌细胞cAMP生成量的增加反应,类似于M受体激动剂Carb,两者效应均通过作用于M2受体途径实现。%Aim:To study the effects of anti-peptide antibodies(M2-Ab) against the second extracellular loop of human muscarinic receptor 2 on the cAMP content in rat atria and ventricles.These effects were compared with those of the muscarinic receptor agonist carbachol (Carb).Methods:Radioimmunoassay technique,was used for determining the effects of anti-pepitide antibodies and carb on cAMP content in rat ventricles.Result:①both Carb and M2-Ab were able to inhibit the isoproterenol (Iso) stimulated cAMP production in rat atria and ventricles.Carb at 2μmol/L,10μmol/L and 50μmol/L decreased Iso-stimulated cAMP production by (8.5±1.2)%,(16.2±1.4)% and (29.5±2.1)%,respectively;whereas M2-Ab at 50nmol/L,100nmol/L and 400nmol/L decreased it by (6.1±0.6)%,(17.3±1.8)% and (31.7±3.1)% (P<0.01),respectively.②Both Carb and M2-Ab

  1. 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.

  2. Identification of the Receptor Binding Domain of the Mouse Mammary Tumor Virus Envelope Protein

    Science.gov (United States)

    Zhang, Yuanming; Rassa, John C.; deObaldia, Maria Elena; Albritton, Lorraine M.; Ross, Susan R.

    2003-01-01

    Mouse mammary tumor virus (MMTV) is a betaretrovirus that infects rodent cells and uses mouse transferrin receptor 1 for cell entry. To characterize the interaction of MMTV with its receptor, we aligned the MMTV envelope surface (SU) protein with that of Friend murine leukemia virus (F-MLV) and identified a putative receptor-binding domain (RBD) that included a receptor binding sequence (RBS) of five amino acids and a heparin-binding domain (HBD). Mutation of the HBD reduced virus infectivity, and soluble heparan sulfate blocked infection of cells by wild-type pseudovirus. Interestingly, some but not all MMTV-like elements found in primary and cultured human breast cancer cell lines, termed h-MTVs, had sequence alterations in the putative RBS. Single substitution of one of the amino acids found in an h-MTV RBS variant in the RBD of MMTV, Phe40 to Ser, did not alter species tropism but abolished both virus binding to cells and infectivity. Neutralizing anti-SU monoclonal antibodies also recognized a glutathione S-transferase fusion protein that contained the five-amino-acid RBS region from MMTV. The critical Phe40 residue is located on a surface of the MMTV RBD model that is distant from and may be structurally more rigid than the region of F-MLV RBD that contains its critical binding site residues. This suggests that, in contrast to other murine retroviruses, binding to its receptor may result in few or no changes in MMTV envelope protein conformation. PMID:12970432

  3. Identification of fibroblast growth factor receptor 3 (FGFR3 as a protein receptor for botulinum neurotoxin serotype A (BoNT/A.

    Directory of Open Access Journals (Sweden)

    Birgitte P S Jacky

    Full Text Available Botulinum neurotoxin serotype A (BoNT/A causes transient muscle paralysis by entering motor nerve terminals (MNTs where it cleaves the SNARE protein Synaptosomal-associated protein 25 (SNAP25206 to yield SNAP25197. Cleavage of SNAP25 results in blockage of synaptic vesicle fusion and inhibition of the release of acetylcholine. The specific uptake of BoNT/A into pre-synaptic nerve terminals is a tightly controlled multistep process, involving a combination of high and low affinity receptors. Interestingly, the C-terminal binding domain region of BoNT/A, HC/A, is homologous to fibroblast growth factors (FGFs, making it a possible ligand for Fibroblast Growth Factor Receptors (FGFRs. Here we present data supporting the identification of Fibroblast Growth Factor Receptor 3 (FGFR3 as a high affinity receptor for BoNT/A in neuronal cells. HC/A binds with high affinity to the two extra-cellular loops of FGFR3 and acts similar to an agonist ligand for FGFR3, resulting in phosphorylation of the receptor. Native ligands for FGFR3; FGF1, FGF2, and FGF9 compete for binding to FGFR3 and block BoNT/A cellular uptake. These findings show that FGFR3 plays a pivotal role in the specific uptake of BoNT/A across the cell membrane being part of a larger receptor complex involving ganglioside- and protein-protein interactions.

  4. Molecular expression of adenosine receptors in OVCAR-3, Caov-4 and SKOV-3 human ovarian cancer cell lines.

    Science.gov (United States)

    Hajiahmadi, S; Panjehpour, M; Aghaei, M; Mousavi, S

    2015-01-01

    Adenosine receptors (A1, A2a, A2b and A3) have several physiological and pathological roles in cancer cell lines. The present study was carried out to evaluate the mRNA and protein expression profile and functional role of adenosine receptors in OVCAR-3, Caov-4 and SKOV-3 ovarian cancer cell lines. The levels of mRNA and protein expression of A1, A2a, A2b and A3 adenosine receptors in the ovarian cancer cell lines were measured by Real-time PCR and western blotting. The functional roles of adenosine receptors were investigated through measurement of cAMP levels after agonist treatment. The mRNA and protein of all adenosine receptors subtypes were expressed in the ovarian cancer cell lines. Our findings demonstrated that A2b and A3 had the most mRNA and protein expression. Moreover, cAMP assay confirmed the functional role of A2b and A3 adenosine receptors. This findings demonstrated that A2b and A3 subtypes are most important adenosine receptors in humn ovarian cancer cell lines. This information provide a strong possibility into the relationship of A2b and A3 adenosine receptor and ovarian cancer. PMID:26430456

  5. Molecular expression of adenosine receptors in OVCAR-3, Caov-4 and SKOV-3 human ovarian cancer cell lines

    Science.gov (United States)

    Hajiahmadi, S.; Panjehpour, M.; Aghaei, M.; Mousavi, S.

    2015-01-01

    Adenosine receptors (A1, A2a, A2b and A3) have several physiological and pathological roles in cancer cell lines. The present study was carried out to evaluate the mRNA and protein expression profile and functional role of adenosine receptors in OVCAR-3, Caov-4 and SKOV-3 ovarian cancer cell lines. The levels of mRNA and protein expression of A1, A2a, A2b and A3 adenosine receptors in the ovarian cancer cell lines were measured by Real-time PCR and western blotting. The functional roles of adenosine receptors were investigated through measurement of cAMP levels after agonist treatment. The mRNA and protein of all adenosine receptors subtypes were expressed in the ovarian cancer cell lines. Our findings demonstrated that A2b and A3 had the most mRNA and protein expression. Moreover, cAMP assay confirmed the functional role of A2b and A3 adenosine receptors. This findings demonstrated that A2b and A3 subtypes are most important adenosine receptors in humn ovarian cancer cell lines. This information provide a strong possibility into the relationship of A2b and A3 adenosine receptor and ovarian cancer. PMID:26430456

  6. The BTB/kelch protein, KRIP6, modulates the interaction of PICK1 with GluR6 kainate receptors

    OpenAIRE

    Laezza, Fernanda; Wilding, Timothy J.; Sequeira, Sunitha; Craig, Ann Marie; Huettner, James E

    2008-01-01

    Neuronal proteins of the BTB/kelch and PDZ domain families interact with different regions of the cytoplasmic C-terminal domain of the GluR6 kainate receptor subunit. The BTB/kelch protein KRIP6 binds within a 58 amino acid segment of GluR6 proximal to the plasma membrane. In contrast, PDZ domain proteins, such as PICK1 and PSD95, interact with the last 4 residues of the GluR6 C-terminus. KRIP6 reduces peak currents mediated by recombinant GluR6 receptors and by native kainate receptors in ne...

  7. 3,4-methylenedioxyamphetamine upregulates p75 neurotrophin receptor protein expression in the rat brain

    Institute of Scientific and Technical Information of China (English)

    Chaomin Wang; Zugui Peng; Weihong Kuang; Hanyu Zheng; Jiang Long; Xue Wang

    2012-01-01

    The p75 neurotrophin receptor, which is a member of the tumor necrosis factor receptor superfamily, facilitates apoptosis during development and following central nervous system injury. Previous stu-dies have shown that programmed cell death is likely involved in the neurotoxic effects of 3, 4-methylenedioxy-N-methylamphetamine (MDMA), because MDMA induces apoptosis of immor-talized neurons through regulation of proteins belonging to the Bcl-2 family. In the present study, intraperitoneal injection of different doses of MDMA (20, 50, and 100 mg/kg) induced significant behavioral changes, such as increased excitability, increased activity, and irritability in rats. Moreover, changes exhibited dose-dependent adaptation. Following MDMA injection in rat brain tissue, the number of apoptotic cells dose-dependently increased and p75 neurotrophin receptor expression significantly increased in the prefrontal cortex, cerebellum, and hippocampus. These findings confirmed that MDMA induced neuronal apoptosis, and results suggested that this effect was related by upregulated protein expression of the p75 neurotrophin receptor.

  8. 3,4-methylenedioxyamphetamine upregulates p75 neurotrophin receptor protein expression in the rat brain.

    Science.gov (United States)

    Wang, Chaomin; Peng, Zugui; Kuang, Weihong; Zheng, Hanyu; Long, Jiang; Wang, Xue

    2012-04-25

    The p75 neurotrophin receptor, which is a member of the tumor necrosis factor receptor superfamily, facilitates apoptosis during development and following central nervous system injury. Previous studies have shown that programmed cell death is likely involved in the neurotoxic effects of 3, 4-methylenedioxy-N-methylamphetamine (MDMA), because MDMA induces apoptosis of immortalized neurons through regulation of proteins belonging to the Bcl-2 family. In the present study, intraperitoneal injection of different doses of MDMA (20, 50, and 100 mg/kg) induced significant behavioral changes, such as increased excitability, increased activity, and irritability in rats. Moreover, changes exhibited dose-dependent adaptation. Following MDMA injection in rat brain tissue, the number of apoptotic cells dose-dependently increased and p75 neurotrophin receptor expression significantly increased in the prefrontal cortex, cerebellum, and hippocampus. These findings confirmed that MDMA induced neuronal apoptosis, and results suggested that this effect was related by upregulated protein expression of the p75 neurotrophin receptor. PMID:25722682

  9. Prediction of G Protein-Coupled Receptors with SVM-Prot Features and Random Forest.

    Science.gov (United States)

    Liao, Zhijun; Ju, Ying; Zou, Quan

    2016-01-01

    G protein-coupled receptors (GPCRs) are the largest receptor superfamily. In this paper, we try to employ physical-chemical properties, which come from SVM-Prot, to represent GPCR. Random Forest was utilized as classifier for distinguishing them from other protein sequences. MEME suite was used to detect the most significant 10 conserved motifs of human GPCRs. In the testing datasets, the average accuracy was 91.61%, and the average AUC was 0.9282. MEME discovery analysis showed that many motifs aggregated in the seven hydrophobic helices transmembrane regions adapt to the characteristic of GPCRs. All of the above indicate that our machine-learning method can successfully distinguish GPCRs from non-GPCRs. PMID:27529053

  10. G-protein-Coupled Receptors and Their (Bio Chemical Significance Win 2012 Nobel Prize in Chemistry

    Directory of Open Access Journals (Sweden)

    Hsi-Hsien Lin

    2013-06-01

    Full Text Available G-protein-coupled receptors (GPCRs are seven transmembrane cell surface proteins specialized in cellular communication. These receptors represent a major gateway through which cells convert external cues into intracellular signals and respond with appropriate actions. While the effects of hormones, neurotransmitters, and drugs on cells, tissues, organs, and even whole organisms are well described, the molecular identity of the direct targets and the diverse signaling mechanisms of these biological ligands have been slow and hard to define. The Nobel Prize in Chemistry for the year 2012 acknowledges the importance of GPCRs in these processes, especially for the contribution of Profs Robert J. Lefkowitz and Brian K. Kobilka to the studies of GPCRs. In this brief review, the seminal works accomplished by the two GPCR pioneers are summarized and the (bio chemical significance of GPCRs in health and disease is discussed.

  11. Enhancement of gene transactivation activity of androgen receptor by hepatitis B virus X protein

    International Nuclear Information System (INIS)

    Hepatitis B virus (HBV) X protein (HBx) is a regulatory protein that is required for efficient replication of HBV in its natural host. In this report, we demonstrate by co-immunoprecipitation experiments that HBx can physically bind to the androgen receptor (AR), which is a nuclear hormone receptor that is expressed in many different tissues including the liver. This observation is further supported by confocal microscopy, which reveals that HBx can alter the subcellular localization of the AR both in the presence and in the absence of dihydrotestosterone (DHT). Further studies indicate that HBx can enhance the gene transactivation activity of AR by enhancing its DNA binding activity in a DHT-dependent manner. However, HBx does not remain associated with AR on the DNA. As AR can regulate the expression of a number of cellular genes, our results raise the possibility that HBV pathogenesis may be mediated in part via the interaction between HBx and AR

  12. Prediction of G Protein-Coupled Receptors with SVM-Prot Features and Random Forest

    Science.gov (United States)

    Ju, Ying

    2016-01-01

    G protein-coupled receptors (GPCRs) are the largest receptor superfamily. In this paper, we try to employ physical-chemical properties, which come from SVM-Prot, to represent GPCR. Random Forest was utilized as classifier for distinguishing them from other protein sequences. MEME suite was used to detect the most significant 10 conserved motifs of human GPCRs. In the testing datasets, the average accuracy was 91.61%, and the average AUC was 0.9282. MEME discovery analysis showed that many motifs aggregated in the seven hydrophobic helices transmembrane regions adapt to the characteristic of GPCRs. All of the above indicate that our machine-learning method can successfully distinguish GPCRs from non-GPCRs. PMID:27529053

  13. A combined LDL receptor/LDL receptor adaptor protein 1 mutation as the cause for severe familial hypercholesterolemia.

    Science.gov (United States)

    Soufi, Muhidien; Rust, Stephan; Walter, Michael; Schaefer, Juergen R

    2013-05-25

    Familial hypercholesterolemia (FH) results from impaired catabolism of plasma low density lipoproteins (LDL), thus leading to high cholesterol, atherosclerosis, and a high risk of premature myocardial infarction. FH is commonly caused by defects of the LDL receptor or its main ligand apoB, together mediating cellular uptake and clearance of plasma LDL. In some cases FH is inherited by mutations in the genes of PCSK9 and LDLRAP1 (ARH) in a dominant or recessive trait. The encoded proteins are required for LDL receptor stability and internalization within the LDLR pathway. To detect the underlying genetic defect in a family of Turkish descent showing unregular inheritance of severe FH, we screened the four candidate genes by denaturing gradient gel electrophoresis (DGGE) mutation analysis. We identified different combinatory mixtures of LDLR- and LDLRAP1-gene defects as the cause for severe familial hypercholesterolemia in this family. We also show for the first time that a heterozygous LDLR mutation combined with a homozygous LDLRAP1 mutation produces a more severe hypercholesterolemia phenotype in the same family than a homozygous LDLR mutation alone. PMID:23510778

  14. Specificity protein 4 (Sp4) transcriptionally regulates inhibitory GABAergic receptors in neurons.

    Science.gov (United States)

    Nair, Bindu; Johar, Kaid; Priya, Anusha; Wong-Riley, Margaret T T

    2016-01-01

    Previous studies in our laboratory have shown that the neuron-specific specificity protein 4 (Sp4) transcriptionally regulates many excitatory neurotransmitter receptor subunit genes, such as those for GluN1, GluN2A, and GluN2B of N-methyl-d-aspartate (NMDA) receptors and Gria2 of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors. It also regulates Atp1a1 and Atp1b1 subunit genes of Na(+)/K(+)-ATPase, a major energy-consuming enzyme, as well as all 13 subunits of cytochrome c oxidase (COX), an important energy-generating enzyme. Thus, there is a tight coupling between energy consumption, energy production, and excitatory neuronal activity at the transcriptional level in neurons. The question is whether inhibitory neurotransmitter receptors are also regulated by Sp4. In the present study, we tested our hypothesis that Sp4 regulates receptor subunit genes of a major inhibitory neurotransmitter, GABA, specifically GABAA receptors. By means of multiple approaches, including in silico analysis, electrophoretic mobility shift and supershift assays, real-time quantitative PCR, chromatin immunoprecipitation, promoter mutational analysis, over-expression and shRNA of Sp4, functional assays, and western blots, we found that Sp4 functionally regulates the transcription of Gabra1 (GABAA α1) and Gabra2 (GABAA α2), but not Gabra3 (GABAA α3) subunit genes. The binding sites of Sp4 are conserved among rats, humans, and mice. Thus, our results substantiate our hypothesis that Sp4 plays a key role in regulating the transcription of GABAA receptor subunit genes. They also indicate that Sp4 is in a position to transcriptionally regulate the balance between excitatory and inhibitory neurochemical expressions in neurons. PMID:26469128

  15. A Perspective on Studying G-Protein-Coupled Receptor Signaling with Resonance Energy Transfer Biosensors in Living Organisms

    NARCIS (Netherlands)

    J. van Unen; J. Woolard; A. Rinken; C. Hoffmann; S.J. Hill; J. Goedhart; M.R. Bruchas; M. Bouvier; M.J.W. Adjobo-Hermans

    2015-01-01

    The last frontier for a complete understanding of G-protein-coupled receptor (GPCR) biology is to be able to assess GPCR activity, interactions, and signaling in vivo, in real time within biologically intact systems. This includes the ability to detect GPCR trafficking, dimerization, protein-protein

  16. Selective autophagy of non-ubiquitylated targets in plants: looking for cognate receptor/adaptor proteins

    Directory of Open Access Journals (Sweden)

    Vasko eVeljanovski

    2014-06-01

    Full Text Available Cellular homeostasis is essential for the physiology of eukaryotic cells. Eukaryotic cells, including plant cells, utilize two main pathways to adjust the level of cytoplasmic components, namely the proteasomal and the lysosomal/vacuolar pathways. Macroautophagy is a lysosomal/vacuolar pathway which, until recently, was thought to be non-specific and a bulk degradation process. However, selective autophagy which can be activated in the cell under various physiological conditions, involves the specific degradation of defined macromolecules or organelles by a conserved molecular mechanism. For this process to be efficient, the mechanisms underlying the recognition and selection of the cargo to be engulfed by the double-membrane autophagosome are critical, and not yet well understood. Ubiquitin (poly-ubiquitin conjugation to the target appears to be a conserved ligand mechanism in many types of selective autophagy, and defined receptors/adaptors recognizing and regulating the autophagosomal capture of the ubiquitylated target have been characterized. However, non-proteinaceous and non-ubiquitylated cargoes are also selectively degraded by this pathway. This ubiquitin-independent selective autophagic pathway also involves receptor and/or adaptor proteins linking the cargo to the autophagic machinery. Some of these receptor/adaptor proteins including accessory autophagy-related (Atg and non-Atg proteins have been described in yeast and animal cells but not yet in plants. In this review we discuss the ubiquitin-independent cargo selection mechanisms in selective autophagy degradation of organelles and macromolecules and speculate on potential plant receptor/adaptor proteins.

  17. Radiation damage to specific complexes of DNA with proteins: estrogen response element DNA - estrogen receptor

    Czech Academy of Sciences Publication Activity Database

    Štísová, Viktorie; Běgusová, Marie; Goffinont, S.; Spotheim-Maurizot, M.

    Legnaro-Padova: INFN - Laboratori Nazionali di Legnaro, 2005. [International Symposium on Microdosimetry /14./. 13.11.2005-18.11.2005, Venezia] R&D Projects: GA MŠk(CZ) 1P05OC085; GA ČR(CZ) GA202/05/H031 Institutional research plan: CEZ:AV0Z10480505 Keywords : DNA-protein complex * radiation damage * estrogen receptor * estrogen response element DNA Subject RIV: BO - Biophysics

  18. Uncoupling of M1 muscarinic receptor/G-protein interaction by amyloid beta(1-42)

    Czech Academy of Sciences Publication Activity Database

    Janíčková, Helena; Rudajev, Vladimír; Zimčík, Pavel; Jakubík, Jan; Tanila, H.; El-Fakahany, E. E.; Doležal, Vladimír

    2013-01-01

    Roč. 67, April (2013), s. 272-283. ISSN 0028-3908 R&D Projects: GA ČR(CZ) GA305/09/0681; GA ČR(CZ) GBP304/12/G069; GA MŠk(CZ) 7E10060 Institutional research plan: CEZ:AV0Z50110509 Institutional support: RVO:67985823 Keywords : Alzheimer ´s Disease * muscarinic receptors * G-proteins Subject RIV: ED - Physiology Impact factor: 4.819, year: 2013

  19. Characterization and comparison of commercially available TNF receptor 2-Fc fusion protein products

    OpenAIRE

    Tan, Qingqiao; Guo, Qingcheng; Fang, Chen; Wang, Chong; Li, Bohua; Wang, Hao; Li, Jing; GUO, YAJUN

    2012-01-01

    Because of rapidly increasing market demand and rising cost pressure, the innovator of etanercept (Enbrel®) will inevitably face competition from biosimilar versions of the product. In this study, to elucidate the differences between the reference etanercept and its biosimilars, we characterized and compared the quality attributes of two commercially available, biosimilar TNF receptor 2-Fc fusion protein products. Biosimilar 1 showed high similarity to Enbrel® in critical quality attributes i...

  20. Inhibition of Receptor Interacting Protein Kinases Attenuates Cardiomyocyte Hypertrophy Induced by Palmitic Acid

    OpenAIRE

    Mingyue Zhao; Lihui Lu; Song Lei; Hua Chai; Siyuan Wu; Xiaoju Tang; Qinxue Bao; Li Chen; Wenchao Wu; Xiaojing Liu

    2016-01-01

    Palmitic acid (PA) is known to cause cardiomyocyte dysfunction. Cardiac hypertrophy is one of the important pathological features of PA-induced lipotoxicity, but the mechanism by which PA induces cardiomyocyte hypertrophy is still unclear. Therefore, our study was to test whether necroptosis, a receptor interacting protein kinase 1 and 3 (RIPK1 and RIPK3-) dependent programmed necrosis, was involved in the PA-induced cardiomyocyte hypertrophy. We used the PA-treated primary neonatal rat cardi...