WorldWideScience

Sample records for acid receptor trafficking

  1. Coxsackievirus and adenovirus receptor (CAR) mediates trafficking of acid sensing ion channel 3 (ASIC3) via PSD-95.

    Science.gov (United States)

    Excoffon, Katherine J D A; Kolawole, Abimbola O; Kusama, Nobuyoshi; Gansemer, Nicholas D; Sharma, Priyanka; Hruska-Hageman, Alesia M; Petroff, Elena; Benson, Christopher J

    2012-08-17

    We have previously shown that the Coxsackievirus and adenovirus receptor (CAR) can interact with post-synaptic density 95 (PSD-95) and localize PSD-95 to cell-cell junctions. We have also shown that activity of the acid sensing ion channel (ASIC3), a H(+)-gated cation channel that plays a role in mechanosensation and pain signaling, is negatively modulated by PSD-95 through a PDZ-based interaction. We asked whether CAR and ASIC3 simultaneously interact with PSD-95, and if so, whether co-expression of these proteins alters their cellular distribution and localization. Results indicate that CAR and ASIC3 co-immunoprecipitate only when co-expressed with PSD-95. CAR also brings both PSD-95 and ASIC3 to the junctions of heterologous cells. Moreover, CAR rescues PSD-95-mediated inhibition of ASIC3 currents. These data suggest that, in addition to activity as a viral receptor and adhesion molecule, CAR can play a role in trafficking proteins, including ion channels, in a PDZ-based scaffolding complex. Copyright © 2012 Elsevier Inc. All rights reserved.

  2. Sucrose ingestion induces rapid AMPA receptor trafficking.

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    Tukey, David S; Ferreira, Jainne M; Antoine, Shannon O; D'amour, James A; Ninan, Ipe; Cabeza de Vaca, Soledad; Incontro, Salvatore; Wincott, Charlotte; Horwitz, Julian K; Hartner, Diana T; Guarini, Carlo B; Khatri, Latika; Goffer, Yossef; Xu, Duo; Titcombe, Roseann F; Khatri, Megna; Marzan, Dave S; Mahajan, Shahana S; Wang, Jing; Froemke, Robert C; Carr, Kenneth D; Aoki, Chiye; Ziff, Edward B

    2013-04-03

    The mechanisms by which natural rewards such as sugar affect synaptic transmission and behavior are largely unexplored. Here, we investigate regulation of nucleus accumbens synapses by sucrose intake. Previous studies have shown that AMPA receptor (AMPAR) trafficking is a major mechanism for regulating synaptic strength, and that in vitro, trafficking of AMPARs containing the GluA1 subunit takes place by a two-step mechanism involving extrasynaptic and then synaptic receptor transport. We report that in rat, repeated daily ingestion of a 25% sucrose solution transiently elevated spontaneous locomotion and potentiated accumbens core synapses through incorporation of Ca(2+)-permeable AMPA receptors (CPARs), which are GluA1-containing, GluA2-lacking AMPARs. Electrophysiological, biochemical, and quantitative electron microscopy studies revealed that sucrose training (7 d) induced a stable (>24 h) intraspinous GluA1 population, and that in these rats a single sucrose stimulus rapidly (5 min) but transiently (<24 h) elevated GluA1 at extrasynaptic sites. CPARs and dopamine D1 receptors were required in vivo for elevated locomotion after sucrose ingestion. Significantly, a 7 d protocol of daily ingestion of a 3% solution of saccharin, a noncaloric sweetener, induced synaptic GluA1 similarly to 25% sucrose ingestion. These findings identify multistep GluA1 trafficking, previously described in vitro, as a mechanism for acute regulation of synaptic transmission in vivo by a natural orosensory reward. Trafficking is stimulated by a chemosensory pathway that is not dependent on the caloric value of sucrose.

  3. Opioid receptor trafficking and interaction in nociceptors

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    Zhang, X; Bao, L; Li, S

    2015-01-01

    Opiate analgesics such as morphine are often used for pain therapy. However, antinociceptive tolerance and dependence may develop with long-term use of these drugs. It was found that μ-opioid receptors can interact with δ-opioid receptors, and morphine antinociceptive tolerance can be reduced by blocking δ-opioid receptors. Recent studies have shown that μ- and δ-opioid receptors are co-expressed in a considerable number of small neurons in the dorsal root ganglion. The interaction of μ-opioid receptors with δ-opioid receptors in the nociceptive afferents is facilitated by the stimulus-induced cell-surface expression of δ-opioid receptors, and contributes to morphine tolerance. Further analysis of the molecular, cellular and neural circuit mechanisms that regulate the trafficking and interaction of opioid receptors and related signalling molecules in the pain pathway would help to elucidate the mechanism of opiate analgesia and improve pain therapy. LINKED ARTICLES This article is part of a themed section on Opioids: New Pathways to Functional Selectivity. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2015.172.issue-2 PMID:24611685

  4. Oxidation inhibits PTH receptor signaling and trafficking.

    Science.gov (United States)

    Ardura, Juan A; Alonso, Verónica; Esbrit, Pedro; Friedman, Peter A

    2017-01-22

    Reactive Oxygen Species (ROS) increase during aging, potentially affecting many tissues including brain, heart, and bone. ROS alter signaling pathways and constitute potential therapeutic targets to limit oxidative damaging effects in aging-associated diseases. Parathyroid hormone receptors (PTHR) are widely expressed and PTH is the only anabolic therapy for osteoporosis. The effects of oxidative stress on PTHR signaling and trafficking have not been elucidated. Here, we used Fluorescence Resonance Energy Transfer (FRET)-based cAMP, ERK, and calcium fluorescent biosensors to analyze the effects of ROS on PTHR signaling and trafficking by live-cell imaging. PTHR internalization and recycling were measured in HEK-293 cells stably transfected with HA-PTHR. PTH increased cAMP production, ERK phosphorylation, and elevated intracellular calcium. Pre-incubation with H2O2 reduced all PTH-dependent signaling pathways. These inhibitory effects were not a result of PTH oxidation since PTH incubated with H2O2 triggered similar responses. PTH promoted internalization and recycling of the PTHR. Both events were significantly reduced by H2O2 pre-incubation. These findings highlight the role of oxidation on PTHR signaling and trafficking, and suggest the relevance of ROS as a putative target in diseases associated with oxidative stress such as age-related osteoporosis. Copyright © 2016 Elsevier Inc. All rights reserved.

  5. Cellular trafficking of nicotinic acetylcholine receptors

    Institute of Scientific and Technical Information of China (English)

    Paul A ST JOHN

    2009-01-01

    Nicotinic acetylcholine receptors (nAChRs) play critical roles throughout the body. Precise regulation of the cellular location and availability of nAChRs on neurons and target cells is critical to their proper function. Dynamic, post-translational regulation of nAChRs, particularly control of their movements among the different compartments of cells, is an important aspect of that regulation. A combination of new information and new techniques has the study of nAChR trafficking poised for new breakthroughs.

  6. Ganglioside Regulation of AMPA Receptor Trafficking

    Science.gov (United States)

    Prendergast, Jillian; Umanah, George K.E.; Yoo, Seung-Wan; Lagerlöf, Olof; Motari, Mary G.; Cole, Robert N.; Huganir, Richard L.; Dawson, Ted M.; Dawson, Valina L.

    2014-01-01

    Gangliosides are major cell-surface determinants on all vertebrate neurons. Human congenital disorders of ganglioside biosynthesis invariably result in intellectual disability and are often associated with intractable seizures. To probe the mechanisms of ganglioside functions, affinity-captured ganglioside-binding proteins from rat cerebellar granule neurons were identified by quantitative proteomic mass spectrometry. Of the six proteins that bound selectively to the major brain ganglioside GT1b (GT1b:GM1 > 4; p < 10−4), three regulate neurotransmitter receptor trafficking: Thorase (ATPase family AAA domain-containing protein 1), soluble N-ethylmaleimide-sensitive factor (NSF) attachment protein (γ-SNAP), and the transmembrane protein Nicalin. Thorase facilitates endocytosis of GluR2 subunit-containing AMPA-type glutamate receptors (AMPARs) in an ATPase-dependent manner; its deletion in mice results in learning and memory deficits (J. Zhang et al., 2011b). GluR2-containing AMPARs did not bind GT1b, but bound specifically to another ganglioside, GM1. Addition of noncleavable ATP (ATPγS) significantly disrupted ganglioside binding, whereas it enhanced AMPAR association with Thorase, NSF, and Nicalin. Mutant mice lacking GT1b expressed markedly higher brain Thorase, whereas Thorase-null mice expressed higher GT1b. Treatment of cultured hippocampal neurons with sialidase, which cleaves GT1b (and other sialoglycans), resulted in a significant reduction in the size of surface GluR2 puncta. These data support a model in which GM1-bound GluR2-containing AMPARs are functionally segregated from GT1b-bound AMPAR-trafficking complexes. Release of ganglioside binding may enhance GluR2-containing AMPAR association with its trafficking complexes, increasing endocytosis. Disrupting ganglioside biosynthesis may result in reduced synaptic expression of GluR2-contianing AMPARs resulting in intellectual deficits and seizure susceptibility in mice and humans. PMID:25253868

  7. Ganglioside regulation of AMPA receptor trafficking.

    Science.gov (United States)

    Prendergast, Jillian; Umanah, George K E; Yoo, Seung-Wan; Lagerlöf, Olof; Motari, Mary G; Cole, Robert N; Huganir, Richard L; Dawson, Ted M; Dawson, Valina L; Schnaar, Ronald L

    2014-09-24

    Gangliosides are major cell-surface determinants on all vertebrate neurons. Human congenital disorders of ganglioside biosynthesis invariably result in intellectual disability and are often associated with intractable seizures. To probe the mechanisms of ganglioside functions, affinity-captured ganglioside-binding proteins from rat cerebellar granule neurons were identified by quantitative proteomic mass spectrometry. Of the six proteins that bound selectively to the major brain ganglioside GT1b (GT1b:GM1 > 4; p Nicalin. Thorase facilitates endocytosis of GluR2 subunit-containing AMPA-type glutamate receptors (AMPARs) in an ATPase-dependent manner; its deletion in mice results in learning and memory deficits (J. Zhang et al., 2011b). GluR2-containing AMPARs did not bind GT1b, but bound specifically to another ganglioside, GM1. Addition of noncleavable ATP (ATPγS) significantly disrupted ganglioside binding, whereas it enhanced AMPAR association with Thorase, NSF, and Nicalin. Mutant mice lacking GT1b expressed markedly higher brain Thorase, whereas Thorase-null mice expressed higher GT1b. Treatment of cultured hippocampal neurons with sialidase, which cleaves GT1b (and other sialoglycans), resulted in a significant reduction in the size of surface GluR2 puncta. These data support a model in which GM1-bound GluR2-containing AMPARs are functionally segregated from GT1b-bound AMPAR-trafficking complexes. Release of ganglioside binding may enhance GluR2-containing AMPAR association with its trafficking complexes, increasing endocytosis. Disrupting ganglioside biosynthesis may result in reduced synaptic expression of GluR2-contianing AMPARs resulting in intellectual deficits and seizure susceptibility in mice and humans.

  8. Hyperinsulinemia and skeletal muscle fatty acid trafficking.

    Science.gov (United States)

    Kanaley, Jill A; Shadid, Samyah; Sheehan, Michael T; Guo, ZengKui; Jensen, Michael D

    2013-08-15

    We hypothesized that insulin alters plasma free fatty acid (FFA) trafficking into intramyocellular (im) long-chain acylcarnitines (imLCAC) and triglycerides (imTG). Overnight-fasted adults (n = 41) received intravenous infusions of [U-¹³C]palmitate (0400-0900 h) and [U-¹³C]oleate (0800-1400 h) to label imTG and imLCAC. A euglycemic-hyperinsulinemic (1.0 mU·kg fat-free mass⁻¹·min⁻¹) clamp (0800-1400 h) and two muscle biopsies (0900 h, 1400 h) were performed. The patterns of [U-¹³C]palmitate incorporation into imTG-palmitate and palmitoylcarnitine were similar to those we reported in overnight postabsorptive adults (saline control); the intramyocellular palmitoylcarnitine enrichment was not different from and correlated with imTG-palmitate enrichment for both the morning (r = 0.38, P = 0.02) and afternoon (r = 0.44, P = 0.006) biopsy samples. Plasma FFA concentrations, flux, and the incorporation of plasma oleate into imTG-oleate during hyperinsulinemia were ~1/10th of that observed in the previous saline control studies (P women than men, suggesting that sex differences in intramyocellular palmitate trafficking may occur under hyperinsulinemic conditions. We conclude that plasma FFA trafficking into imTG during hyperinsulinemia is markedly suppressed, and these newly incorporated FFA fatty acids do not readily enter the LCAC preoxidative pools. Hyperinsulinemia does not seem to inhibit the entry of fatty acids from imTG pools that were labeled under fasting conditions, possibly reflecting the presence of two distinct imTG pools that are differentially regulated by insulin.

  9. Hyperinsulinemia and skeletal muscle fatty acid trafficking

    OpenAIRE

    Kanaley, Jill A.; Shadid, Samyah; Sheehan, Michael T.; Guo, ZengKui; Jensen, Michael D

    2013-01-01

    We hypothesized that insulin alters plasma free fatty acid (FFA) trafficking into intramyocellular (im) long-chain acylcarnitines (imLCAC) and triglycerides (imTG). Overnight-fasted adults (n = 41) received intravenous infusions of [U-13C]palmitate (0400–0900 h) and [U-13C]oleate (0800–1400 h) to label imTG and imLCAC. A euglycemic-hyperinsulinemic (1.0 mU·kg fat-free mass−1·min−1) clamp (0800–1400 h) and two muscle biopsies (0900 h, 1400 h) were performed. The patterns of [U-13C]palmitate in...

  10. Hyperinsulinemia and skeletal muscle fatty acid trafficking

    Science.gov (United States)

    Kanaley, Jill A.; Shadid, Samyah; Sheehan, Michael T.; Guo, ZengKui

    2013-01-01

    We hypothesized that insulin alters plasma free fatty acid (FFA) trafficking into intramyocellular (im) long-chain acylcarnitines (imLCAC) and triglycerides (imTG). Overnight-fasted adults (n = 41) received intravenous infusions of [U-13C]palmitate (0400–0900 h) and [U-13C]oleate (0800–1400 h) to label imTG and imLCAC. A euglycemic-hyperinsulinemic (1.0 mU·kg fat-free mass−1·min−1) clamp (0800–1400 h) and two muscle biopsies (0900 h, 1400 h) were performed. The patterns of [U-13C]palmitate incorporation into imTG-palmitate and palmitoylcarnitine were similar to those we reported in overnight postabsorptive adults (saline control); the intramyocellular palmitoylcarnitine enrichment was not different from and correlated with imTG-palmitate enrichment for both the morning (r = 0.38, P = 0.02) and afternoon (r = 0.44, P = 0.006) biopsy samples. Plasma FFA concentrations, flux, and the incorporation of plasma oleate into imTG-oleate during hyperinsulinemia were ∼1/10th of that observed in the previous saline control studies (P < 0.001). At the time of the second biopsy, the enrichment in oleoylcarnitine was <25% of that in imTG-oleate and was not correlated with imTG-oleate enrichment. The intramyocellular nonesterified fatty acid-palmitate-to-imTG-palmitate enrichment ratio was greater (P < 0.05) in women than men, suggesting that sex differences in intramyocellular palmitate trafficking may occur under hyperinsulinemic conditions. We conclude that plasma FFA trafficking into imTG during hyperinsulinemia is markedly suppressed, and these newly incorporated FFA fatty acids do not readily enter the LCAC preoxidative pools. Hyperinsulinemia does not seem to inhibit the entry of fatty acids from imTG pools that were labeled under fasting conditions, possibly reflecting the presence of two distinct imTG pools that are differentially regulated by insulin. PMID:23820622

  11. Ca2+-permeable AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid) receptors and dopamine D1 receptors regulate GluA1 trafficking in striatal neurons.

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    Tukey, David S; Ziff, Edward B

    2013-12-06

    Regulation of striatal medium spiny neuron synapses underlies forms of motivated behavior and pathological drug seeking. A primary mechanism for increasing synaptic strength is the trafficking of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs) into the postsynapse, a process mediated by GluA1 AMPAR subunit phosphorylation. We have examined the role of converging glutamate and dopamine inputs in regulating biochemical cascades upstream of GluA1 phosphorylation. We focused on the role of Ca(2+)-permeable AMPARs (CPARs), which lack the GluA2 AMPAR subunit. Under conditions that prevented depolarization, stimulation of CPARs activated neuronal nitric oxide synthase and production of cGMP. CPAR-dependent cGMP production was sufficient to induce synaptic insertion of GluA1, detected by confocal microscopy, through a mechanism dependent on GluA1 Ser-845 phosphorylation. Dopamine D1 receptors, in contrast, stimulate GluA1 extra synaptic insertion. Simultaneous activation of dopamine D1 receptors and CPARs induced additive increases in GluA1 membrane insertion, but only CPAR stimulation augmented CPAR-dependent GluA1 synaptic insertion. This incorporation into the synapse proceeded through a sequential two-step mechanism; that is, cGMP-dependent protein kinase II facilitated membrane insertion and/or retention, and protein kinase C activity was necessary for synaptic insertion. These data suggest a feed-forward mechanism for synaptic priming whereby an initial stimulus acting independently of voltage-gated conductance increases striatal neuron excitability, facilitating greater neuronal excitation by a subsequent stimulus.

  12. Dynamic DNA methylation controls glutamate receptor trafficking and synaptic scaling.

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    Sweatt, J David

    2016-05-01

    Hebbian plasticity, including long-term potentiation and long-term depression, has long been regarded as important for local circuit refinement in the context of memory formation and stabilization. However, circuit development and stabilization additionally relies on non-Hebbian, homeostatic, forms of plasticity such as synaptic scaling. Synaptic scaling is induced by chronic increases or decreases in neuronal activity. Synaptic scaling is associated with cell-wide adjustments in postsynaptic receptor density, and can occur in a multiplicative manner resulting in preservation of relative synaptic strengths across the entire neuron's population of synapses. Both active DNA methylation and demethylation have been validated as crucial regulators of gene transcription during learning, and synaptic scaling is known to be transcriptionally dependent. However, it has been unclear whether homeostatic forms of plasticity such as synaptic scaling are regulated via epigenetic mechanisms. This review describes exciting recent work that has demonstrated a role for active changes in neuronal DNA methylation and demethylation as a controller of synaptic scaling and glutamate receptor trafficking. These findings bring together three major categories of memory-associated mechanisms that were previously largely considered separately: DNA methylation, homeostatic plasticity, and glutamate receptor trafficking. This review describes exciting recent work that has demonstrated a role for active changes in neuronal DNA methylation and demethylation as a controller of synaptic scaling and glutamate receptor trafficking. These findings bring together three major categories of memory-associated mechanisms that were previously considered separately: glutamate receptor trafficking, DNA methylation, and homeostatic plasticity.

  13. Ubiquitin-dependent trafficking and turnover of ionotropic glutamate receptors

    Directory of Open Access Journals (Sweden)

    Marisa S Goo

    2015-10-01

    Full Text Available Changes in synaptic strength underlie the basis of learning and memory and are controlled, in part, by the insertion or removal of AMPA-type glutamate receptors at the postsynaptic membrane of excitatory synapses. Once internalized, these receptors may be recycled back to the plasma membrane by subunit-specific interactions with other proteins or by post-translational modifications such as phosphorylation. Alternatively, these receptors may be targeted for destruction by multiple degradation pathways in the cell. Ubiquitination, another post-translational modification, has recently emerged as a key signal that regulates the recycling and trafficking of glutamate receptors. In this review, we will discuss recent findings on the role of ubiquitination in the trafficking and turnover of ionotropic glutamate receptors and plasticity of excitatory synapses.

  14. Membrane Trafficking of Death Receptors: Implications on Signalling

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    Wulf Schneider-Brachert

    2013-07-01

    Full Text Available Death receptors were initially recognised as potent inducers of apoptotic cell death and soon ambitious attempts were made to exploit selective ignition of controlled cellular suicide as therapeutic strategy in malignant diseases. However, the complexity of death receptor signalling has increased substantially during recent years. Beyond activation of the apoptotic cascade, involvement in a variety of cellular processes including inflammation, proliferation and immune response was recognised. Mechanistically, these findings raised the question how multipurpose receptors can ensure selective activation of a particular pathway. A growing body of evidence points to an elegant spatiotemporal regulation of composition and assembly of the receptor-associated signalling complex. Upon ligand binding, receptor recruitment in specialized membrane compartments, formation of receptor-ligand clusters and internalisation processes constitute key regulatory elements. In this review, we will summarise the current concepts of death receptor trafficking and its implications on receptor-associated signalling events.

  15. Rapid glutamate receptor 2 trafficking during retinal degeneration

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    Lin Yanhua

    2012-02-01

    Full Text Available Abstract Background Retinal degenerations, such as age-related macular degeneration (AMD and retinitis pigmentosa (RP, are characterized by photoreceptor loss and anomalous remodeling of the surviving retina that corrupts visual processing and poses a barrier to late-stage therapeutic interventions in particular. However, the molecular events associated with retinal remodeling remain largely unknown. Given our prior evidence of ionotropic glutamate receptor (iGluR reprogramming in retinal degenerations, we hypothesized that the edited glutamate receptor 2 (GluR2 subunit and its trafficking may be modulated in retinal degenerations. Results Adult albino Balb/C mice were exposed to intense light for 24 h to induce light-induced retinal degeneration (LIRD. We found that prior to the onset of photoreceptor loss, protein levels of GluR2 and related trafficking proteins, including glutamate receptor-interacting protein 1 (GRIP1 and postsynaptic density protein 95 (PSD-95, were rapidly increased. LIRD triggered neuritogenesis in photoreceptor survival regions, where GluR2 and its trafficking proteins were expressed in the anomalous dendrites. Immunoprecipitation analysis showed interaction between KIF3A and GRIP1 as well as PSD-95, suggesting that KIF3A may mediate transport of GluR2 and its trafficking proteins to the novel dendrites. However, in areas of photoreceptor loss, GluR2 along with its trafficking proteins nearly vanished in retracted retinal neurites. Conclusions All together, LIRD rapidly triggers GluR2 plasticity, which is a potential mechanism behind functionally phenotypic revisions of retinal neurons and neuritogenesis during retinal degenerations.

  16. Molecular mechanisms of somatostatin receptor trafficking.

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    Csaba, Zsolt; Peineau, Stéphane; Dournaud, Pascal

    2012-02-01

    The neuropeptide somatostatin (SRIF) is an important modulator of neurotransmission in the central nervous system and acts as a potent inhibitor of hormone and exocrine secretion. In addition, SRIF regulates cell proliferation in normal and tumorous tissues. The six somatostatin receptor subtypes (sst1, sst2A, sst2B, sst3, sst4, and sst5), which belong to the G protein-coupled receptor (GPCR) family, share a common molecular topology: a hydrophobic core of seven transmembrane-spanning α-helices, three intracellular loops, three extracellular loops, an amino-terminus outside the cell, and a carboxyl-terminus inside the cell. For most of the GPCRs, intracytosolic sequences, and more particularly the C-terminus, are believed to interact with proteins that are mandatory for either exporting neosynthesized receptor, anchoring receptor at the plasma membrane, internalization, recycling, or degradation after ligand binding. Accordingly, most of the SRIF receptors can traffic not only in vitro within different cell types but also in vivo. A picture of the pathways and proteins involved in these processes is beginning to emerge.

  17. AMPA receptor trafficking in inflammation-induced dorsal horn central sensitization

    Institute of Scientific and Technical Information of China (English)

    Yuan-Xiang Tao

    2012-01-01

    Activity-dependent postsynaptic receptor trafficking is critical for long-term synaptic plasticity in the brain,but it is unclear whether this mechanism actually mediates the spinal cord dorsal horn central sensitization (a specific form of synaptic plasticity) that is associated with persistent pain.Recent studies have shown that peripheral inflammation drives changes in α-amino-3-hydroxy-5-methy1-4-isoxazolepropionic acid receptor (AMPAR) subunit trafficking in the dorsal horn and that such changes contribute to the hypersensitivity that underlies persistent pain.Here,we review current evidence to illustrate how spinal cord AMPARs participate in the dorsal horn central sensitization associated with persistent pain.Understanding these mechanisms may allow the development of novel therapeutic strategies for treating persistent pain.

  18. C. elegans SMA-10 regulates BMP receptor trafficking.

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    Gleason, Ryan J; Vora, Mehul; Li, Ying; Kane, Nanci S; Liao, Kelvin; Padgett, Richard W

    2017-01-01

    Signal transduction of the conserved transforming growth factor-β (TGFβ) family signaling pathway functions through two distinct serine/threonine transmembrane receptors, the type I and type II receptors. Endocytosis orchestrates the assembly of signaling complexes by coordinating the entry of receptors with their downstream signaling mediators. Recently, we showed that the C. elegans type I bone morphogenetic protein (BMP) receptor SMA-6, part of the TGFβ family, is recycled through the retromer complex while the type II receptor, DAF-4 is recycled in a retromer-independent, ARF-6 dependent manner. From genetic screens in C. elegans aimed at identifying new modifiers of BMP signaling, we reported on SMA-10, a conserved LRIG (leucine-rich and immunoglobulin-like domains) transmembrane protein. It is a positive regulator of BMP signaling that binds to the SMA-6 receptor. Here we show that the loss of sma-10 leads to aberrant endocytic trafficking of SMA-6, resulting in its accumulation in distinct intracellular endosomes including the early endosome, multivesicular bodies (MVB), and the late endosome with a reduction in signaling strength. Our studies show that trafficking defects caused by the loss of sma-10 are not universal, but affect only a limited set of receptors. Likewise, in Drosophila, we find that the fly homolog of sma-10, lambik (lbk), reduces signaling strength of the BMP pathway, consistent with its function in C. elegans and suggesting evolutionary conservation of function. Loss of sma-10 results in reduced ubiquitination of the type I receptor SMA-6, suggesting a possible mechanism for its regulation of BMP signaling.

  19. Actin-dependent mechanisms in AMPA receptor trafficking

    Directory of Open Access Journals (Sweden)

    Jonathan G Hanley

    2014-11-01

    Full Text Available The precise regulation of AMPA receptor (AMPAR number and subtype at the synapse is crucial for the regulation of excitatory neurotransmission, synaptic plasticity and the consequent formation of appropriate neural circuits during learning and memory. AMPAR trafficking involves the dynamic processes of exocytosis, endocytosis and endosomal recycling, all of which involve the actin cytoskeleton. The actin cytoskeleton is highly dynamic and highly regulated by an abundance of actin-binding proteins and upstream signalling pathways that modulate actin polymerization and depolymerisation. Actin dynamics generate forces that manipulate membranes in the process of vesicle biogenesis, and also for propelling vesicles through the cytoplasm to reach their destination. In addition, trafficking mechanisms exploit more stable aspects of the actin cytoskeleton by using actin-based motor proteins to traffic vesicular cargo along actin filaments. Numerous studies have shown that actin dynamics are critical for AMPAR localization and function. The identification of actin-binding proteins that physically interact with AMPAR subunits, and research into their mode of action is starting to shed light on the mechanisms involved. Such proteins either regulate actin dynamics to modulate mechanical forces exerted on AMPAR-containing membranes, or associate with actin filaments to target or transport AMPAR-containing vesicles to specific subcellular regions. In addition, actin-regulatory proteins that do not physically interact with AMPARs may influence AMPAR trafficking by regulating the local actin environment in the dendritic spine.

  20. Signal Transduction and Intracellular Trafficking by the Interleukin 36 Receptor*

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    Saha, Siddhartha S.; Singh, Divyendu; Raymond, Ernest L.; Ganesan, Rajkumar; Caviness, Gary; Grimaldi, Christine; Woska, Joseph R.; Mennerich, Detlev; Brown, Su-Ellen; Mbow, M. Lamine; Kao, C. Cheng

    2015-01-01

    Improper signaling of the IL-36 receptor (IL-36R), a member of the IL-1 receptor family, has been associated with various inflammation-associated diseases. However, the requirements for IL-36R signal transduction remain poorly characterized. This work seeks to define the requirements for IL-36R signaling and intracellular trafficking. In the absence of cognate agonists, IL-36R was endocytosed and recycled to the plasma membrane. In the presence of IL-36, IL-36R increased accumulation in LAMP1+ lysosomes. Endocytosis predominantly used a clathrin-mediated pathway, and the accumulation of the IL-36R in lysosomes did not result in increased receptor turnover. The ubiquitin-binding Tollip protein contributed to IL-36R signaling and increased the accumulation of both subunits of the IL-36R. PMID:26269592

  1. Sushi domains confer distinct trafficking profiles on GABAB receptors

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    Hannan, Saad; Wilkins, Megan E.; Smart, Trevor G.

    2012-01-01

    GABAB receptors mediate slow inhibitory neurotransmission in the brain and feature during excitatory synaptic plasticity, as well as various neurological conditions. These receptors are obligate heterodimers composed of GABABR1 and R2 subunits. The two predominant R1 isoforms differ by the presence of two complement control protein modules or Sushi domains (SDs) in the N terminus of R1a. By using live imaging, with an α-bungarotoxin-binding site (BBS) and fluorophore-linked bungarotoxin, we studied how R2 stabilizes R1b subunits at the cell surface. Heterodimerization with R2 reduced the rate of internalization of R1b, compared with R1b homomers. However, R1aR2 heteromers exhibited increased cell surface stability compared with R1bR2 receptors in hippocampal neurons, suggesting that for receptors containing the R1a subunit, the SDs play an additional role in the surface stability of GABAB receptors. Both SDs were necessary to increase the stability of R1aR2 because single deletions caused the receptors to be internalized at the same rate and extent as R1bR2 receptors. Consistent with these findings, a chimera formed from the metabotropic glutamate receptor (mGluR)2 and the SDs from R1a increased the surface stability of mGluR2. These results suggest a role for SDs in stabilizing cell surface receptors that could impart different pre- and postsynaptic trafficking itineraries on GABAB receptors, thereby contributing to their physiological and pathological roles. PMID:22778417

  2. Retromer-Mediated Trafficking of Transmembrane Receptors and Transporters

    Directory of Open Access Journals (Sweden)

    Stine C. Klinger

    2015-07-01

    Full Text Available Transport between the endoplasmatic reticulum, the Golgi-network, the endo-lysosomal system and the cell surface can be categorized as anterograde or retrograde, describing traffic that goes forward or backward, respectively. Traffic going from the plasma membrane to endosomes and lysosomes or the trans-Golgi network (TGN constitutes the major retrograde transport routes. Several transmembrane proteins undergo retrograde transport as part of a recycling mechanism that contributes to reutilization and maintenance of a steady-state protein localization. In addition, some receptors are hijacked by exotoxins and used for entry and intracellular transport. The physiological relevance of retrograde transport cannot be overstated. Retrograde trafficking of the amyloid precursor protein determines the distribution between organelles, and hence the possibility of cleavage by γ-secretase. Right balancing of the pathways is critical for protection against Alzheimer’s disease. During embryonic development, retrograde transport of Wntless to the TGN is essential for the following release of Wnt from the plasma membrane. Furthermore, overexpression of Wntless has been linked to oncogenesis. Here, we review relevant aspects of the retrograde trafficking of mammalian transmembrane receptors and transporters, with focus on the retromer-mediated transport between endosomes and the TGN.

  3. Real-time trafficking and signaling of the glucagon-like peptide-1 receptor

    DEFF Research Database (Denmark)

    Roed, Sarah Noerklit; Wismann, Pernille; Underwood, Christina Rye

    2014-01-01

    . A fundamental mechanism controlling the signaling capacity of GPCRs is the post-endocytic trafficking of receptors between recycling and degradative fates. Here, we combined microscopy with novel real-time assays to monitor both receptor trafficking and signaling in living cells. We find that the human GLP-1R...

  4. Structures of human folate receptors reveal biological trafficking states and diversity in folate and antifolate recognition.

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    Wibowo, Ardian S; Singh, Mirage; Reeder, Kristen M; Carter, Joshua J; Kovach, Alexander R; Meng, Wuyi; Ratnam, Manohar; Zhang, Faming; Dann, Charles E

    2013-09-17

    Antifolates, folate analogs that inhibit vitamin B9 (folic acid)-using cellular enzymes, have been used over several decades for the treatment of cancer and inflammatory diseases. Cellular uptake of the antifolates in clinical use occurs primarily via widely expressed facilitative membrane transporters. More recently, human folate receptors (FRs), high affinity receptors that transport folate via endocytosis, have been proposed as targets for the specific delivery of new classes of antifolates or folate conjugates to tumors or sites of inflammation. The development of specific, FR-targeted antifolates would be accelerated if additional biophysical data, particularly structural models of the receptors, were available. Here we describe six distinct crystallographic models that provide insight into biological trafficking of FRs and distinct binding modes of folate and antifolates to these receptors. From comparison of the structures, we delineate discrete structural conformations representative of key stages in the endocytic trafficking of FRs and propose models for pH-dependent conformational changes. Additionally, we describe the molecular details of human FR in complex with three clinically prevalent antifolates, pemetrexed (also Alimta), aminopterin, and methotrexate. On the whole, our data form the basis for rapid design and implementation of unique, FR-targeted, folate-based drugs for the treatment of cancer and inflammatory diseases.

  5. Modulation of cell surface GABA B receptors by desensitization,trafficking and regulated degradation

    Institute of Scientific and Technical Information of China (English)

    Dietmar; Benke; Khaled; Zemoura; Patrick; J; Maier

    2012-01-01

    Inhibitory neurotransmission ensures normal brain function by counteracting and integrating excitatory activity.-Aminobutyric acid (GABA) is the main inhibitory neurotransmitter in the mammalian central nervous system,and mediates its effects via two classes of receptors:the GABA A and GABA B receptors.GABA A receptors are heteropentameric GABA-gated chloride channels and responsible for fast inhibitory neurotransmission.GABA B receptors are heterodimeric G protein coupled receptors (GPCR) that mediate slow and prolonged inhibitory transmission.The extent of inhibitory neurotransmission is determined by a variety of factors,such as the degree of transmitter release and changes in receptor activity by posttranslational modifications (e.g.,phosphorylation),as well as by the number of receptors present in the plasma membrane available for signal transduction.The level of GABA B receptors at the cell surface critically depends on the residence time at the cell surface and finally the rates of endocytosis and degradation.In this review we focus primarily on recent advances in the understanding of trafficking mechanisms that determine the expression level of GABA B receptors in the plasma membrane,and thereby signaling strength.

  6. Synaptic AMPA receptor subunit trafficking is independent of the C terminus in the GluR2-lacking mouse.

    Science.gov (United States)

    Panicker, Sandip; Brown, Keith; Nicoll, Roger A

    2008-01-22

    Glutamate is the primary excitatory neurotransmitter in the brain, and alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) type glutamate receptors mediate most fast synaptic transmission. AMPA receptors are tetrameric assemblies composed from four possible subunits (GluR1-4). In hippocampal pyramidal cells, AMPA receptors are heteromeric receptors containing the GluR2 subunit and either GluR1 or GluR3. It is generally accepted that the trafficking of GluR1/GluR2 receptors to synapses requires activity, whereas GluR2/GluR3 receptors traffic constitutively. It has been suggested that the trafficking is governed by the cytoplasmic C termini of the subunits. Because the basis for this theory relied on the introduction of unnatural, homomeric, calcium-permeable AMPA receptors, we have used the GluR2(-/-) knock out mouse to determine whether the expression of mutated forms of GluR2 can rescue WT synaptic responses. We find that GluR2, lacking its entire C terminus, or a GluR2 chimera containing the C terminus of GluR1, is capable of trafficking to the synapse in the absence of activity. These findings suggest that the GluR2 C terminus is not required for GluR2 synaptic insertion.

  7. Functional Consequences of Glucagon-like Peptide-1 Receptor Cross-talk and Trafficking

    DEFF Research Database (Denmark)

    Roed, Sarah Noerklit; Nøhr, Anne Cathrine; Wismann, Pernille

    2015-01-01

    The signaling capacity of seven-transmembrane/G-protein-coupled receptors (7TM/GPCRs) can be regulated through ligand-mediated receptor trafficking. Classically, the recycling of internalized receptors is associated with resensitization, whereas receptor degradation terminates signaling. We have......) and glucagon (GCGR) receptors. The interaction and cross-talk between coexpressed receptors is a wide phenomenon of the 7TM/GPCR superfamily. Numerous reports show functional consequences for signaling and trafficking of the involved receptors. On the basis of the high structural similarity and tissue...... coexpression, we here investigated the potential cross-talk between GLP-1R and GIPR or GCGR in both trafficking and signaling pathways. Using a real-time time-resolved FRET-based internalization assay, we show that GLP-1R, GIPR, and GCGR internalize with differential properties. Remarkably, upon coexpression...

  8. Concurrent Imaging of Receptor Trafficking and Calcium Dynamics by Spinning Disk Confocal Microscopy.

    Science.gov (United States)

    Larsen, DeLaine D; Choy, Regina Wai-Yan; Park, Minjong

    2017-01-01

    Synaptic activity is modulated by the activation of neuromodulator receptors present in dendrites of neurons. The majority of neuromodulator receptors are G protein coupled receptors (GPCRs), in which membrane trafficking regulates their activities. Membrane trafficking of neuromodulator receptors and their signaling occurs on a rapid time scale and emerging studies indicate that neuromodulator receptors function not just from the plasma membrane but also from the endocytic compartments. Here, we describe a live cell imaging approach using spinning disk confocal microscopy to investigate the effect of neuromodulator receptor activation on synaptic activity by measuring calcium dynamics in primary rat striatal neurons. The advantages of spinning disk confocal microscopy and recent improvements in the genetically encoded calcium sensor, GCaMP6, provide an imaging approach to image both the receptor membrane trafficking to endocytic compartments, and calcium dynamics at a high spatial and temporal resolution. We believe this approach of imaging both the neuromodulator receptor membrane trafficking and synaptic activity using GCaMP6 is a powerful tool to address many questions regarding possible roles of membrane trafficking of neuromodulator receptors in synaptic activity.

  9. Mutations of Vasopressin Receptor 2 Including Novel L312S Have Differential Effects on Trafficking.

    Science.gov (United States)

    Tiulpakov, Anatoly; White, Carl W; Abhayawardana, Rekhati S; See, Heng B; Chan, Audrey S; Seeber, Ruth M; Heng, Julian I; Dedov, Ivan; Pavlos, Nathan J; Pfleger, Kevin D G

    2016-08-01

    Nephrogenic syndrome of inappropriate antidiuresis (NSIAD) is a genetic disease first described in 2 unrelated male infants with severe symptomatic hyponatremia. Despite undetectable arginine vasopressin levels, patients have inappropriately concentrated urine resulting in hyponatremia, hypoosmolality, and natriuresis. Here, we describe and functionally characterize a novel vasopressin type 2 receptor (V2R) gain-of-function mutation. An L312S substitution in the seventh transmembrane domain was identified in a boy presenting with water-induced hyponatremic seizures at the age of 5.8 years. We show that, compared with wild-type V2R, the L312S mutation results in the constitutive production of cAMP, indicative of the gain-of-function NSIAD profile. Interestingly, like the previously described F229V and I130N NSIAD-causing mutants, this appears to both occur in the absence of notable constitutive β-arrestin2 recruitment and can be reduced by the inverse agonist Tolvaptan. In addition, to understand the effect of various V2R substitutions on the full receptor "life-cycle," we have used and further developed a bioluminescence resonance energy transfer intracellular localization assay using multiple localization markers validated with confocal microscopy. This allowed us to characterize differences in the constitutive and ligand-induced localization and trafficking profiles of the novel L312S mutation as well as for previously described V2R gain-of-function mutants (NSIAD; R137C and R137L), loss-of-function mutants (nephrogenic diabetes insipidus; R137H, R181C, and M311V), and a putative silent V266A V2R polymorphism. In doing so, we describe differences in trafficking between unique V2R substitutions, even at the same amino acid position, therefore highlighting the value of full and thorough characterization of receptor function beyond simple signaling pathway analysis.

  10. 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.

  11. NMDA-receptor trafficking and targeting: implications for synaptic transmission and plasticity.

    Science.gov (United States)

    Carroll, Reed C; Zukin, R Suzanne

    2002-11-01

    Dynamic regulation of synaptic efficacy is thought to play a crucial role in formation of neuronal connections and in experience-dependent modification of neural circuitry. The molecular and cellular mechanisms by which synaptic changes are triggered and expressed are the focus of intense interest. This articles reviews recent evidence that NMDA receptors undergo dynamically regulated targeting and trafficking, and that the physical transport of NMDA receptors in and out of the synaptic membrane contributes to several forms of long-lasting synaptic plasticity. The identification of targeting and internalization sequences in NMDA-receptor subunits has begun the unraveling of some mechanisms that underlie activity-dependent redistribution of NMDA receptors. Given that NMDA receptors are widely expressed throughout the CNS, regulation of NMDA-receptor trafficking provides a potentially important way to modulate efficacy of synaptic transmission.

  12. An Integrated Model of Epidermal Growth Factor Receptor Trafficking and Signal Transduction

    Energy Technology Data Exchange (ETDEWEB)

    Resat, Haluk; Ewald, Jonathan A.; Dixon, David A.; Wiley, H. S.

    2003-08-01

    Endocytic trafficking of many types of receptors can have profound effects on subsequent signaling events. Quantitative models of these processes, however, have usually considered trafficking and signaling independently. Here, we present an integrated model of both the trafficking and signaling pathway of the epidermal growth factor receptor (EGFR) using a probability weighted-dynamic Monte Carlo simulation. Our model consists of hundreds of distinct endocytic compartments and about 13,000 reactions/events that occur over a broad spatio-temporal range. By using a realistic multi-compartment model, we can investigate the distribution of the receptors among cellular compartments as well as their potential signal transduction characteristics. Our new model also allows the incorporation of physio-chemical aspects of ligand-receptor interactions, such as pH-dependent binding in different endosomal compartments. To determine the utility of this approach, we simulated the differential activation of the EGFR by two of its ligands, epidermal growth factor (EGF) and transforming growth factor- alpha (TGF-a). Our simulations predict that when EGFR is activated with TGF-a, receptor activation is biased toward the cell surface whereas EGF produces a signaling bias towards the endosomal compartment. Experiments confirm these predictions from our model and simulations. Our model accurately predicts the kinetics and extent of receptor down-regulation induced by either EGF or TGF-a. Our results suggest that receptor trafficking controls the compartmental bias of signal transduction, rather than simply modulating signal magnitude. Our model provides a new approach to evaluating the complex effect of receptor trafficking on signal transduction. Importantly, the stochastic and compartmental nature of the simulation allows these models to be directly tested by high-throughput approaches, such as quantitative image analysis.

  13. The role of ubiquitination in lysosomal trafficking of δ-opioid receptors.

    Science.gov (United States)

    Henry, Anastasia G; White, Ian J; Marsh, Mark; von Zastrow, Mark; Hislop, James N

    2011-02-01

    The δ-opioid receptor (DOR) undergoes ligand-induced downregulation by endosomal sorting complex required for transport (ESCRT)-dependent endocytic trafficking to lysosomes. In contrast to a number of other signaling receptors, the DOR can downregulate effectively when its ubiquitination is prevented. We explored the membrane trafficking basis of this behavior. First, we show that internalized DORs traverse the canonical multivesicular body (MVB) pathway and localize to intralumenal vesicles (ILVs). Second, we show that DOR ubiquitination stimulates, but is not essential for, receptor transfer to ILVs and proteolysis of the receptor endodomain. Third, we show that receptor ubiquitination plays no detectable role in the early sorting of internalized DORs out of the recycling pathway. Finally, we show that DORs undergo extensive proteolytic fragmentation in the ectodomain, even when receptor ubiquitination is prevented or ILV formation itself is blocked. Together, these results are sufficient to explain why DORs downregulate effectively in the absence of ubiquitination, and they place a discrete molecular sorting operation in the MVB pathway effectively upstream of the ESCRT. More generally, these findings support the hypothesis that mammalian cells can control the cytoplasmic accessibility of internalized signaling receptors independently from their ultimate trafficking fate.

  14. Multiple motifs regulate the trafficking of GABA(B) receptors at distinct checkpoints within the secretory pathway.

    Science.gov (United States)

    Restituito, Sophie; Couve, Andrés; Bawagan, Hinayana; Jourdain, Sabine; Pangalos, Menelas N; Calver, Andrew R; Freeman, Katie B; Moss, Stephen J

    2005-04-01

    gamma-Aminobutyric acid type B receptors (GABA(B)) are G-protein-coupled receptors that mediate GABAergic inhibition in the brain. Their functional expression is dependent upon the formation of heterodimers between GABA(B)R1 and GABA(B)R2 subunits, a process that occurs within the endoplasmic reticulum (ER). However, the mechanisms that regulate receptor surface expression remain largely unknown. Here, we demonstrate that access to the cell surface for GABA(B)R1 is sequentially controlled by an RSR(R) motif and a LL motif within its cytoplasmic domain. In addition, we reveal that msec7-1, a guanine-nucleotide-exchange factor (GEF) for the ADP-ribosylation factor (ARF) family of GTPases, critical regulators of vesicular membrane trafficking, interacts with GABA(B)R1 via the LL motif in this subunit. Finally, we establish that msec7-1 modulates the cell surface expression of GABA(B) receptors, a process that is dependent upon the integrity of the LL motif in GABA(B)R1. Together, our results demonstrate that the cell surface expression of the GABA(B)R1 subunit is regulated by multiple motifs, which act at distinct checkpoints in the secretory pathway, and also suggest a novel role for msec7-1 in regulating the membrane trafficking of GABA(B)R1 subunits.

  15. Early Growth Response 1 (Egr-1) Regulates N-Methyl-d-aspartate Receptor (NMDAR)-dependent Transcription of PSD-95 and α-Amino-3-hydroxy-5-methyl-4-isoxazole Propionic Acid Receptor (AMPAR) Trafficking in Hippocampal Primary Neurons*

    Science.gov (United States)

    Qin, Xike; Jiang, Yongjun; Tse, Yiu Chung; Wang, Yunling; Wong, Tak Pan; Paudel, Hemant K.

    2015-01-01

    The N-methyl-d-aspartate receptor (NMDAR) controls synaptic plasticity and memory function and is one of the major inducers of transcription factor Egr-1 in the hippocampus. However, how Egr-1 mediates the NMDAR signal in neurons has remained unclear. Here, we show that the hippocampus of mice lacking Egr-1 displays electrophysiology properties and ultrastructure that are similar to mice overexpressing PSD-95, a major scaffolding protein of postsynaptic density involved in synapse formation, synaptic plasticity, and synaptic targeting of AMPA receptors (AMPARs), which mediate the vast majority of excitatory transmission in the CNS. We demonstrate that Egr-1 is a transcription repressor of the PSD-95 gene and is recruited to the PSD-95 promoter in response to NMDAR activation. Knockdown of Egr-1 in rat hippocampal primary neurons blocks NMDAR-induced PSD-95 down-regulation and AMPAR endocytosis. Likewise, overexpression of Egr-1 in rat hippocampal primary neurons causes reduction in PSD-95 protein level and promotes AMPAR endocytosis. Our data indicate that Egr-1 is involved in NMDAR-mediated PSD-95 down-regulation and AMPAR endocytosis, a process important in the expression of long term depression. PMID:26475861

  16. Trafficking of ErbB receptors and its influence on signaling

    Energy Technology Data Exchange (ETDEWEB)

    Wiley, H. S.

    2003-03-10

    Although members of the ErbB receptor family are found predominatly at the cell surface, these receptors undergo constant cycling between the plasma membrane and the endosomal compartment. In the absence of an activating ligand, these receptors are slowly internalized (t 1/2 ~ 30 min) are quickly recycled.The constitutive degradation rate of the epidermal growth factor (EGF) receptor (EGFR) is slower than other ErbB family members and only the EGFR appears to alter its trafficking pattern in response to ligand binding. Continued...

  17. Nuclear functions and subcellular trafficking mechanisms of the epidermal growth factor receptor family

    Science.gov (United States)

    2012-01-01

    Accumulating evidence suggests that various diseases, including many types of cancer, result from alteration of subcellular protein localization and compartmentalization. Therefore, it is worthwhile to expand our knowledge in subcellular trafficking of proteins, such as epidermal growth factor receptor (EGFR) and ErbB-2 of the receptor tyrosine kinases, which are highly expressed and activated in human malignancies and frequently correlated with poor prognosis. The well-characterized trafficking of cell surface EGFR is routed, via endocytosis and endosomal sorting, to either the lysosomes for degradation or back to the plasma membrane for recycling. A novel nuclear mode of EGFR signaling pathway has been gradually deciphered in which EGFR is shuttled from the cell surface to the nucleus after endocytosis, and there, it acts as a transcriptional regulator, transmits signals, and is involved in multiple biological functions, including cell proliferation, tumor progression, DNA repair and replication, and chemo- and radio-resistance. Internalized EGFR can also be transported from the cell surface to several intracellular compartments, such as the Golgi apparatus, the endoplasmic reticulum, and the mitochondria, in addition to the nucleus. In this review, we will summarize the functions of nuclear EGFR family and the potential pathways by which EGFR is trafficked from the cell surface to a variety of cellular organelles. A better understanding of the molecular mechanism of EGFR trafficking will shed light on both the receptor biology and potential therapeutic targets of anti-EGFR therapies for clinical application. PMID:22520625

  18. Nuclear functions and subcellular trafficking mechanisms of the epidermal growth factor receptor family

    Directory of Open Access Journals (Sweden)

    Wang Ying-Nai

    2012-04-01

    Full Text Available Abstract Accumulating evidence suggests that various diseases, including many types of cancer, result from alteration of subcellular protein localization and compartmentalization. Therefore, it is worthwhile to expand our knowledge in subcellular trafficking of proteins, such as epidermal growth factor receptor (EGFR and ErbB-2 of the receptor tyrosine kinases, which are highly expressed and activated in human malignancies and frequently correlated with poor prognosis. The well-characterized trafficking of cell surface EGFR is routed, via endocytosis and endosomal sorting, to either the lysosomes for degradation or back to the plasma membrane for recycling. A novel nuclear mode of EGFR signaling pathway has been gradually deciphered in which EGFR is shuttled from the cell surface to the nucleus after endocytosis, and there, it acts as a transcriptional regulator, transmits signals, and is involved in multiple biological functions, including cell proliferation, tumor progression, DNA repair and replication, and chemo- and radio-resistance. Internalized EGFR can also be transported from the cell surface to several intracellular compartments, such as the Golgi apparatus, the endoplasmic reticulum, and the mitochondria, in addition to the nucleus. In this review, we will summarize the functions of nuclear EGFR family and the potential pathways by which EGFR is trafficked from the cell surface to a variety of cellular organelles. A better understanding of the molecular mechanism of EGFR trafficking will shed light on both the receptor biology and potential therapeutic targets of anti-EGFR therapies for clinical application.

  19. REGULATION OF Fc RECEPTOR ENDOCYTIC TRAFFICKING BY UBIQUITINATION

    Directory of Open Access Journals (Sweden)

    Rosa eMolfetta

    2014-09-01

    Full Text Available Most immune cells, particularly phagocytes, express various receptors for the Fc-portion of the different immunoglobulin isotypes (Fc receptors, FcRs. By binding to the antibody, they provide a link between the adaptive immune system and the powerful effector functions triggered by innate immune cells such as mast cells, neutrophils, macrophages, and NK cells. Upon ligation of the immune complexes, the downstream signalling pathways initiated by the different receptors are quite similar for different FcR classes leading to the secretion of preformed and de novo synthesized pro-inflammatory mediators. FcR engagement also promotes negative signals through the combined action of several molecules that limit the extent and duration of positive signalling. To this regard, ligand-induced ubiquitination of Fc receptors for IgE (FcεR and IgG (FcγR has become recognized as a key modification that generates signals for the internalization and/or delivery of engaged receptor complexes to lysosomes or cytoplasmic proteasomes for degradation, providing negative-feedback regulation of Fc receptor activity.In this review, we discuss recent advances in our understanding of the molecular mechanisms that ensure the clearance of engaged Fcε and Fcγ receptor complexes from the cell surface with an emphasis given to the cooperation between the ubiquitin pathway and endosomal adaptors including the endosomal sorting complex required for transport (ESCRT in controlling receptor internalization and sorting along the endocytic compartments.

  20. Ferlins: regulators of vesicle fusion for auditory neurotransmission, receptor trafficking and membrane repair.

    Science.gov (United States)

    Lek, Angela; Evesson, Frances J; Sutton, R Bryan; North, Kathryn N; Cooper, Sandra T

    2012-02-01

    Ferlins are a family of multiple C2 domain proteins with emerging roles in vesicle fusion and membrane trafficking. Ferlin mutations are associated with muscular dystrophy (dysferlin) and deafness (otoferlin) in humans, and infertility in Caenorhabditis elegans (Fer-1) and Drosophila (misfire), demonstrating their importance for normal cellular functioning. Ferlins show ancient origins in eukaryotic evolution and are detected in all eukaryotic kingdoms, including unicellular eukaryotes and apicomplexian protists, suggesting origins in a common ancestor predating eukaryotic evolutionary branching. The characteristic feature of the ferlin family is their multiple tandem cytosolic C2 domains (five to seven C2 domains), the most of any protein family, and an extremely rare feature amongst eukaryotic proteins. Ferlins also bear a unique nested DysF domain and small conserved 60-70 residue ferlin-specific sequences (Fer domains). Ferlins segregate into two subtypes based on the presence (type I ferlin) or absence (type II ferlin) of the DysF and FerA domains. Ferlins have diverse tissue-specific and developmental expression patterns, with ferlin animal models united by pathologies arising from defects in vesicle fusion. Consistent with their proposed role in vesicle trafficking, ferlin interaction partners include cytoskeletal motors, other vesicle-associated trafficking proteins and transmembrane receptors or channels. Herein we summarize the research history of the ferlins, an intriguing family of structurally conserved proteins with a preserved ancestral function as regulators of vesicle fusion and receptor trafficking.

  1. The role of the prostaglandin D2 receptor, DP, in eosinophil trafficking

    DEFF Research Database (Denmark)

    Schratl, Petra; Royer, Julia F; Kostenis, Evi

    2007-01-01

    of DP has remained unclear. We report in this study that, in addition to CRTH2, the DP receptor plays an important role in eosinophil trafficking. First, we investigated the release of eosinophils from bone marrow using the in situ perfused guinea pig hind limb preparation. PGD2 induced the rapid...... that eosinophils in human bone marrow specimens expressed DP and CRTH2 receptors at similar levels. Eosinophils isolated from human peripheral blood likewise expressed DP receptor protein but at lower levels than CRTH2. In agreement with this, the chemotaxis of human peripheral blood eosinophils was inhibited both...

  2. Identification of Phosphorylation Sites Regulating sst3 Somatostatin Receptor Trafficking.

    Science.gov (United States)

    Lehmann, Andreas; Kliewer, Andrea; Günther, Thomas; Nagel, Falko; Schulz, Stefan

    2016-06-01

    The human somatostatin receptor 3 (sst3) is expressed in about 50% of all neuroendocrine tumors and hence a promising target for multireceptor somatostatin analogs. The sst3 receptor is unique among ssts in that it exhibits a very long intracellular C-terminal tail containing a huge number of potential phosphate acceptor sites. Consequently, our knowledge about the functional role of the C-terminal tail in sst3 receptor regulation is very limited. Here, we have generated a series of phosphorylation-deficient mutants that enabled us to determine crucial sites for its agonist-induced β-arrestin mobilization, internalization, and down-regulation. Based on this information, we generated phosphosite-specific antibodies for C-terminal Ser(337)/Thr(341), Thr(348), and Ser(361) that enabled us to investigate the temporal patterns of sst3 phosphorylation and dephosphorylation. We found that the endogenous ligand somatostatin induced a rapid and robust phosphorylation that was completely blocked by the sst3 antagonist NVP-ACQ090. The stable somatostatin analogs pasireotide and octreotide promoted clearly less phosphorylation compared with somatostatin. We also show that sst3 phosphorylation occurred within seconds to minutes, whereas dephosphorylation of the sst3 receptor occurred at a considerable slower rate. In addition, we also identified G protein-coupled receptor kinases 2 and 3 and protein phosphatase 1α and 1β as key regulators of sst3 phosphorylation and dephosphorylation, respectively. Thus, we here define the C-terminal phosphorylation motif of the human sst3 receptor that regulates its agonist-promoted phosphorylation, β-arrestin recruitment, and internalization of this clinically relevant receptor.

  3. PSD-95 expression controls l-DOPA dyskinesia through dopamine D1 receptor trafficking

    Science.gov (United States)

    Porras, Gregory; Berthet, Amandine; Dehay, Benjamin; Li, Qin; Ladepeche, Laurent; Normand, Elisabeth; Dovero, Sandra; Martinez, Audrey; Doudnikoff, Evelyne; Martin-Négrier, Marie-Laure; Chuan, Qin; Bloch, Bertrand; Choquet, Daniel; Boué-Grabot, Eric; Groc, Laurent; Bezard, Erwan

    2012-01-01

    l-DOPA–induced dyskinesia (LID), a detrimental consequence of dopamine replacement therapy for Parkinson’s disease, is associated with an alteration in dopamine D1 receptor (D1R) and glutamate receptor interactions. We hypothesized that the synaptic scaffolding protein PSD-95 plays a pivotal role in this process, as it interacts with D1R, regulates its trafficking and function, and is overexpressed in LID. Here, we demonstrate in rat and macaque models that disrupting the interaction between D1R and PSD-95 in the striatum reduces LID development and severity. Single quantum dot imaging revealed that this benefit was achieved primarily by destabilizing D1R localization, via increased lateral diffusion followed by increased internalization and diminished surface expression. These findings indicate that altering D1R trafficking via synapse-associated scaffolding proteins may be useful in the treatment of dyskinesia in Parkinson’s patients. PMID:23041629

  4. Linking supply to demand: the neuronal monocarboxylate transporter MCT2 and the alpha-amino-3-hydroxyl-5-methyl-4-isoxazole-propionic acid receptor GluR2/3 subunit are associated in a common trafficking process.

    Science.gov (United States)

    Pierre, Karin; Chatton, Jean-Yves; Parent, Annabelle; Repond, Cendrine; Gardoni, Fabrizio; Di Luca, Monica; Pellerin, Luc

    2009-05-01

    MCT2 is the major neuronal monocarboxylate transporter (MCT) that allows the supply of alternative energy substrates such as lactate to neurons. Recent evidence obtained by electron microscopy has demonstrated that MCT2, like alpha-amino-3-hydroxyl-5-methyl-4-isoxazole-propionic acid (AMPA) receptors, is localized in dendritic spines of glutamatergic synapses. Using immunofluorescence, we show in this study that MCT2 colocalizes extensively with GluR2/3 subunits of AMPA receptors in neurons from various mouse brain regions as well as in cultured neurons. It also colocalizes with GluR2/3-interacting proteins, such as C-kinase-interacting protein 1, glutamate receptor-interacting protein 1 and clathrin adaptor protein. Coimmunoprecipitation of MCT2 with GluR2/3 and C-kinase-interacting protein 1 suggests their close interaction within spines. Parallel changes in the localization of both MCT2 and GluR2/3 subunits at and beneath the plasma membrane upon various stimulation paradigms were unraveled using an original immunocytochemical and transfection approach combined with three-dimensional image reconstruction. Cell culture incubation with AMPA or insulin triggered a marked intracellular accumulation of both MCT2 and GluR2/3, whereas both tumor necrosis factor alpha and glycine (with glutamate) increased their cell surface immunolabeling. Similar results were obtained using Western blots performed on membrane or cytoplasm-enriched cell fractions. Finally, an enhanced lactate flux into neurons was demonstrated after MCT2 translocation on the cell surface. These observations provide unequivocal evidence that MCT2 is linked to AMPA receptor GluR2/3 subunits and undergoes a similar translocation process in neurons upon activation. MCT2 emerges as a novel component of the synaptic machinery putatively linking neuroenergetics to synaptic transmission.

  5. Drug Trafficking into Macrophages via the Endocytotic Receptor CD163.

    Science.gov (United States)

    Graversen, Jonas Heilskov; Moestrup, Søren Kragh

    2015-06-23

    In inflammatory diseases, macrophages are a main producer of a range of cytokines regulating the inflammatory state. This also includes inflammation induced by tumor growth, which recruits so-called tumor-associated macrophages supporting tumor growth. Macrophages are therefore relevant targets for cytotoxic or phenotype-modulating drugs in the treatment of inflammatory and cancerous diseases. Such targeting of macrophages has been tried using the natural propensity of macrophages to non-specifically phagocytose circulating foreign particulate material. In addition, the specific targeting of macrophage-expressed receptors has been used in order to obtain a selective uptake in macrophages and reduce adverse effects of off-target delivery of drugs. CD163 is a highly expressed macrophage-specific endocytic receptor that has been studied for intracellular delivery of small molecule drugs to macrophages using targeted liposomes or antibody drug conjugates. This review will focus on the biology of CD163 and its potential role as a target for selective macrophage targeting compared with other macrophage targeting approaches.

  6. Drug Trafficking into Macrophages via the Endocytotic Receptor CD163

    Science.gov (United States)

    Graversen, Jonas Heilskov; Moestrup, Søren Kragh

    2015-01-01

    In inflammatory diseases, macrophages are a main producer of a range of cytokines regulating the inflammatory state. This also includes inflammation induced by tumor growth, which recruits so-called tumor-associated macrophages supporting tumor growth. Macrophages are therefore relevant targets for cytotoxic or phenotype-modulating drugs in the treatment of inflammatory and cancerous diseases. Such targeting of macrophages has been tried using the natural propensity of macrophages to non-specifically phagocytose circulating foreign particulate material. In addition, the specific targeting of macrophage-expressed receptors has been used in order to obtain a selective uptake in macrophages and reduce adverse effects of off-target delivery of drugs. CD163 is a highly expressed macrophage-specific endocytic receptor that has been studied for intracellular delivery of small molecule drugs to macrophages using targeted liposomes or antibody drug conjugates. This review will focus on the biology of CD163 and its potential role as a target for selective macrophage targeting compared with other macrophage targeting approaches. PMID:26111002

  7. Hormonal regulation of AMPA receptor trafficking and memory formation

    Directory of Open Access Journals (Sweden)

    Harmen J Krugers

    2009-10-01

    Full Text Available Humans and rodents retain memories for stressful events very well. The facilitated retention of these memories is normally very useful. However, in susceptible individuals a variety of pathological conditions may develop in which memories related to stressful events remain inappropriately present, such as in post-traumatic stress disorder. The memory enhancing effects of stress are mediated by hormones, such as norepinephrine and glucocorticoids which are released during stressful experiences. Here we review recently identified molecular mechanisms that underlie the effects of stress hormones on synaptic efficacy and learning and memory. We discuss AMPA receptors as major target for stress hormones and describe a model in which norepinephrine and glucocorticoids are able to strengthen and prolong different phases of stressful memories.

  8. Calcium-Sensing Receptor: Trafficking, Endocytosis, Recycling, and Importance of Interacting Proteins.

    Science.gov (United States)

    Ray, Kausik

    2015-01-01

    The cloning of the extracellular calcium-sensing receptor (CaSR) provided a new paradigm in G-protein-coupled receptor (GPCR) signaling in which principal physiological ligand is a cation, namely, extracellular calcium (Ca(o)(2+)). A wealth of information has accumulated in the past two decades about the CaSR's structure and function, its contribution to pathology in disorders of calcium in humans, and CaSR-based therapeutics. The CaSR unlike many other GPCRs must function in the presence of its ligand, thus understanding the mechanisms such as anterograde trafficking and endocytic pathways of this receptor are complex and fallen behind other classical GPCRs. Factors controlling CaSR signaling include various proteins affecting the expression of the CaSR as well as modulation of its trafficking to and from the cell surface. The dimeric cell-surface CaSR links to various heterotrimeric G-proteins (G(q/11), G(i/o), G(12/13), and G(s)) to regulate intracellular second messengers, lipid kinases, various protein kinases, and transcription factors that are part of the machinery enabling the receptor to modulate the functions of the wide variety of cells in which it is expressed. This chapter describes key features of CaSR structure and function and discusses novel mechanisms by which the level of cell-surface receptor expression can be regulated including forward trafficking during biosynthesis, desensitization, internalization and recycling from the cell surface, and degradation. These processes are impacted by its interactions with several proteins in addition to signaling molecules per se (i.e., G-proteins, protein kinases, inositol phosphates, etc.) and include small molecular weight G-proteins (Sar1, Rabs, ARF, P24A, RAMPs, filamin A, 14-3-3 proteins, calmodulin, and caveolin-1). Moreover, CaSR signaling seems compartmentalized in cell-type-specific manner, and caveolin and filamin A likely act as scaffolds that bind signaling components and other key cellular

  9. GABA type a receptor trafficking and the architecture of synaptic inhibition.

    Science.gov (United States)

    Lorenz-Guertin, Joshua M; Jacob, Tija C

    2017-09-13

    Ubiquitous expression of GABA type A receptors (GABAA R) in the central nervous system establishes their central role in coordinating most aspects of neural function and development. Dysregulation of GABAergic neurotransmission manifests in a number of human health disorders and conditions that in certain cases can be alleviated by drugs targeting these receptors. Precise changes in the quantity or activity of GABAA Rs localized at the cell surface and at GABAergic postsynaptic sites directly impact the strength of inhibition. The molecular mechanisms constituting receptor trafficking to and from these compartments therefore dictate the efficacy of GABAA R function. Here we review the current understanding of how GABAA Rs traffic through biogenesis, plasma membrane transport, and degradation. Emphasis is placed on discussing novel GABAergic synaptic proteins, receptor and scaffolding post-translational modifications, activity-dependent changes in GABAA R confinement, and neuropeptide and neurosteroid mediated changes. We further highlight modern techniques currently advancing the knowledge of GABAA R trafficking and clinically relevant neurodevelopmental diseases connected to GABAergic dysfunction. © 2017 Wiley Periodicals, Inc. Develop Neurobiol, 2017. © 2017 Wiley Periodicals, Inc.

  10. Mu opioid receptor-effector coupling and trafficking in dorsal root ganglia neurons.

    Science.gov (United States)

    Walwyn, W M; Wei, W; Xie, C-W; Chiu, K; Kieffer, B L; Evans, C J; Maidment, N T

    2006-10-13

    Morphine induces profound analgesic tolerance in vivo despite inducing little internalization of the mu opioid receptor (muOR). Previously proposed explanations suggest that this lack of internalization could either lead to prolonged signaling and associated compensatory changes in downstream signaling systems, or that the receptor is unable to recycle and resensitize and so loses efficacy, either mechanism resulting in tolerance. We therefore examined, in cultured neurons, the relationship between muOR internalization and desensitization in response to two agonists, D-Ala2, N-MePhe4, Gly5-ol-enkephalin (DAMGO) and morphine. In addition, we studied the chimeric mu/delta opioid receptor (mu/ partial differentialOR) which could affect internalization and desensitization in neurons. Dorsal root ganglia neurons from muOR knockout mice were transduced with an adenovirus expressing either receptor and their respective internalization, desensitization and trafficking profiles determined. Both receptors desensitized equally, measured by Ca2+ current inhibition, during the first 5 min of agonist exposure to DAMGO or morphine treatment, although the mu/partial differentialOR desensitized more extensively. Such rapid desensitization was unrelated to internalization as DAMGO, but not morphine, internalized both receptors after 20 min. In response to DAMGO the mu/partial differentialOR internalized more rapidly than the muOR and was trafficked through Rab4-positive endosomes and lysosomal-associated membrane protein-1-labeled lysosomes whereas the muOR was trafficked through Rab4 and Rab11-positive endosomes. Chronic desensitization of the Ca2+ current response, after 24 h of morphine or DAMGO incubation, was seen in the DAMGO, but not morphine-treated, muOR-expressing cells. Such persistence of signaling after chronic morphine treatment suggests that compensation of downstream signaling systems, rather than loss of efficacy due to poor receptor recycling, is a more likely

  11. Dynamic Na+-H+ Exchanger Regulatory Factor-1 Association and Dissociation Regulate Parathyroid Hormone Receptor Trafficking at Membrane Microdomains*

    Science.gov (United States)

    Ardura, Juan A.; Wang, Bin; Watkins, Simon C.; Vilardaga, Jean-Pierre; Friedman, Peter A.

    2011-01-01

    Na/H exchanger regulatory factor-1 (NHERF1) is a cytoplasmic PDZ (postsynaptic density 95/disc large/zona occludens) protein that assembles macromolecular complexes and determines the localization, trafficking, and signaling of select G protein-coupled receptors and other membrane-delimited proteins. The parathyroid hormone receptor (PTHR), which regulates mineral ion homeostasis and bone turnover, is a G protein-coupled receptor harboring a PDZ-binding motif that enables association with NHERF1 and tethering to the actin cytoskeleton. NHERF1 interactions with the PTHR modify its trafficking and signaling. Here, we characterized by live cell imaging the mechanism whereby NHERF1 coordinates the interactions of multiple proteins, as well as the fate of NHERF1 itself upon receptor activation. Upon PTHR stimulation, NHERF1 rapidly dissociates from the receptor and induces receptor aggregation in long lasting clusters that are enriched with the actin-binding protein ezrin and with clathrin. After NHERF1 dissociates from the PTHR, ezrin then directly interacts with the PTHR to stabilize the PTHR at the cell membrane. Recruitment of β-arrestins to the PTHR is delayed until NHERF1 dissociates from the receptor, which is then trafficked to clathrin for internalization. The ability of NHERF to interact dynamically with the PTHR and cognate adapter proteins regulates receptor trafficking and signaling in a spatially and temporally coordinated manner. PMID:21832055

  12. Dynamic Na+-H+ exchanger regulatory factor-1 association and dissociation regulate parathyroid hormone receptor trafficking at membrane microdomains.

    Science.gov (United States)

    Ardura, Juan A; Wang, Bin; Watkins, Simon C; Vilardaga, Jean-Pierre; Friedman, Peter A

    2011-10-07

    Na/H exchanger regulatory factor-1 (NHERF1) is a cytoplasmic PDZ (postsynaptic density 95/disc large/zona occludens) protein that assembles macromolecular complexes and determines the localization, trafficking, and signaling of select G protein-coupled receptors and other membrane-delimited proteins. The parathyroid hormone receptor (PTHR), which regulates mineral ion homeostasis and bone turnover, is a G protein-coupled receptor harboring a PDZ-binding motif that enables association with NHERF1 and tethering to the actin cytoskeleton. NHERF1 interactions with the PTHR modify its trafficking and signaling. Here, we characterized by live cell imaging the mechanism whereby NHERF1 coordinates the interactions of multiple proteins, as well as the fate of NHERF1 itself upon receptor activation. Upon PTHR stimulation, NHERF1 rapidly dissociates from the receptor and induces receptor aggregation in long lasting clusters that are enriched with the actin-binding protein ezrin and with clathrin. After NHERF1 dissociates from the PTHR, ezrin then directly interacts with the PTHR to stabilize the PTHR at the cell membrane. Recruitment of β-arrestins to the PTHR is delayed until NHERF1 dissociates from the receptor, which is then trafficked to clathrin for internalization. The ability of NHERF to interact dynamically with the PTHR and cognate adapter proteins regulates receptor trafficking and signaling in a spatially and temporally coordinated manner.

  13. The deubiquitinating enzyme USP8 promotes trafficking and degradation of the chemokine receptor 4 at the sorting endosome.

    Science.gov (United States)

    Berlin, Ilana; Higginbotham, Katherine M; Dise, Rebecca S; Sierra, Maria I; Nash, Piers D

    2010-11-26

    Reversible ubiquitination orchestrated by the opposition of ubiquitin ligases and deubiquitinating enzymes mediates endocytic trafficking of cell surface receptors for lysosomal degradation. Ubiquitin-specific protease 8 (USP8) has previously been implicated in endocytosis of several receptors by virtue of their deubiquitination. The present study explores an indirect role for USP8 in cargo trafficking through its regulation of the chemokine receptor 4 (CXCR4). Contrary to the effects of USP8 loss on enhanced green fluorescent protein, we find that USP8 depletion stabilizes CXCR4 on the cell surface and attenuates receptor degradation without affecting its ubiquitination status. In the presence of ligand, diminished CXCR4 turnover is accompanied by receptor accumulation on enlarged early endosomes and leads to enhancement of phospho-ERK signaling. Perturbation in CXCR4 trafficking, resulting from USP8 inactivation, occurs at the ESCRT-0 checkpoint, and catalytic mutation of USP8 specifically targeted to the ESCRT-0 complex impairs the spatial and temporal organization of the sorting endosome. USP8 functionally opposes the ubiquitin ligase AIP4 with respect to ESCRT-0 ubiquitination, thereby promoting trafficking of CXCR4. Collectively, our findings demonstrate a functional cooperation between USP8, AIP4, and the ESCRT-0 machinery at the early sorting phase of CXCR4 and underscore the versatility of USP8 in shaping trafficking events at the early-to-late endosome transition.

  14. The binding of NCAM to FGFR1 induces a specific cellular response mediated by receptor trafficking

    DEFF Research Database (Denmark)

    Francavilla, Chiara; Cattaneo, Paola; Berezin, Vladimir

    2009-01-01

    different from that elicited by FGF-2. In contrast to FGF-induced degradation of endocytic FGFR1, NCAM promotes the stabilization of the receptor, which is recycled to the cell surface in a Rab11- and Src-dependent manner. In turn, FGFR1 recycling is required for NCAM-induced sustained activation of various...... effectors. Furthermore, NCAM, but not FGF-2, promotes cell migration, and this response depends on FGFR1 recycling and sustained Src activation. Our results implicate NCAM as a nonconventional ligand for FGFR1 that exerts a peculiar control on the intracellular trafficking of the receptor, resulting...... in a specific cellular response. Besides introducing a further level of complexity in the regulation of FGFR1 function, our findings highlight the link of FGFR recycling with sustained signaling and cell migration and the critical role of these events in dictating the cellular response evoked by receptor...

  15. PICK1 interacts with ABP/GRIP to regulate AMPA receptor trafficking.

    Science.gov (United States)

    Lu, Wei; Ziff, Edward B

    2005-08-04

    PICK1 and ABP/GRIP bind to the AMPA receptor (AMPAR) GluR2 subunit C terminus. Transfer of the receptor from ABP/GRIP to PICK1, facilitated by GluR2 S880 phosphorylation, may initiate receptor trafficking. Here we report protein interactions that regulate these steps. The PICK1 BAR domain interacts intermolecularly with the ABP/GRIP linker II region and intramolecularly with the PICK1 PDZ domain. Binding of PKCalpha or GluR2 to the PICK1 PDZ domain disrupts the intramolecular interaction and facilitates the PICK1 BAR domain association with ABP/GRIP. Interference with the PICK1-ABP/GRIP interaction impairs S880 phosphorylation of GluR2 by PKC and decreases the constitutive surface expression of GluR2, the NMDA-induced endocytosis of GluR2, and recycling of internalized GluR2. We suggest that the PICK1 interaction with ABP/GRIP is a critical step in controlling GluR2 trafficking.

  16. In silico investigation of ADAM12 effect on TGF-β receptors trafficking

    Directory of Open Access Journals (Sweden)

    LeMeur Nolwenn

    2009-09-01

    Full Text Available Abstract Background The transforming growth factor beta is known to have pleiotropic effects, including differentiation, proliferation and apoptosis. However the underlying mechanisms remain poorly understood. The regulation and effect of TGF-β signaling is complex and highly depends on specific protein context. In liver, we have recently showed that the disintegrin and metalloproteinase ADAM12 interacts with TGF-β receptors and modulates their trafficking among membranes, a crucial point in TGF-β signaling and development of fibrosis. The present study aims to better understand how ADAM12 impacts on TGF-β receptors trafficking and TGF-β signaling. Findings We extracted qualitative biological observations from experimental data and defined a family of models producing a behavior compatible with the presence of ADAM12. We computationally explored the properties of this family of models which allowed us to make novel predictions. We predict that ADAM12 increases TGF-β receptors internalization rate between the cell surface and the endosomal membrane. It also appears that ADAM12 modifies TGF-β signaling shape favoring a permanent response by removing the transient component observed under physiological conditions. Conclusion In this work, confronting differential models with qualitative biological observations, we obtained predictions giving new insights into the role of ADAM12 in TGF-β signaling and hepatic fibrosis process.

  17. Distinct human and mouse membrane trafficking systems for sweet taste receptors T1r2 and T1r3.

    Science.gov (United States)

    Shimizu, Madoka; Goto, Masao; Kawai, Takayuki; Yamashita, Atsuko; Kusakabe, Yuko

    2014-01-01

    The sweet taste receptors T1r2 and T1r3 are included in the T1r taste receptor family that belongs to class C of the G protein-coupled receptors. Heterodimerization of T1r2 and T1r3 is required for the perception of sweet substances, but little is known about the mechanisms underlying this heterodimerization, including membrane trafficking. We developed tagged mouse T1r2 and T1r3, and human T1R2 and T1R3 and evaluated membrane trafficking in human embryonic kidney 293 (HEK293) cells. We found that human T1R3 surface expression was only observed when human T1R3 was coexpressed with human T1R2, whereas mouse T1r3 was expressed without mouse T1r2 expression. A domain-swapped chimera and truncated human T1R3 mutant showed that the Venus flytrap module and cysteine-rich domain (CRD) of human T1R3 contain a region related to the inhibition of human T1R3 membrane trafficking and coordinated regulation of human T1R3 membrane trafficking. We also found that the Venus flytrap module of both human T1R2 and T1R3 are needed for membrane trafficking, suggesting that the coexpression of human T1R2 and T1R3 is required for this event. These results suggest that the Venus flytrap module and CRD receive taste substances and play roles in membrane trafficking of human T1R2 and T1R3. These features are different from those of mouse receptors, indicating that human T1R2 and T1R3 are likely to have a novel membrane trafficking system.

  18. The Role of Palmitoylation in Signalling, Cellular Trafficking and Plasma Membrane Localization of Protease-Activated Receptor-2

    OpenAIRE

    2011-01-01

    Protease-activated receptor-2 (PAR2) is a G protein coupled receptor (GPCR) activated by proteolytic cleavage of its amino terminal domain by trypsin-like serine proteases. This irreversible activation mechanism leads to rapid receptor desensitization by internalisation and degradation. We have explored the role of palmitoylation, the post-translational addition of palmitate, in PAR2 signalling, trafficking, cell surface expression and desensitization. Experiments using the palmitoylation inh...

  19. A naturally occurring isoform inhibits parathyroid hormone receptor trafficking and signaling.

    Science.gov (United States)

    Alonso, Verónica; Ardura, Juan A; Wang, Bin; Sneddon, W Bruce; Friedman, Peter A

    2011-01-01

    Parathyroid hormone (PTH) regulates calcium homeostasis and bone remodeling through its cognitive receptor (PTHR). We describe here a PTHR isoform harboring an in-frame 42-bp deletion of exon 14 (Δe14-PTHR) that encodes transmembrane domain 7. Δe14-PTHR was detected in human kidney and buccal epithelial cells. We characterized its topology, cellular localization, and signaling, as well as its interactions with PTHR. The C-terminus of the Δe14-PTHR is extracellular, and cell surface expression is strikingly reduced compared with the PTHR. Δe14-PTHR displayed impaired trafficking and accumulated in endoplasmic reticulum. Signaling and activation of cAMP and ERK by Δe14-PTHR was decreased significantly compared with PTHR. Δe14-PTHR acts as a functional dominant-negative by suppressing the action of PTHR. Cells cotransfected with both receptors exhibit markedly reduced PTHR cell membrane expression, colocalization with Δe14-PTHR in endoplasmic reticulum, and diminished cAMP activation and ERK phosphorylation in response to challenge with PTH. Δe14-PTHR forms heterodimers with PTHR, which may account for cytoplasmic retention of PTHR in the presence of Δe14-PTHR. Analysis of the PTHR heteronuclear RNA suggests that base-pair complementarity in introns surrounding exon 14 causes exon skipping and accounts for generation of the Δe14-PTHR isoform. Thus Δe14-PTHR is a poorly functional receptor that acts as a dominant-negative of PTHR trafficking and signaling and may contribute to PTH resistance. © 2011 American Society for Bone and Mineral Research.

  20. Regulation of N-Formyl Peptide Receptor Signaling and Trafficking by Arrestin-Src Kinase Interaction.

    Science.gov (United States)

    Wagener, Brant M; Marjon, Nicole A; Prossnitz, Eric R

    2016-01-01

    Arrestins were originally described as proteins recruited to ligand-activated, phosphorylated G protein-coupled receptors (GPCRs) to attenuate G protein-mediated signaling. It was later revealed that arrestins also mediate GPCR internalization and recruit a number of signaling proteins including, but not limited to, Src family kinases, ERK1/2, and JNK3. GPCR-arrestin binding and trafficking control the spatial and temporal activity of these multi-protein complexes. In previous reports, we concluded that N-formyl peptide receptor (FPR)-mediated apoptosis, which occurs upon receptor stimulation in the absence of arrestins, is associated with FPR accumulation in perinuclear recycling endosomes. Under these conditions, inhibition of Src kinase and ERK1/2 prevented FPR-mediated apoptosis. To better understand the role of Src kinase in this process, in the current study we employed a previously described arrestin-2 (arr2) mutant deficient in Src kinase binding (arr2-P91G/P121E). Unlike wild type arrestin, arr2-P91G/P121E did not inhibit FPR-mediated apoptosis, suggesting that Src binding to arrestin-2 prevents apoptotic signaling. However, in cells expressing this mutant, FPR-mediated apoptosis was still blocked by inhibition of Src kinase activity, suggesting that activation of Src independent of arrestin-2 binding is involved in FPR-mediated apoptosis. Finally, while Src kinase inhibition prevented FPR-mediated-apoptosis in the presence of arr2-P91G/P121E, it did not prevent FPR-arr2-P91G/P121E accumulation in the perinuclear recycling endosome. On the contrary, inhibition of Src kinase activity mediated the accumulation of activated FPR-wild type arrestin-2 in recycling endosomes without initiating FPR-mediated apoptosis. Based on these observations, we conclude that Src kinase has two independent roles following FPR activation that regulate both FPR-arrestin-2 signaling and trafficking.

  1. Regulation of N-Formyl Peptide Receptor Signaling and Trafficking by Arrestin-Src Kinase Interaction.

    Directory of Open Access Journals (Sweden)

    Brant M Wagener

    Full Text Available Arrestins were originally described as proteins recruited to ligand-activated, phosphorylated G protein-coupled receptors (GPCRs to attenuate G protein-mediated signaling. It was later revealed that arrestins also mediate GPCR internalization and recruit a number of signaling proteins including, but not limited to, Src family kinases, ERK1/2, and JNK3. GPCR-arrestin binding and trafficking control the spatial and temporal activity of these multi-protein complexes. In previous reports, we concluded that N-formyl peptide receptor (FPR-mediated apoptosis, which occurs upon receptor stimulation in the absence of arrestins, is associated with FPR accumulation in perinuclear recycling endosomes. Under these conditions, inhibition of Src kinase and ERK1/2 prevented FPR-mediated apoptosis. To better understand the role of Src kinase in this process, in the current study we employed a previously described arrestin-2 (arr2 mutant deficient in Src kinase binding (arr2-P91G/P121E. Unlike wild type arrestin, arr2-P91G/P121E did not inhibit FPR-mediated apoptosis, suggesting that Src binding to arrestin-2 prevents apoptotic signaling. However, in cells expressing this mutant, FPR-mediated apoptosis was still blocked by inhibition of Src kinase activity, suggesting that activation of Src independent of arrestin-2 binding is involved in FPR-mediated apoptosis. Finally, while Src kinase inhibition prevented FPR-mediated-apoptosis in the presence of arr2-P91G/P121E, it did not prevent FPR-arr2-P91G/P121E accumulation in the perinuclear recycling endosome. On the contrary, inhibition of Src kinase activity mediated the accumulation of activated FPR-wild type arrestin-2 in recycling endosomes without initiating FPR-mediated apoptosis. Based on these observations, we conclude that Src kinase has two independent roles following FPR activation that regulate both FPR-arrestin-2 signaling and trafficking.

  2. LRRK2 affects vesicle trafficking, neurotransmitter extracellular level and membrane receptor localization.

    Directory of Open Access Journals (Sweden)

    Rossana Migheli

    Full Text Available The leucine-rich repeat kinase 2 (LRRK2 gene was found to play a role in the pathogenesis of both familial and sporadic Parkinson's disease (PD. LRRK2 encodes a large multi-domain protein that is expressed in different tissues. To date, the physiological and pathological functions of LRRK2 are not clearly defined. In this study we have explored the role of LRRK2 in controlling vesicle trafficking in different cellular or animal models and using various readouts. In neuronal cells, the presence of LRRK2(G2019S pathological mutant determines increased extracellular dopamine levels either under basal conditions or upon nicotine stimulation. Moreover, mutant LRRK2 affects the levels of dopamine receptor D1 on the membrane surface in neuronal cells or animal models. Ultrastructural analysis of PC12-derived cells expressing mutant LRRK2(G2019S shows an altered intracellular vesicle distribution. Taken together, our results point to the key role of LRRK2 to control vesicle trafficking in neuronal cells.

  3. LRRK2 Affects Vesicle Trafficking, Neurotransmitter Extracellular Level and Membrane Receptor Localization

    Science.gov (United States)

    Spissu, Ylenia; Sanna, Giovanna; Xiong, Yulan; Dawson, Ted M.; Dawson, Valina L.; Galioto, Manuela; Rocchitta, Gaia; Biosa, Alice; Serra, Pier Andrea; Carri, Maria Teresa; Crosio, Claudia; Iaccarino, Ciro

    2013-01-01

    The leucine-rich repeat kinase 2 (LRRK2) gene was found to play a role in the pathogenesis of both familial and sporadic Parkinson’s disease (PD). LRRK2 encodes a large multi-domain protein that is expressed in different tissues. To date, the physiological and pathological functions of LRRK2 are not clearly defined. In this study we have explored the role of LRRK2 in controlling vesicle trafficking in different cellular or animal models and using various readouts. In neuronal cells, the presence of LRRK2G2019S pathological mutant determines increased extracellular dopamine levels either under basal conditions or upon nicotine stimulation. Moreover, mutant LRRK2 affects the levels of dopamine receptor D1 on the membrane surface in neuronal cells or animal models. Ultrastructural analysis of PC12-derived cells expressing mutant LRRK2G2019S shows an altered intracellular vesicle distribution. Taken together, our results point to the key role of LRRK2 to control vesicle trafficking in neuronal cells. PMID:24167564

  4. The role of palmitoylation in signalling, cellular trafficking and plasma membrane localization of protease-activated receptor-2.

    Directory of Open Access Journals (Sweden)

    Mark N Adams

    Full Text Available Protease-activated receptor-2 (PAR2 is a G protein coupled receptor (GPCR activated by proteolytic cleavage of its amino terminal domain by trypsin-like serine proteases. This irreversible activation mechanism leads to rapid receptor desensitization by internalisation and degradation. We have explored the role of palmitoylation, the post-translational addition of palmitate, in PAR2 signalling, trafficking, cell surface expression and desensitization. Experiments using the palmitoylation inhibitor 2-bromopalmitate indicated that palmitate addition is important in trafficking of PAR2 endogenously expressed by prostate cancer cell lines. This was supported by palmitate labelling using two approaches, which showed that PAR2 stably expressed by CHO-K1 cells is palmitoylated and that palmitoylation occurs on cysteine 361. Palmitoylation is required for optimal PAR2 signalling as Ca²⁺ flux assays indicated that in response to trypsin agonism, palmitoylation deficient PAR2 is ∼9 fold less potent than wildtype receptor with a reduction of about 33% in the maximum signal induced via the mutant receptor. Confocal microscopy, flow cytometry and cell surface biotinylation analyses demonstrated that palmitoylation is required for efficient cell surface expression of PAR2. We also show that receptor palmitoylation occurs within the Golgi apparatus and is required for efficient agonist-induced rab11a-mediated trafficking of PAR2 to the cell surface. Palmitoylation is also required for receptor desensitization, as agonist-induced β-arrestin recruitment and receptor endocytosis and degradation were markedly reduced in CHO-PAR2-C361A cells compared with CHO-PAR2 cells. These data provide new insights on the life cycle of PAR2 and demonstrate that palmitoylation is critical for efficient signalling, trafficking, cell surface localization and degradation of this receptor.

  5. The Role of Palmitoylation in Signalling, Cellular Trafficking and Plasma Membrane Localization of Protease-Activated Receptor-2

    Science.gov (United States)

    Adams, Mark N.; Christensen, Melinda E.; He, Yaowu; Waterhouse, Nigel J.; Hooper, John D.

    2011-01-01

    Protease-activated receptor-2 (PAR2) is a G protein coupled receptor (GPCR) activated by proteolytic cleavage of its amino terminal domain by trypsin-like serine proteases. This irreversible activation mechanism leads to rapid receptor desensitization by internalisation and degradation. We have explored the role of palmitoylation, the post-translational addition of palmitate, in PAR2 signalling, trafficking, cell surface expression and desensitization. Experiments using the palmitoylation inhibitor 2-bromopalmitate indicated that palmitate addition is important in trafficking of PAR2 endogenously expressed by prostate cancer cell lines. This was supported by palmitate labelling using two approaches, which showed that PAR2 stably expressed by CHO-K1 cells is palmitoylated and that palmitoylation occurs on cysteine 361. Palmitoylation is required for optimal PAR2 signalling as Ca2+ flux assays indicated that in response to trypsin agonism, palmitoylation deficient PAR2 is ∼9 fold less potent than wildtype receptor with a reduction of about 33% in the maximum signal induced via the mutant receptor. Confocal microscopy, flow cytometry and cell surface biotinylation analyses demonstrated that palmitoylation is required for efficient cell surface expression of PAR2. We also show that receptor palmitoylation occurs within the Golgi apparatus and is required for efficient agonist-induced rab11a-mediated trafficking of PAR2 to the cell surface. Palmitoylation is also required for receptor desensitization, as agonist-induced β-arrestin recruitment and receptor endocytosis and degradation were markedly reduced in CHO-PAR2-C361A cells compared with CHO-PAR2 cells. These data provide new insights on the life cycle of PAR2 and demonstrate that palmitoylation is critical for efficient signalling, trafficking, cell surface localization and degradation of this receptor. PMID:22140500

  6. Brain Region-Specific Effects of cGMP-Dependent Kinase II Knockout on AMPA Receptor Trafficking and Animal Behavior

    Science.gov (United States)

    Kim, Seonil; Pick, Joseph E.; Abera, Sinedu; Khatri, Latika; Ferreira, Danielle D. P.; Sathler, Matheus F.; Morison, Sage L.; Hofmann, Franz; Ziff, Edward B.

    2016-01-01

    Phosphorylation of GluA1, a subunit of AMPA receptors (AMPARs), is critical for AMPAR synaptic trafficking and control of synaptic transmission. cGMP-dependent protein kinase II (cGKII) mediates this phosphorylation, and cGKII knockout (KO) affects GluA1 phosphorylation and alters animal behavior. Notably, GluA1 phosphorylation in the KO…

  7. Brain Region-Specific Effects of cGMP-Dependent Kinase II Knockout on AMPA Receptor Trafficking and Animal Behavior

    Science.gov (United States)

    Kim, Seonil; Pick, Joseph E.; Abera, Sinedu; Khatri, Latika; Ferreira, Danielle D. P.; Sathler, Matheus F.; Morison, Sage L.; Hofmann, Franz; Ziff, Edward B.

    2016-01-01

    Phosphorylation of GluA1, a subunit of AMPA receptors (AMPARs), is critical for AMPAR synaptic trafficking and control of synaptic transmission. cGMP-dependent protein kinase II (cGKII) mediates this phosphorylation, and cGKII knockout (KO) affects GluA1 phosphorylation and alters animal behavior. Notably, GluA1 phosphorylation in the KO…

  8. Regulation of endocytic trafficking of transferrin receptor by optineurin and its impairment by a glaucoma-associated mutant

    Directory of Open Access Journals (Sweden)

    Rangaraj Nandini

    2010-01-01

    Full Text Available Abstract Background Optineurin is a multifunctional protein involved in several functions such as vesicular trafficking from the Golgi to the plasma membrane, NF-κB regulation, signal transduction and gene expression. Mutations in optineurin are associated with glaucoma, a neurodegenerative eye disease that causes blindness. Genetic evidence suggests that the E50K (Glu50Lys is a dominant disease-causing mutation of optineurin. However, functional alterations caused by mutations in optineurin are not known. Here, we have analyzed the role of optineurin in endocytic recycling and the effect of E50K mutant on this process. Results We show that the knockdown of optineurin impairs trafficking of transferrin receptor to the juxtanuclear region. A point mutation (D474N in the ubiquitin-binding domain abrogates localization of optineurin to the recycling endosomes and interaction with transferrin receptor. The function of ubiquitin-binding domain of optineurin is also needed for trafficking of transferrin to the juxtanuclear region. A disease causing mutation, E50K, impairs endocytic recycling of transferrin receptor as shown by enlarged recycling endosomes, slower dynamics of E50K vesicles and decreased transferrin uptake by the E50K-expressing cells. This impaired trafficking by the E50K mutant requires the function of its ubiquitin-binding domain. Compared to wild type optineurin, the E50K optineurin shows enhanced interaction and colocalization with transferrin receptor and Rab8. The velocity of Rab8 vesicles is reduced by co-expression of the E50K mutant. These results suggest that the E50K mutant affects Rab8-mediated transferrin receptor trafficking. Conclusions Our results suggest that optineurin regulates endocytic trafficking of transferrin receptor to the juxtanuclear region. The E50K mutant impairs trafficking at the recycling endosomes due to altered interactions with Rab8 and transferrin receptor. These results also have implications for

  9. Monitoring G protein-coupled receptor and β-arrestin trafficking in live cells using enhanced bystander BRET

    Science.gov (United States)

    Namkung, Yoon; Le Gouill, Christian; Lukashova, Viktoria; Kobayashi, Hiroyuki; Hogue, Mireille; Khoury, Etienne; Song, Mideum; Bouvier, Michel; Laporte, Stéphane A.

    2016-01-01

    Endocytosis and intracellular trafficking of receptors are pivotal to maintain physiological functions and drug action; however, robust quantitative approaches are lacking to study such processes in live cells. Here we present new bioluminescence resonance energy transfer (BRET) sensors to quantitatively monitor G protein-coupled receptors (GPCRs) and β-arrestin trafficking. These sensors are based on bystander BRET and use the naturally interacting chromophores luciferase (RLuc) and green fluorescent protein (rGFP) from Renilla. The versatility and robustness of this approach are exemplified by anchoring rGFP at the plasma membrane or in endosomes to generate high dynamic spectrometric BRET signals on ligand-promoted recruitment or sequestration of RLuc-tagged proteins to, or from, specific cell compartments, as well as sensitive subcellular BRET imaging for protein translocation visualization. These sensors are scalable to high-throughput formats and allow quantitative pharmacological studies of GPCR trafficking in real time, in live cells, revealing ligand-dependent biased trafficking of receptor/β-arrestin complexes. PMID:27397672

  10. Acid-sensing ion channels: trafficking and synaptic function

    Directory of Open Access Journals (Sweden)

    Zha Xiang-ming

    2013-01-01

    Full Text Available Abstract Extracellular acidification occurs in the brain with elevated neural activity, increased metabolism, and neuronal injury. This reduction in pH can have profound effects on brain function because pH regulates essentially every single biochemical reaction. Therefore, it is not surprising to see that Nature evolves a family of proteins, the acid-sensing ion channels (ASICs, to sense extracellular pH reduction. ASICs are proton-gated cation channels that are mainly expressed in the nervous system. In recent years, a growing body of literature has shown that acidosis, through activating ASICs, contributes to multiple diseases, including ischemia, multiple sclerosis, and seizures. In addition, ASICs play a key role in fear and anxiety related psychiatric disorders. Several recent reviews have summarized the importance and therapeutic potential of ASICs in neurological diseases, as well as the structure-function relationship of ASICs. However, there is little focused coverage on either the basic biology of ASICs or their contribution to neural plasticity. This review will center on these topics, with an emphasis on the synaptic role of ASICs and molecular mechanisms regulating the spatial distribution and function of these ion channels.

  11. Functional characterization of protease-activated receptor -1 palmitoylation in receptor signaling and trafficking /

    OpenAIRE

    2014-01-01

    G protein-coupled receptors (GPCRs) are the largest family of signaling receptors that respond to diverse stimuli and regulate many physiological responses. GPCRs elicit their cellular responses by coupling to distinct subtypes of heterotrimeric G-proteins composed of G[alpha] and G[beta][gamma] subunits. Activated GPCRs undergo conformational changes that allow the receptor to exchange GDP for GTP on the G[alpha] subunit, which induces dissociation from the [beta][gamma] subunits and subsequ...

  12. Vesicular Trafficking to the Immune Synapse: How to Assemble Receptor-Tailored Pathways from a Basic Building Set.

    Science.gov (United States)

    Onnis, Anna; Finetti, Francesca; Baldari, Cosima T

    2016-01-01

    The signals that orchestrate T-cell activation are coordinated within a highly organized interface with the antigen-presenting cell (APC), known as the immune synapse (IS). IS assembly depends on T-cell antigen receptor engagement by a specific peptide antigen-major histocompatibility complex ligand. This primary event leads to polarized trafficking of receptors and signaling mediators associated with recycling endosomes to the cellular interface, which contributes to IS assembly as well as signal termination and favors information transfer from T cells to APCs. Here, we will review recent advances on the vesicular pathways implicated in IS assembly and maintenance, focusing on the spatiotemporal regulation of the traffic of specific receptors by Rab GTPases. Based on accumulating evidence that the IS is a functional homolog of the primary cilium, which coordinates several central signaling pathways in ciliated cells, we will also discuss the similarities in the mechanisms regulating vesicular trafficking to these specialized membrane domains.

  13. Modulation of β-amyloid precursor protein trafficking and processing by the low density lipoprotein receptor family

    Directory of Open Access Journals (Sweden)

    Cam Judy A

    2006-08-01

    Full Text Available Abstract Amyloid-β peptide (Aβ accumulation in the brain is an early, toxic event in the pathogenesis of Alzheimer's disease (AD. Aβ is produced by proteolytic processing of a transmembrane protein, β-amyloid precursor protein (APP, by β- and γ-secretases. Mounting evidence has demonstrated that alterations in APP cellular trafficking and localization directly impact its processing to Aβ. Recent studies have shown that members of the low-density lipoprotein receptor family, including LRP, LRP1B, SorLA/LR11, and apolipoprotein E (apoE receptor 2, interact with APP and regulate its endocytic trafficking. Another common feature of these receptors is their ability to bind apoE, which exists in three isoforms in humans and the presence of the ε4 allele represents a genetic risk factor for AD. In this review, we summarize the current understanding of the function of these apoE receptors with a focus on their role in APP trafficking and processing. Knowledge of the interactions between these distinct low-density lipoprotein receptor family members and APP may ultimately influence future therapies for AD.

  14. Cellular Recognition and Trafficking of Amorphous Silica Nanoparticles by Macrophage Scavenger Receptor A

    Energy Technology Data Exchange (ETDEWEB)

    Orr, Galya; Chrisler, William B.; Cassens, Kaylyn J.; Tan, Ruimin; Tarasevich, Barbara J.; Markillie, Lye Meng; Zangar, Richard C.; Thrall, Brian D.

    2011-09-01

    The internalization of engineered nanoparticles (ENPs) into cells is known to involve active transport mechanisms, yet the precise biological molecules involved are poorly understood. We demonstrate that the uptake of amorphous silica ENPs (92 nm) by macrophage cells is strongly inhibited by silencing expression of scavenger receptor A (SR-A). In addition, ENP uptake is augmented by introducing SR-A expression into human cells that are normally non-phagocytic. Confocal fluorescent microscopy analyses show that the majority of single or small clusters of silica ENPs co-localize intracellularly with SR-A and are internalized through a pathway characteristic of clathrin-dependent endocytosis. In contrast, larger silica NP agglomerates (>500 nm) are poorly co-localized with the receptor, suggesting independent trafficking or internalization pathways are involved. SR-A silencing also caused decreased cellular secretion of pro-inflammatory cytokines in response to silica ENPs. As SR-A is expressed in macrophages throughout the reticulo-endothelial system, this pathway is likely an important determinant of the biodistribution of, and cellular response to ENPs.

  15. Heat shock protein 27 is required for sex steroid receptor trafficking to and functioning at the plasma membrane.

    Science.gov (United States)

    Razandi, Mahnaz; Pedram, Ali; Levin, Ellis R

    2010-07-01

    Classical sex steroid receptors (SRs) localize at the plasma membranes (PMs) of cells, initiating signal transduction through kinase cascades that contribute to steroid hormone action. Palmitoylation of the SRs is required for membrane localization and function, but the proteins that facilitate this modification and subsequent receptor trafficking are unknown. Initially using a proteomic approach, we identified that heat shock protein 27 (Hsp27) binds to a motif in estrogen receptor alpha (ERalpha) and promotes palmitoylation of the SR. Hsp27-induced acylation occurred on the ERalpha monomer and augmented caveolin-1 interactions with ERalpha, resulting in membrane localization, kinase activation, and DNA synthesis in breast cancer cells. Oligomerization of Hsp27 was required, and similar results were found for the trafficking of endogenous progesterone and androgen receptors to the PMs of breast and prostate cancer cells, respectively. Small interfering RNA (siRNA) knockdown of Hsp27 prevented sex SR trafficking to and signaling from the membrane. These results identify a conserved and novel function for Hsp27 with potential as a target for interrupting signaling from membrane sex SRs to tumor biology in hormone-responsive cancers.

  16. Slit-Dependent Endocytic Trafficking of the Robo Receptor Is Required for Son of Sevenless Recruitment and Midline Axon Repulsion.

    Directory of Open Access Journals (Sweden)

    Rebecca K Chance

    2015-09-01

    Full Text Available Understanding how axon guidance receptors are activated by their extracellular ligands to regulate growth cone motility is critical to learning how proper wiring is established during development. Roundabout (Robo is one such guidance receptor that mediates repulsion from its ligand Slit in both invertebrates and vertebrates. Here we show that endocytic trafficking of the Robo receptor in response to Slit-binding is necessary for its repulsive signaling output. Dose-dependent genetic interactions and in vitro Robo activation assays support a role for Clathrin-dependent endocytosis, and entry into both the early and late endosomes as positive regulators of Slit-Robo signaling. We identify two conserved motifs in Robo's cytoplasmic domain that are required for its Clathrin-dependent endocytosis and activation in vitro; gain of function and genetic rescue experiments provide strong evidence that these trafficking events are required for Robo repulsive guidance activity in vivo. Our data support a model in which Robo's ligand-dependent internalization from the cell surface to the late endosome is essential for receptor activation and proper repulsive guidance at the midline by allowing recruitment of the downstream effector Son of Sevenless in a spatially constrained endocytic trafficking compartment.

  17. Slit-Dependent Endocytic Trafficking of the Robo Receptor Is Required for Son of Sevenless Recruitment and Midline Axon Repulsion.

    Science.gov (United States)

    Chance, Rebecca K; Bashaw, Greg J

    2015-09-01

    Understanding how axon guidance receptors are activated by their extracellular ligands to regulate growth cone motility is critical to learning how proper wiring is established during development. Roundabout (Robo) is one such guidance receptor that mediates repulsion from its ligand Slit in both invertebrates and vertebrates. Here we show that endocytic trafficking of the Robo receptor in response to Slit-binding is necessary for its repulsive signaling output. Dose-dependent genetic interactions and in vitro Robo activation assays support a role for Clathrin-dependent endocytosis, and entry into both the early and late endosomes as positive regulators of Slit-Robo signaling. We identify two conserved motifs in Robo's cytoplasmic domain that are required for its Clathrin-dependent endocytosis and activation in vitro; gain of function and genetic rescue experiments provide strong evidence that these trafficking events are required for Robo repulsive guidance activity in vivo. Our data support a model in which Robo's ligand-dependent internalization from the cell surface to the late endosome is essential for receptor activation and proper repulsive guidance at the midline by allowing recruitment of the downstream effector Son of Sevenless in a spatially constrained endocytic trafficking compartment.

  18. Biphasic coupling of neuronal nitric oxide synthase phosphorylation to the NMDA receptor regulates AMPA receptor trafficking and neuronal cell death.

    Science.gov (United States)

    Rameau, Gerald A; Tukey, David S; Garcin-Hosfield, Elsa D; Titcombe, Roseann F; Misra, Charu; Khatri, Latika; Getzoff, Elizabeth D; Ziff, Edward B

    2007-03-28

    Postsynaptic nitric oxide (NO) production affects synaptic plasticity and neuronal cell death. Ca2+ fluxes through the NMDA receptor (NMDAR) stimulate the production of NO by neuronal nitric oxide synthase (nNOS). However, the mechanisms by which nNOS activity is regulated are poorly understood. We evaluated the effect of neuronal stimulation with glutamate on the phosphorylation of nNOS. We show that, in cortical neurons, a low glutamate concentration (30 microM) induces rapid and transient NMDAR-dependent phosphorylation of S1412 by Akt, followed by sustained phosphorylation of S847 by CaMKII (calcium-calmodulin-dependent kinase II). We demonstrate that phosphorylation of S1412 by Akt is necessary for activation of nNOS by the NMDAR. nNOS mutagenesis confirms that these phosphorylations respectively activate and inhibit nNOS and, thus, transiently activate NO production. A constitutively active (S1412D), but not a constitutively repressed (S847D) nNOS mutant elevated surface glutamate receptor 2 levels, demonstrating that these phosphorylations can control AMPA receptor trafficking via NO. Notably, an excitotoxic stimulus (150 microM glutamate) induced S1412, but not S847 phosphorylation, leading to deregulated nNOS activation. S1412D did not kill neurons; however, it enhanced the excitotoxicity of a concomitant glutamate stimulus. We propose a swinging domain model for the regulation of nNOS: S1412 phosphorylation facilitates electron flow within the reductase module of nNOS, increasing nNOS sensitivity to Ca2+-calmodulin. These findings suggest a critical role for a kinetically complex and novel series of regulatory nNOS phosphorylations induced by the NMDA receptor for the in vivo control of nNOS.

  19. The Influence of Polyunsaturated Fatty Acids on the Phospholipase D Isoforms Trafficking and Activity in Mast Cells

    Directory of Open Access Journals (Sweden)

    Julia Schumann

    2013-04-01

    Full Text Available The impact of polyunsaturated fatty acid (PUFA supplementation on phospholipase D (PLD trafficking and activity in mast cells was investigated. The enrichment of mast cells with different PUFA including α-linolenic acid (LNA, eicosapentaenoic acid (EPA, docosahexaenoic acid (DHA, linoleic acid (LA or arachidonic acid (AA revealed a PUFA-mediated modulation of the mastoparan-stimulated PLD trafficking and activity. All PUFA examined, except AA, prevented the migration of the PLD1 to the plasma membrane. For PLD2 no PUFA effects on trafficking could be observed. Moreover, PUFA supplementation resulted in an increase of mastoparan-stimulated total PLD activity, which correlated with the number of double bonds of the supplemented fatty acids. To investigate, which PLD isoform was affected by PUFA, stimulated mast cells were supplemented with DHA or AA in the presence of specific PLD-isoform inhibitors. It was found that both DHA and AA diminished the inhibition of PLD activity in the presence of a PLD1 inhibitor. By contrast, only AA diminished the inhibition of PLD activity in the presence of a PLD2 inhibitor. Thus, PUFA modulate the trafficking and activity of PLD isoforms in mast cells differently. This may, in part, account for the immunomodulatory effect of unsaturated fatty acids and contributes to our understanding of the modulation of mast cell activity by PUFA.

  20. Clavulanic acid increases dopamine release in neuronal cells through a mechanism involving enhanced vesicle trafficking

    Science.gov (United States)

    Kost, Gina Chun; Selvaraj, Senthil; Lee, Young Bok; Kim, Deog Joong; Ahn, Chang-Ho; Singh, Brij B

    2011-01-01

    Clavulanic acid is a CNS-modulating compound with exceptional blood-brain barrier permeability and safety profile. Clavulanic acid has been proposed to have anti-depressant activity and is currently entering Phase IIb clinical trials for the treatment of Major Depressive Disorder (MDD). Studies have also shown that clavulanic acid suppresses anxiety and enhances sexual functions in rodent and primate models by a mechanism involving central nervous system (CNS) modulation, although its detailed mechanism of action has yet to be elucidated. To further examine its potential as a CNS modulating agent as well as its mechanism of action, we investigated the effects of clavulanic acid in neuronal cells. Our results indicate that clavulanic acid enhances dopamine release in PC12 and SH-SY5Y cells without affecting dopamine synthesis. Furthermore, using affinity chromatography we were able to identify two proteins, Munc18-1 and Rab4 that potentially bind to clavulanic acid and play a critical role in neurosecretion and the vesicle trafficking process. Consistent with this result, an increase in the translocation of Munc18-1 and Rab4 from the cytoplasm to the plasma membrane was observed in clavulanic acid treated cells. Overall, these data suggest that clavulanic acid enhances dopamine release in a mechanism involving Munc18-1 and Rab4 modulation and warrants further investigation of its therapeutic use in CNS disorders, such as depression. PMID:21964384

  1. Clavulanic acid increases dopamine release in neuronal cells through a mechanism involving enhanced vesicle trafficking.

    Science.gov (United States)

    Kost, Gina Chun; Selvaraj, Senthil; Lee, Young Bok; Kim, Deog Joong; Ahn, Chang-Ho; Singh, Brij B

    2011-10-24

    Clavulanic acid is a CNS-modulating compound with exceptional blood-brain barrier permeability and safety profile. Clavulanic acid has been proposed to have anti-depressant activity and is currently entering Phase IIb clinical trials for the treatment of Major Depressive Disorder (MDD). Studies have also shown that clavulanic acid suppresses anxiety and enhances sexual functions in rodent and primate models by a mechanism involving central nervous system (CNS) modulation, although its detailed mechanism of action has yet to be elucidated. To further examine its potential as a CNS modulating agent as well as its mechanism of action, we investigated the effects of clavulanic acid in neuronal cells. Our results indicate that clavulanic acid enhances dopamine release in PC12 and SH-SY5Y cells without affecting dopamine synthesis. Furthermore, using affinity chromatography we were able to identify two proteins, Munc18-1 and Rab4 that potentially bind to clavulanic acid and play a critical role in neurosecretion and the vesicle trafficking process. Consistent with this result, an increase in the translocation of Munc18-1 and Rab4 from the cytoplasm to the plasma membrane was observed in clavulanic acid treated cells. Overall, these data suggest that clavulanic acid enhances dopamine release in a mechanism involving Munc18-1 and Rab4 modulation and warrants further investigation of its therapeutic use in CNS disorders, such as depression.

  2. Cyclic GMP signaling in cardiomyocytes modulates fatty acid trafficking and prevents triglyceride accumulation.

    Science.gov (United States)

    Khairallah, Ramzi J; Khairallah, Maya; Gélinas, Roselle; Bouchard, Bertrand; Young, Martin E; Allen, Bruce G; Lopaschuk, Gary D; Deschepper, Christian F; Des Rosiers, Christine

    2008-08-01

    While the balance between carbohydrates and fatty acids for energy production appears to be crucial for cardiac homeostasis, much remains to be learned about the molecular mechanisms underlying this relationship. Given the reported benefits of cGMP signaling on the myocardium, we investigated the impact of its chronic activation on cardiac energy metabolism using mice overexpressing a constitutively active cytoplasmic guanylate cyclase (GC(+/0)) in cardiomyocytes. Ex vivo working GC(+/0) heart perfusions with (13)C-labeled substrates revealed an altered pattern of exogenous substrate fuel selection compared to controls, namely a 38+/-9% lower contribution of exogenous fatty acids to acetyl-CoA formation, while that of carbohydrates remains unchanged despite a two-fold increase in glycolysis. The lower contribution of exogenous fatty acids to energy production is not associated with changes in energy demand or supply (contractile function, oxygen consumption, tissue acetyl-CoA or CoA levels, citric acid cycle flux rate) or in the regulation of beta-oxidation (acetyl-CoA carboxylase activity, tissue malonyl-CoA levels). However, GC(+/0) hearts show a two-fold increase in the incorporation of exogenous oleate into triglycerides. Furthermore, the following molecular data are consistent with a concomitant increase in triglyceride hydrolysis: (i) increased abundance of hormone sensitive lipase (HSL) protein (24+/-11%) and mRNA (22+/-4%) as well as (ii) several phosphorylation events related to HSL inhibitory (AMPK) and activation (ERK 1/2) sites, which should contribute to enhance its activity. These changes in exogenous fatty acid trafficking in GC(+/0) hearts appear to be functionally relevant, as demonstrated by their resistance to fasting-induced triglyceride accumulation. While the documented metabolic profile of GC(+/0) mouse hearts is partly reminiscent of hypertrophied hearts, the observed changes in lipid trafficking have not been previously documented, and may

  3. One-way membrane trafficking of SOS in receptor-triggered Ras activation.

    Science.gov (United States)

    Christensen, Sune M; Tu, Hsiung-Lin; Jun, Jesse E; Alvarez, Steven; Triplet, Meredith G; Iwig, Jeffrey S; Yadav, Kamlesh K; Bar-Sagi, Dafna; Roose, Jeroen P; Groves, Jay T

    2016-09-01

    SOS is a key activator of the small GTPase Ras. In cells, SOS-Ras signaling is thought to be initiated predominantly by membrane recruitment of SOS via the adaptor Grb2 and balanced by rapidly reversible Grb2-SOS binding kinetics. However, SOS has multiple protein and lipid interactions that provide linkage to the membrane. In reconstituted-membrane experiments, these Grb2-independent interactions were sufficient to retain human SOS on the membrane for many minutes, during which a single SOS molecule could processively activate thousands of Ras molecules. These observations raised questions concerning how receptors maintain control of SOS in cells and how membrane-recruited SOS is ultimately released. We addressed these questions in quantitative assays of reconstituted SOS-deficient chicken B-cell signaling systems combined with single-molecule measurements in supported membranes. These studies revealed an essentially one-way trafficking process in which membrane-recruited SOS remains trapped on the membrane and continuously activates Ras until being actively removed via endocytosis.

  4. G protein-coupled receptor regulation: The role of protein interactions and receptor trafficking

    OpenAIRE

    Sandén, Caroline

    2009-01-01

    The superfamily of G protein-coupled receptors (GPCR) is the largest gene family in the human genome. GPCR-mediated signaling operates in every human cell, and about 50% of existing clinically useful drugs act through GPCR. Kinins are proinflammatory peptides that are rapidly produced extracellularly following pathological insults and tissue damage. These peptides act through two GPCR subtypes, B1 (B1R) and B2 (B2R), to elicit numerous inflammatory responses including vasodilatiation, increas...

  5. Excitatory amino acid receptor antagonists

    DEFF Research Database (Denmark)

    Johansen, T N; Frydenvang, Karla Andrea; Ebert, B

    1997-01-01

    We have previously shown that (RS)-2-amino-2-(5-tert-butyl-3-hydroxyisoxazol-4-yl)acetic acid (ATAA) is an antagonist at N-methyl-D-aspartic acid (NMDA) and (RS)-2-amino-3-(3-hydroxy-5-methylisoxazol-4-yl)propionic acid (AMPA) receptors. We have now resolved ATAA via diastereomeric salt formation......)-phenylethylamine salt of N-BOC-(R)-ATAA. Like ATAA, neither (R)- nor (S)-ATAA significantly affected (IC50 > 100 microM) the receptor binding of tritiated AMPA, kainic acid, or (RS)-3-(2-carboxypiperazin-4-yl)propyl-1-phosphonic acid, the latter being a competitive NMDA antagonist. Electrophysiological experiments......, using the rat cortical wedge preparation, showed the NMDA antagonist effect as well as the AMPA antagonist effect of ATAA to reside exclusively in the (R)-enantiomer (Ki = 75 +/- 5 microM and 57 +/- 1 microM, respectively). Neither (R)- nor (S)-ATAA significantly reduced kainic acid-induced excitation...

  6. Ligand-directed trafficking of the δ-opioid receptor in vivo: two paths toward analgesic tolerance.

    Science.gov (United States)

    Pradhan, Amynah A A; Walwyn, Wendy; Nozaki, Chihiro; Filliol, Dominique; Erbs, Eric; Matifas, Audrey; Evans, Christopher; Kieffer, Brigitte L

    2010-12-08

    δ-Opioid receptors are G-protein-coupled receptors that regulate nociceptive and emotional responses. It has been well established that distinct agonists acting at the same G-protein-coupled receptor can engage different signaling or regulatory responses. This concept, known as biased agonism, has important biological and therapeutic implications. Ligand-biased responses are well described in cellular models, however, demonstrating the physiological relevance of biased agonism in vivo remains a major challenge. The aim of this study was to investigate the long-term consequences of ligand-biased trafficking of the δ-opioid receptor, at both the cellular and behavioral level. We used δ agonists with similar binding and analgesic properties, but high [SNC80 ((+)-4-[(αR)-α-((2S,5R)-4-allyl-2,5-dimethyl-1-piperazinyl)-3-methoxybenzyl]-N,N-diethylbenzamide)]- or low [ARM390 (N,N-diethyl-4-(phenyl-piperidin-4-ylidenemethyl)-benzamide)]-internalization potencies. As we found previously, a single SNC80-but not ARM390-administration triggered acute desensitization of the analgesic response in mice. However, daily injections of either compound over 5 d produced full analgesic tolerance. SNC80-tolerant animals showed widespread receptor downregulation, and tolerance to analgesic, locomotor and anxiolytic effects of the agonist. Hence, internalization-dependent tolerance developed, as a result of generalized receptor degradation. In contrast, ARM390-tolerant mice showed intact receptor expression, but δ-opioid receptor coupling to Ca²+ channels was abolished in dorsal root ganglia. Concomitantly, tolerance developed for agonist-induced analgesia, but not locomotor or anxiolytic responses. Therefore, internalization-independent tolerance was produced by anatomically restricted adaptations leading to pain-specific tolerance. Hence, ligand-directed receptor trafficking of the δ-opioid receptor engages distinct adaptive responses, and this study reveals a novel aspect of

  7. Role of endosomal trafficking dynamics on the regulation of hepatic insulin receptor activity: models for Fao cells.

    Science.gov (United States)

    Hori, Sharon S; Kurland, Irwin J; DiStefano, Joseph J

    2006-05-01

    Evidence indicates that endosomal insulin receptor (IR) trafficking plays a role in regulating insulin signal transduction. To evaluate its importance, we developed a series of biokinetic models for quantifying activated surface and endosomal IR dynamics from published experimental data. Starting with a published two-compartment Fao hepatoma model, a four-pool model was formulated that depicts IR autophosphorylation after receptor binding, IR endosomal internalization/trafficking, insulin dissociation from and dephosphorylation of internalized IR, and recycling of unliganded, dephosphorylated IR to the plasma membrane. Quantification required three additional data sets, two measured, but unmodeled by the same group. A five-pool model created to include endosomal trafficking of the nonphosphorylated insulin-IR complex was fitted using the same data sets, augmented with another published data set. Creation of a six-pool model added the physiologically relevant dissociation of insulin ligand from the activated endosomal IR. More importantly, all three models, validated against additional data not used in model fitting, predict that, mechanistically, internalization of activated IR is a rate-limiting step, at least under the receptor saturating conditions of the fitting data. This rate includes the transit time to a site where insulin dissociation from and/or dephosphorylation of the IR occurs by docking with protein-tyrosine phosphatases (PTPases), or where a sufficient conformational change occurs in the IR, perhaps due to insulin-IR dissociation, where associated PTPases may complete IR dephosphorylation. Our new models indicate that key events in endosomal IR trafficking have significance in mediating IR activity, possibly serving to regulate insulin signal transduction.

  8. Real-time studies of the interactions between epidermal growth factor and its receptor during endocytic trafficking.

    Science.gov (United States)

    Martin-Fernandez, M L; Clarke, D T; Tobin, M J; Jones, G R

    2000-09-01

    The interactions of growth factors with cell surface receptors regulate fundamental cell processes, such as growth, differentiation and transformation. Understanding the nature of these interactions at the molecular level is of fundamental importance in cell biology. This is not only from the point of view of basic science, but also because of the repercussions such knowledge might have in understanding the mode of action of drugs in cells. Receptor mediated endocytosis has been implicated in the downregulation of the mitogenic signal. However, no data are thus far available on how growth factor/receptor interactions might control endocytic trafficking. Here we show that information on modes of binding and receptor conformational changes can be obtained using time-resolved fluorescence methods. We have found that fluorescent probes bound to epidermal growth factor (EGF) show dynamic fluorescence quenching when EGF is bound to internalising EGF receptors (EGFR). We propose that this dynamic quenching takes place because EGF-bound probes interact with tryptophan residues in the extracellular domain of the EGF-EGFR complex. Real-time accumulation of fluorescent decays has also allowed us to follow the time course of a conformational change in EGFR occurring during endocytosis, and correlate this information with endosomal trafficking and EGFR recycling.

  9. Dissociation and trafficking of rat GABAB receptor heterodimer upon chronic capsaicin stimulation.

    Science.gov (United States)

    Laffray, Sophie; Tan, Kelly; Dulluc, Josette; Bouali-Benazzouz, Rabia; Calver, Andrew R; Nagy, Frédéric; Landry, Marc

    2007-03-01

    Gamma-aminobutyric acid type B receptors (GABAB) are G-protein-coupled receptors that mediate GABAergic inhibition in the brain. Their functional expression is dependent upon the formation of heterodimers between GABAB1 and GABAB2 subunits, a process that occurs within the endoplasmic reticulum. However, the mechanisms that regulate GABAB receptor oligomerization at the plasma membrane remain largely unknown. We first characterized the functional cytoarchitecture of an organotypic co-culture model of rat dorsal root ganglia and spinal cord. Subsequently, we studied the interactions between GABAB subunits after chronic stimulation of sensory fibres with capsaicin. Surface labelling of recombinant proteins showed a decrease in subunit co-localization and GABAB2 labelling, after capsaicin treatment. In these conditions, fluorescence lifetime imaging measurements further demonstrated a loss of interactions between green fluorescent protein-GABAB1b and t-dimer discosoma sp red fluorescent protein-GABAB2 subunits. Finally, we established that the GABAB receptor undergoes clathrin-dependent internalization and rapid recycling to the plasma membrane following activation with baclofen, a GABAB agonist. However, in cultures chronically stimulated with capsaicin, the agonist-induced endocytosis was decreased, reflecting changes in the dimeric state of the receptor. Taken together, our results indicate that the chronic stimulation of sensory fibres can dissociate the GABAB heterodimer and alters its responsiveness to the endogenous ligand. Chronic stimulation thus modulates receptor oligomerization, providing additional levels of control of signalling.

  10. Quantitative analysis of agonist-dependent parathyroid hormone receptor trafficking in whole cells using a functional green fluorescent protein conjugate.

    Science.gov (United States)

    Conway, B R; Minor, L K; Xu, J Z; D'Andrea, M R; Ghosh, R N; Demarest, K T

    2001-12-01

    Many G-protein coupled receptors (GPCRs) undergo ligand-dependent internalization upon activation. The parathyroid hormone (PTH) receptor undergoes endocytosis following prolonged exposure to ligand although the ultimate fate of the receptor following internalization is largely unknown. To investigate compartmentalization of the PTH receptor, we have established a stable cell line expressing a PTH receptor-green fluorescent protein (PTHR-GFP) conjugate and an algorithm to quantify PTH receptor internalization. HEK 293 cells expressing the PTHR-GFP were compared with cells expressing the wild-type PTH receptor in whole-cell binding and functional assays. 125I-PTH binding studies revealed similar Bmax and kD values in cells expressing either the PTHR-GFP or the wild-type PTH receptor. PTH-induced cAMP accumulation was similar in both cell lines suggesting that addition of the GFP to the cytoplasmic tail of the PTH receptor does not alter the ligand binding or G-protein coupling properties of the receptor. Using confocal fluorescence microscopy, we demonstrated that PTH treatment of cells expressing the PTHR-GFP conjugate produced a time-dependent redistribution of the receptor to the endosomal compartment which was blocked by pretreatment with PTH antagonist peptides. Treatment with hypertonic sucrose prevented PTH-induced receptor internalization, suggesting that the PTH receptor internalizes via a clathrin-dependent mechanism. Moreover, co-localization with internalized transferrin showed that PTHR-GFP trafficking utilized the endocytic recycling compartment. Experiments using cycloheximide to inhibit protein synthesis demonstrated that recycling of the PTHR-GFP back to the plasma membrane was complete within 1-2 h of ligand removal and was partially blocked by pretreatment with cytochalasin D, but not nocodazole. We also demonstrated that the PTH receptor, upon recycling to the plasma membrane, is capable of undergoing a second round of internalization, a finding

  11. Diversion of phagosome trafficking by pathogenic Rhodococcus equi depends on mycolic acid chain length.

    Science.gov (United States)

    Sydor, Tobias; von Bargen, Kristine; Hsu, Fong-Fu; Huth, Gitta; Holst, Otto; Wohlmann, Jens; Becken, Ulrike; Dykstra, Tobias; Söhl, Kristina; Lindner, Buko; Prescott, John F; Schaible, Ulrich E; Utermöhlen, Olaf; Haas, Albert

    2013-03-01

    Rhodococcus equi is a close relative of Mycobacterium spp. and a facultative intracellular pathogen which arrests phagosome maturation in macrophages before the late endocytic stage. We have screened a transposon mutant library of R. equi for mutants with decreased capability to prevent phagolysosome formation. This screen yielded a mutant in the gene for β-ketoacyl-(acyl carrier protein)-synthase A (KasA), a key enzyme of the long-chain mycolic acid synthesizing FAS-II system. The longest kasA mutant mycolic acid chains were 10 carbon units shorter than those of wild-type bacteria. Coating of non-pathogenic E. coli with purified wild-type trehalose dimycolate reduced phagolysosome formation substantially which was not the case with shorter kasA mutant-derived trehalose dimycolate. The mutant was moderately attenuated in macrophages and in a mouse infection model, but was fully cytotoxic.Whereas loss of KasA is lethal in mycobacteria, R. equi kasA mutant multiplication in broth was normal proving that long-chain mycolic acid compounds are not necessarily required for cellular integrity and viability of the bacteria that typically produce them. This study demonstrates a central role of mycolic acid chain length in diversion of trafficking by R. equi. © 2012 Blackwell Publishing Ltd.

  12. Flagellin peptide flg22 gains access to long-distance trafficking in Arabidopsis via its receptor, FLS2

    Energy Technology Data Exchange (ETDEWEB)

    Jelenska, Joanna [University of Chicago; Greenberg, Jean T. [University of Chicago; Mirzadeh, Saed [ORNL; Davern, Sandra M. [ORNL; Standaert, Robert F. [ORNL

    2017-03-01

    Diverse pathogen-derived molecules, such as bacterial flagellin and its conserved peptide flg22, are recognized in plants via plasma membrane receptors and induce both local and systemic immune responses. The fate of such ligands was unknown: whether and by what mechanism(s) they enter plant cells and whether they are transported to distal tissues. We used biologically active fluorophore and radiolabeled peptides to establish that flg22 moves to distal organs with the closest vascular connections. Remarkably, entry into the plant cell via endocytosis together with the FLS2 receptor is needed for delivery to vascular tissue and long-distance transport of flg22. This contrasts with known routes of long distance transport of other non-cell-permeant molecules in plants, which require membrane-localized transporters for entry to vascular tissue. Thus, a plasma membrane receptor acts as a transporter to enable access of its ligand to distal trafficking routes.

  13. Rab8 modulates metabotropic glutamate receptor subtype 1 intracellular trafficking and signaling in a protein kinase C-dependent manner.

    Science.gov (United States)

    Esseltine, Jessica L; Ribeiro, Fabiola M; Ferguson, Stephen S G

    2012-11-21

    Metabotropic glutamate receptors (mGluRs) are G protein-coupled receptors (GPCRs) that are activated by glutamate, the primary excitatory neurotransmitter in the CNS. Alterations in glutamate receptor signaling are implicated in neuropathologies such as Alzheimer's disease, ischemia, and Huntington's disease among others. Group 1 mGluRs (mGluR1 and mGluR5) are primarily coupled to Gα(q/11) leading to the activation of phospholipase C and the formation of diacylglycerol and inositol 1,4,5-trisphosphate, which results in the release of intracellular calcium stores and protein kinase C (PKC) activation. Desensitization, endocytosis, and recycling are major mechanisms of GPCR regulation, and the intracellular trafficking of GPCRs is linked to the Rab family of small G proteins. Rab8 is a small GTPase that is specifically involved in the regulation of secretory/recycling vesicles, modulation of the actin cytoskeleton, and cell polarity. Rab8 has been shown to regulate the synaptic delivery of AMPA receptors during long-term potentiation and during constitutive receptor recycling. We show here that Rab8 interacts with the C-terminal tail of mGluR1a in an agonist-dependent manner and plays a role in regulating of mGluR1a signaling and intracellular trafficking in human embryonic kidney 293 cells. Specifically, Rab8 expression attenuates mGluR1a-mediated inositol phosphate formation and calcium release from mouse neurons in a PKC-dependent manner, while increasing cell surface mGluR1a expression via decreased receptor endocytosis. These experiments provide us with an understanding of the role Rabs play in coordinated regulation of mGluR1a and how this impacts mGluR1a signaling.

  14. Amino acids regulate the intracellular trafficking of the general amino acid permease of Saccharomycescerevisiae.

    Science.gov (United States)

    Chen, Esther J; Kaiser, Chris A

    2002-11-12

    The delivery to the plasma membrane of the general amino acid permease, Gap1p, of Saccharomyces cerevisiae is regulated by the quality of the nitrogen source in the growth medium. In an effort to define how different nitrogen sources control Gap1p sorting, we find that mutations in GDH1 and GLN1 that decrease the flux through the glutamate and glutamine synthesis pathways result in increased Gap1p sorting to the plasma membrane. Conversely, deletion of MKS1, which increases glutamate and glutamine synthesis, decreases Gap1p sorting to the plasma membrane. Glutamate and glutamine are not unusual in their ability to regulate Gap1p sorting, because the addition of all natural amino acids and many amino acid analogs to the growth medium results in increased Gap1p sorting to the vacuole. Importantly, amino acids have the capacity to signal Gap1p sorting to the vacuole regardless of whether they can be used as a source of nitrogen. Finally, we show that rapamycin does not affect Gap1p sorting, indicating that Gap1p sorting is not directly influenced by the TOR pathway. Together, these data show that amino acids are a signal for sorting Gap1p to the vacuole and imply that the nitrogen-regulated Gap1p sorting machinery responds to amino acid-like compounds rather than to the overall nutritional status associated with growth on a particular nitrogen source.

  15. Subtype selective kainic acid receptor agonists

    DEFF Research Database (Denmark)

    Bunch, Lennart; Krogsgaard-Larsen, Povl

    2009-01-01

    (S)-Glutamic acid (Glu) is the major excitatory neurotransmitter in the mammalian central nervous system, activating the plethora of glutamate receptors (GluRs). In broad lines, the GluRs are divided into two major classes: the ionotropic Glu receptors (iGluRs) and the metabotropic Glu receptors (m......GluRs). Within the iGluRs, five subtypes (KA1, KA2, iGluR5-7) show high affinity and express full agonist activity upon binding of the naturally occurring amino acid kainic acid (KA). Thus these receptors have been named the KA receptors. This review describes all-to our knowledge-published KA receptor agonists...

  16. Novel role of sorting nexin 5 in renal D(1) dopamine receptor trafficking and function: implications for hypertension.

    Science.gov (United States)

    Villar, Van Anthony M; Armando, Ines; Sanada, Hironobu; Frazer, Lauren C; Russo, Christen M; Notario, Patricia M; Lee, Hewang; Comisky, Lauren; Russell, Holly Ann; Yang, Yu; Jurgens, Julie A; Jose, Pedro A; Jones, John E

    2013-05-01

    The D1 dopamine receptor (D1R) is widely expressed in the kidney and plays a crucial role in blood pressure regulation. Although much is known about D1R desensitization, especially through G-protein-coupled receptor kinase 4 (GRK4), comparatively little is known about other aspects of D1R trafficking and the proteins involved in the process. We now report the discovery of a dynamic interaction between sorting nexin 5 (SNX5), a component of the mammalian retromer, and D1R in human renal epithelial cells. We show that internalization of agonist-activated D1R is regulated by both SNX5 and GRK4, and that SNX5 is critical to the recycling of the receptor to the plasma membrane. SNX5 depletion increases agonist-activated D1R phosphorylation (>50% at basal condition), prevents D1R internalization and cAMP response, and delays receptor recycling compared to mock siRNA-transfected controls. Moreover, renal restricted subcapsular infusion of Snx5-specific siRNA (vs. mock siRNA) decreases sodium excretion (Δ=-0.2±0.005 mEq/mg creatinine) and further elevates the systolic blood pressure (Δ=48±5 mm Hg) in spontaneously hypertensive rats, indicating that SNX5 depletion impairs renal D1R function. These studies demonstrate an essential role for SNX5 in regulating D1R function, which may have important diagnostic, prognostic, and therapeutic implications in the management of essential hypertension.

  17. siRNA screen of ES cell-derived motor neurons identifies novel regulators of tetanus toxin and neurotrophin receptor trafficking

    Directory of Open Access Journals (Sweden)

    Marco eTerenzio

    2014-05-01

    Full Text Available Neurons rely on the long-range transport of several signalling molecules such as neurotrophins and their receptors, which are required for neuronal development, function and survival. However, the nature of the machinery controlling the trafficking of signalling endosomes containing activated neurotrophin receptors has not yet been completely elucidated. We aimed to identify new players involved in the dynamics of neurotrophin signalling endosomes using a high-throughput unbiased siRNA screening approach to quantify the intracellular accumulation of two fluorescently tagged reporters: the binding fragment of tetanus neurotoxin (HCT, and an antibody directed against the neurotrophin receptor p75NTR. This screen performed in motor neurons differentiated from mouse embryonic stem (ES cells identified a number of candidate genes encoding molecular motors and motor adaptor proteins involved in regulating the intracellular trafficking of these probes. Bicaudal D homolog 1 (BICD1, a molecular motor adaptor with pleiotropic roles in intracellular trafficking, was selected for further analyses, which revealed that BICD1 regulates the intracellular trafficking of HCT and neurotrophin receptors and likely plays an important role in nervous system development and function.

  18. Endocytic Trafficking towards the Vacuole Plays a Key Role in the Auxin Receptor SCFTIR-Independent Mechanism of Lateral Root Formation in A.thaliana

    Institute of Scientific and Technical Information of China (English)

    Patricio Pérez-Henríquez; Natasha V.Raikhel; Lorena Norambuena

    2012-01-01

    Plants' developmental plasticity plays a pivotal role in responding to environmental conditions.One of the most plastic plant organs is the root system.Different environmental stimuli such as nutrients and water deficiency may induce lateral root formation to compensate for a low level of water and/or nutrients.It has been shown that the hormone auxin tunes lateral root development and components for its signaling pathway have been identified.Using chemical biology,we discovered an Arabidopsis thaliana lateral root formation mechanism that is independent of the auxin receptor SCFTIR.The bioactive compound Sortin2 increased lateral root occurrence by acting upstream from the morphological marker of lateral root primordium formation,the mitotic activity.The compound did not display auxin activity.At the cellular level,Sortin2 accelerated endosomal trafficking,resulting in increased trafficking of plasma membrane recycling proteins to the vacuole.Sortin2 affected Late endosome/PVC/MVB trafficking and morphology.Combining Sortin2 with well-known drugs showed that endocytic trafficking of Late E/PVC/MVB towards the vacuole is pivotal for Sortin2induced SCFTIR-independent lateral root initiation.Our results revealed a distinctive role for endosomal trafficking in the promotion of lateral root formation via a process that does not rely on the auxin receptor complex SCFTIR.

  19. Endolysosomal trafficking of viral G protein-coupled receptor functions in innate immunity and control of viral oncogenesis.

    Science.gov (United States)

    Dong, Xiaonan; Cheng, Adam; Zou, Zhongju; Yang, Yih-Sheng; Sumpter, Rhea M; Huang, Chou-Long; Bhagat, Govind; Virgin, Herbert W; Lira, Sergio A; Levine, Beth

    2016-03-15

    The ubiquitin-proteasome system degrades viral oncoproteins and other microbial virulence factors; however, the role of endolysosomal degradation pathways in these processes is unclear. Kaposi's sarcoma-associated herpesvirus (KSHV) is the causative agent of Kaposi's sarcoma, and a constitutively active viral G protein-coupled receptor (vGPCR) contributes to the pathogenesis of KSHV-induced tumors. We report that a recently discovered autophagy-related protein, Beclin 2, interacts with KSHV GPCR, facilitates its endolysosomal degradation, and inhibits vGPCR-driven oncogenic signaling. Furthermore, monoallelic loss of Becn2 in mice accelerates the progression of vGPCR-induced lesions that resemble human Kaposi's sarcoma. Taken together, these findings indicate that Beclin 2 is a host antiviral molecule that protects against the pathogenic effects of KSHV GPCR by facilitating its endolysosomal degradation. More broadly, our data suggest a role for host endolysosomal trafficking pathways in regulating viral pathogenesis and oncogenic signaling.

  20. Catecholamine stress alters neutrophil trafficking and impairs wound healing by β2-adrenergic receptor-mediated upregulation of IL-6.

    Science.gov (United States)

    Kim, Min-Ho; Gorouhi, Farzam; Ramirez, Sandra; Granick, Jennifer L; Byrne, Barbara A; Soulika, Athena M; Simon, Scott I; Isseroff, R Rivkah

    2014-03-01

    Stress-induced hormones can alter the inflammatory response to tissue injury; however, the precise mechanism by which epinephrine influences inflammatory response and wound healing is not well defined. Here we demonstrate that epinephrine alters the neutrophil (polymorphonuclear leukocyte (PMN))-dependent inflammatory response to a cutaneous wound. Using noninvasive real-time imaging of genetically tagged PMNs in a murine skin wound, chronic, epinephrine-mediated stress was modeled by sustained delivery of epinephrine. Prolonged systemic exposure of epinephrine resulted in persistent PMN trafficking to the wound site via an IL-6-mediated mechanism, and this in turn impaired wound repair. Further, we demonstrate that β2-adrenergic receptor-dependent activation of proinflammatory macrophages is critical for epinephrine-mediated IL-6 production. This study expands our current understanding of stress hormone-mediated impairment of wound healing and provides an important mechanistic link to explain how epinephrine stress exacerbates inflammation via increased number and lifetime of PMNs.

  1. The role of the prostaglandin D2 receptor, DP, in eosinophil trafficking

    DEFF Research Database (Denmark)

    Schratl, Petra; Royer, Julia F; Kostenis, Evi

    2007-01-01

    Prostaglandin (PG) D2 is a major mast cell product that acts via two receptors, the D-type prostanoid (DP) and the chemoattractant receptor-homologous molecule expressed on Th2 cells (CRTH2) receptors. Whereas CRTH2 mediates the chemotaxis of eosinophils, basophils, and Th2 lymphocytes, the role...

  2. Recognition of pathogen-associated nucleic acids by endosomal nucleic acid-sensing toll-like receptors

    Institute of Scientific and Technical Information of China (English)

    Xiaobing He; Huaijie Jia; Zhizhong Jing; Dingxiang Liu

    2013-01-01

    Foreign nucleic acids,the essential signature molecules of invading pathogens that act as danger signals for host cells,are detected by endosomal nucleic acid-sensing tolllike receptors (TLRs) 3,7,8,9,and 13.These TLRs have evolved to recognize ‘non-self' nucleic acids within endosomal compartments and rapidly initiate innate immune responses to ensure host protection through induction of type Ⅰ interferons,inflammatory cytokines,chemokines,and co-stimulatory molecules and maturation of immune cells.In this review,we highlight our understanding of the recognition of pathogen-associated nucleic acids and activation of corresponding signaling pathways through endosomal nucleic acid-sensing TLRs 3,7,8,9,and 13 for an enormous diversity of pathogens,with particular emphasis on their compartmentalization,intracellular trafficking,proteolytic cleavage,autophagy,and regulatory programs.

  3. Localisation Microscopy of Breast Epithelial ErbB-2 Receptors and Gap Junctions: Trafficking after γ-Irradiation, Neuregulin-1β, and Trastuzumab Application

    Science.gov (United States)

    Pilarczyk, Götz; Nesnidal, Ines; Gunkel, Manuel; Bach, Margund; Bestvater, Felix; Hausmann, Michael

    2017-01-01

    In cancer, vulnerable breast epithelium malignance tendency correlates with number and activation of ErbB receptor tyrosine kinases. In the presented work, we observe ErbB receptors activated by irradiation-induced DNA injury or neuregulin-1β application, or alternatively, attenuated by a therapeutic antibody using high resolution fluorescence localization microscopy. The gap junction turnover coinciding with ErbB receptor activation and co-transport is simultaneously recorded. DNA injury caused by 4 Gray of 6 MeV photon γ-irradiation or alternatively neuregulin-1β application mobilized ErbB receptors in a nucleograde fashion—a process attenuated by trastuzumab antibody application. This was accompanied by increased receptor density, indicating packing into transport units. Factors mobilizing ErbB receptors also mobilized plasma membrane resident gap junction channels. The time course of ErbB receptor activation and gap junction mobilization recapitulates the time course of non-homologous end-joining DNA repair. We explain our findings under terms of DNA injury-induced membrane receptor tyrosine kinase activation and retrograde trafficking. In addition, we interpret the phenomenon of retrograde co-trafficking of gap junction connexons stimulated by ErbB receptor activation. PMID:28208769

  4. REEPs are membrane shaping adapter proteins that modulate specific g protein-coupled receptor trafficking by affecting ER cargo capacity.

    Science.gov (United States)

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

    2013-01-01

    Receptor expression enhancing proteins (REEPs) were identified by their ability to enhance cell surface expression of a subset of G protein-coupled receptors (GPCRs), specifically GPCRs that have proven difficult to express in heterologous cell systems. Further analysis revealed that they belong to the Yip (Ypt-interacting protein) family and that some REEP subtypes affect ER structure. Yip family comparisons have established other potential roles for REEPs, including regulation of ER-Golgi transport and processing/neuronal localization of cargo proteins. However, these other potential REEP functions and the mechanism by which they selectively enhance GPCR cell surface expression have not been clarified. By utilizing several REEP family members (REEP1, REEP2, and REEP6) and model GPCRs (α2A and α2C adrenergic receptors), we examined REEP regulation of GPCR plasma membrane expression, intracellular processing, and trafficking. Using a combination of immunolocalization and biochemical methods, we demonstrated that this REEP subset is localized primarily to ER, but not plasma membranes. Single cell analysis demonstrated that these REEPs do not specifically enhance surface expression of all GPCRs, but affect ER cargo capacity of specific GPCRs and thus their surface expression. REEP co-expression with α2 adrenergic receptors (ARs) revealed that this REEP subset interacts with and alter glycosidic processing of α2C, but not α2A ARs, demonstrating selective interaction with cargo proteins. Specifically, these REEPs enhanced expression of and interacted with minimally/non-glycosylated forms of α2C ARs. Most importantly, expression of a mutant REEP1 allele (hereditary spastic paraplegia SPG31) lacking the carboxyl terminus led to loss of this interaction. Thus specific REEP isoforms have additional intracellular functions besides altering ER structure, such as enhancing ER cargo capacity, regulating ER-Golgi processing, and interacting with select cargo proteins

  5. Regulation of Epidermal Growth Factor Receptor Trafficking by Lysine Deacetylase HDAC6

    DEFF Research Database (Denmark)

    Lissanu Deribe, Yonathan; Wild, Philipp; Chandrashaker, Akhila;

    2009-01-01

    Binding of epidermal growth factor (EGF) to its receptor leads to receptor dimerization, assembly of protein complexes, and activation of signaling networks that control key cellular responses. Despite their fundamental role in cell biology, little is known about protein complexes associated...

  6. UNC50 prompts G1/S transition and proliferation in HCC by regulation of epidermal growth factor receptor trafficking.

    Directory of Open Access Journals (Sweden)

    Zhou Fang

    Full Text Available UNC50 has long been recognized as a Golgi apparatus protein in yeast, and is involved in nicotinic receptor trafficking in Caenorhabditis elegans, but little is known about UNC50 gene function in human biology despite it being conserved from yeast to high eukaryotes.We investigated the relation between UNC50 and human hepatocellular carcinoma (HCC and the potential mechanisms underlying HCC development.UNC50 mRNA expression patterns in 12 HCC and adjacent non-cancerous tissues determined using northern blotting were confirmed by real-time PCR in another 44 paired tissues. Microarray experiments were used to screen for global effects of UNC50 knockdown in the Hep3B cell line, and were confirmed by real-time PCR, western blotting, flow cytometry, and tetrazolium assay in both UNC50 overexpression and knockdown Hep3B cells.UNC50 expression levels were upregulated in HCC tissues in comparison with the adjacent non-cancerous tissues. UNC50 knockdown reduced mRNA levels of the downstream targets of the epidermal growth factor receptor (EGFR pathway: cyclin D1 (CCND1, EGF, matrix metalloproteinase-7 (MMP7, aldose reductase-like 1 (AKR1B10, cell surface-associated mucin 1 (MUC1, and gastrin (GAST. Moreover, UNC50 influenced EGF, inducing cell cycle entry by affecting cell surface EGFR amounts.UNC50 may plays some roles in HCC progression by affecting the EGFR pathway.

  7. Phosphorylation of threonine 333 regulates trafficking of the human sst5 somatostatin receptor.

    Science.gov (United States)

    Petrich, Aline; Mann, Anika; Kliewer, Andrea; Nagel, Falko; Strigli, Anne; Märtens, Jan Carlo; Pöll, Florian; Schulz, Stefan

    2013-04-01

    The frequent overexpression of the somatostatin receptors sst2 and sst5 in neuroendocrine tumors provides the molecular basis for therapeutic application of novel multireceptor somatostatin analogs. Although the phosphorylation of the carboxyl-terminal region of the sst2 receptor has been studied in detail, little is known about the agonist-induced regulation of the human sst5 receptor. Here, we have generated phosphosite-specific antibodies for the carboxyl-terminal threonines 333 (T333) and 347 (T347), which enabled us to selectively detect either the T333-phosphorylated or the T347-phosphorylated form of sst5. We show that agonist-mediated phosphorylation occurs at T333, whereas T347 is constitutively phosphorylated in the absence of agonist. We further demonstrate that the multireceptor somatostatin analog pasireotide and the sst5-selective ligand L-817,818 but not octreotide or KE108 were able to promote a detectable T333 phosphorylation. Interestingly, BIM-23268 was the only sst5 agonist that was able to stimulate T333 phosphorylation to the same extent as natural somatostatin. Agonist-induced T333 phosphorylation was dose-dependent and selectively mediated by G protein-coupled receptor kinase 2. Similar to that observed for the sst2 receptor, phosphorylation of sst5 occurred within seconds. However, unlike that seen for the sst2 receptor, dephosphorylation and recycling of sst5 were rapidly completed within minutes. We also identify protein phosphatase 1γ as G protein-coupled receptor phosphatase for the sst5 receptor. Together, we provide direct evidence for agonist-selective phosphorylation of carboxyl-terminal T333. In addition, we identify G protein-coupled receptor kinase 2-mediated phosphorylation and protein phosphatase 1γ-mediated dephosphorylation of T333 as key regulators of rapid internalization and recycling of the human sst5 receptor.

  8. Distinct functional domains within the acidic cluster of tegument protein pp28 required for trafficking and cytoplasmic envelopment of human cytomegalovirus.

    Science.gov (United States)

    Seo, Jun-Young; Jeon, Hyejin; Hong, Sookyung; Britt, William J

    2016-10-01

    Human cytomegalovirus UL99-encoded tegument protein pp28 contains a 16 aa acidic cluster that is required for pp28 trafficking to the assembly compartment (AC) and the virus assembly. However, functional signals within the acidic cluster of pp28 remain undefined. Here, we demonstrated that an acidic cluster rather than specific sorting signals was required for trafficking to the AC. Recombinant viruses with chimeric pp28 proteins expressing non-native acidic clusters exhibited delayed viral growth kinetics and decreased production of infectious virus, indicating that the native acidic cluster of pp28 was essential for wild-type virus assembly. These results suggested that the acidic cluster of pp28 has distinct functional domains required for trafficking and for efficient virus assembly. The first half (aa 44-50) of the acidic cluster was sufficient for pp28 trafficking, whereas the native acidic cluster consisting of aa 51-59 was required for the assembly of wild-type levels of infectious virus.

  9. Differential regulation of δ-opioid receptor trafficking after internalization by TIPP and DPDPE

    Institute of Scientific and Technical Information of China (English)

    Min-huaHONG; Yi-minTAO; Xue-junXU; Zhi-qiangCHI; Jing-genLIU

    2004-01-01

    AIM: To explore the mechanisms underlying the difference between TIPP and DPDPE in desensitization of the δ-opioidreceptors. METHODS: GH3 cells stably expressing HA-tagged δ-opioid receptors were treated with TIPP, DPDPE (1 μmol/L)or morphine (10 μmol/L) for different periods of time in the presence or absence of 50 μmol/L monensin or 10 nmol/L OA.Internalization of δ-opioid receptor was assessed using confocal

  10. Abscisic Acid Receptors: Past, Present and Future

    Institute of Scientific and Technical Information of China (English)

    Jianjun Guo; Xiaohan Yang; David J. Weston; Jin-Gui Chen

    2011-01-01

    Abscisic acid (ABA) is the key plant stress hormone. Consistent with the earlier studies in support of the presence of both membrane- and cytoplasm-localized ABA receptors, recent studies have identified multiple ABA receptors located in various subcellular locations. These include a chloroplast envelope-localized receptor (the H subunit of Chloroplast Mg2+-chelatase/ABA Receptor), two plasma membrane-localized receptors (G-protein Coupled Receptor 2 and GPCR-type G proteins),and one cytosol/nucleus-localized Pyrabactin Resistant (PYR)/PYR-Like (PYL)/Regulatory Component of ABA Receptor 1 (RCAR). Although the downstream molecular events for most of the identified ABA receptors are currently unknown, one of them, PYR/PYL/RCAR was found to directly bind and regulate the activity of a long-known central regulator of ABA signaling, the A-group protein phosphatase 2C (PP2C). Together with the Sucrose Non-fermentation Kinase Subfamily 2 (SnRK2s) protein kinases, a central signaling complex (ABA-PYR-PP2Cs-SnRK2s) that is responsible for ABA signal perception and transduction is supported by abundant genetic, physiological, biochemical and structural evidence. The identification of multiple ABA receptors has advanced our understanding of ABA signal perception and transduction while adding an extra layer of complexity.

  11. Importance of constitutive activity and arrestin-independent mechanisms for intracellular trafficking of the ghrelin receptor

    DEFF Research Database (Denmark)

    Holliday, Nicholas D; Holst, Birgitte; Rodionova, Elena A

    2007-01-01

    . Furthermore the interaction between phosphorylated receptors and beta-arrestin adaptor proteins has been examined. Replacement of the FLAG-tagged GhrelinR C tail with the equivalent GPR39 domain (GhR-39 chimera) preserved G(q) signaling. However in contrast to the GhrelinR, GhR-39 receptors exhibited no basal...... and substantially decreased agonist-induced internalization in transiently transfected HEK293 cells. Internalized GhrelinR and GhR-39 were predominantly localized to recycling compartments, identified with transferrin and the monomeric G proteins Rab5 and Rab11. Both the inverse agonist [d-Arg(1), d-Phe(5), d-Trp(7....... In contrast, agonist-stimulated GhrelinRs recruited the clathrin adaptor green fluorescent protein-tagged beta-arrestin2 to endosomes, coincident with increased receptor phosphorylation. Thus, GhrelinR internalization to recycling compartments depends on C-terminal motifs and constitutive activity...

  12. Functional Proteomics Defines the Molecular Switch Underlying FGF Receptor Trafficking and Cellular Outputs

    DEFF Research Database (Denmark)

    Francavilla, Chiara; Rigbolt, Kristoffer T.G.; Emdal, Kristina B

    2013-01-01

    The stimulation of fibroblast growth factor receptors (FGFRs) with distinct FGF ligands generates specific cellular responses. However, the mechanisms underlying this paradigm have remained elusive. Here, we show that FGF-7 stimulation leads to FGFR2b degradation and, ultimately, cell proliferation......, whereas FGF-10 promotes receptor recycling and cell migration. By combining mass-spectrometry-based quantitative proteomics with fluorescence microscopy and biochemical methods, we find that FGF-10 specifically induces the rapid phosphorylation of tyrosine (Y) 734 on FGFR2b, which leads to PI3K and SH3BP4...... recruitment. This complex is crucial for FGFR2b recycling and responses, given that FGF-10 stimulation of either FGFR2b_Y734F mutant- or SH3BP4-depleted cells switches the receptor endocytic route to degradation, resulting in decreased breast cancer cell migration and the inhibition of epithelial branching...

  13. Complex Pharmacology of Free Fatty Acid Receptors

    DEFF Research Database (Denmark)

    Milligan, Graeme; Shimpukade, Bharat; Ulven, Trond

    2017-01-01

    G protein-coupled receptors (GPCRs) are historically the most successful family of drug targets. In recent times it has become clear that the pharmacology of these receptors is far more complex than previously imagined. Understanding of the pharmacological regulation of GPCRs now extends beyond...... pharmacology have shaped understanding of the complex pharmacology of receptors that recognize and are activated by nonesterified or "free" fatty acids (FFAs). The FFA family of receptors is a recently deorphanized set of GPCRs, the members of which are now receiving substantial interest as novel targets...... for the treatment of metabolic and inflammatory diseases. Further understanding of the complex pharmacology of these receptors will be critical to unlocking their ultimate therapeutic potential....

  14. Effect of C-Terminal S-Palmitoylation on D2 Dopamine Receptor Trafficking and Stability

    OpenAIRE

    2015-01-01

    We have used bioorthogonal click chemistry (BCC), a sensitive non-isotopic labeling method, to analyze the palmitoylation status of the D2 dopamine receptor (D2R), a G protein-coupled receptor (GPCR) crucial for regulation of processes such as mood, reward, and motor control. By analyzing a series of D2R constructs containing mutations in cysteine residues, we found that palmitoylation of the D2R most likely occurs on the C-terminal cysteine residue (C443) of the polypeptide. D2Rs in which C4...

  15. New hyperekplexia mutations provide insight into glycine receptor assembly, trafficking, and activation mechanisms

    DEFF Research Database (Denmark)

    Bode, Anna; Wood, Sian-Elin; Mullins, Jonathan G L

    2013-01-01

    to hyperekplexia. Most hyperekplexia cases are caused by mutations in the α1 subunit of the human glycine receptor (hGlyR) gene (GLRA1). Here we analyzed 68 new unrelated hyperekplexia probands for GLRA1 mutations and identified 19 mutations, of which 9 were novel. Electrophysiological analysis demonstrated...

  16. Recent advances on the δ opioid receptor: from trafficking to function.

    Science.gov (United States)

    Gendron, Louis; Mittal, Nitish; Beaudry, Hélène; Walwyn, Wendy

    2015-01-01

    Within the opioid family of receptors, δ (DOPrs) and μ opioid receptors (MOPrs) are typical GPCRs that activate canonical second-messenger signalling cascades to influence diverse cellular functions in neuronal and non-neuronal cell types. These receptors activate well-known pathways to influence ion channel function and pathways such as the map kinase cascade, AC and PI3K. In addition new information regarding opioid receptor-interacting proteins, downstream signalling pathways and resultant functional effects has recently come to light. In this review, we will examine these novel findings focusing on the DOPr and, in doing so, will contrast and compare DOPrs with MOPrs in terms of differences and similarities in function, signalling pathways, distribution and interactions. We will also discuss and clarify issues that have recently surfaced regarding the expression and function of DOPrs in different cell types and analgesia. This article is part of a themed section on Opioids: New Pathways to Functional Selectivity. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2015.172.issue-2. © 2014 The British Pharmacological Society.

  17. Effect of C-Terminal S-Palmitoylation on D2 Dopamine Receptor Trafficking and Stability.

    Science.gov (United States)

    Ebersole, Brittany; Petko, Jessica; Woll, Matthew; Murakami, Shoko; Sokolina, Kate; Wong, Victoria; Stagljar, Igor; Lüscher, Bernhard; Levenson, Robert

    2015-01-01

    We have used bioorthogonal click chemistry (BCC), a sensitive non-isotopic labeling method, to analyze the palmitoylation status of the D2 dopamine receptor (D2R), a G protein-coupled receptor (GPCR) crucial for regulation of processes such as mood, reward, and motor control. By analyzing a series of D2R constructs containing mutations in cysteine residues, we found that palmitoylation of the D2R most likely occurs on the C-terminal cysteine residue (C443) of the polypeptide. D2Rs in which C443 was deleted showed significantly reduced palmitoylation levels, plasma membrane expression, and protein stability compared to wild-type D2Rs. Rather, the C443 deletion mutant appeared to accumulate in the Golgi, indicating that palmitoylation of the D2R is important for cell surface expression of the receptor. Using the full-length D2R as bait in a membrane yeast two-hybrid (MYTH) screen, we identified the palmitoyl acyltransferase (PAT) zDHHC4 as a D2R interacting protein. Co-immunoprecipitation analysis revealed that several other PATs, including zDHHC3 and zDHHC8, also interacted with the D2R and that each of the three PATs was capable of affecting the palmitoylation status of the D2R. Finally, biochemical analyses using D2R mutants and the palmitoylation blocker, 2-bromopalmitate indicate that palmitoylation of the receptor plays a role in stability of the D2R.

  18. Effect of C-Terminal S-Palmitoylation on D2 Dopamine Receptor Trafficking and Stability.

    Directory of Open Access Journals (Sweden)

    Brittany Ebersole

    Full Text Available We have used bioorthogonal click chemistry (BCC, a sensitive non-isotopic labeling method, to analyze the palmitoylation status of the D2 dopamine receptor (D2R, a G protein-coupled receptor (GPCR crucial for regulation of processes such as mood, reward, and motor control. By analyzing a series of D2R constructs containing mutations in cysteine residues, we found that palmitoylation of the D2R most likely occurs on the C-terminal cysteine residue (C443 of the polypeptide. D2Rs in which C443 was deleted showed significantly reduced palmitoylation levels, plasma membrane expression, and protein stability compared to wild-type D2Rs. Rather, the C443 deletion mutant appeared to accumulate in the Golgi, indicating that palmitoylation of the D2R is important for cell surface expression of the receptor. Using the full-length D2R as bait in a membrane yeast two-hybrid (MYTH screen, we identified the palmitoyl acyltransferase (PAT zDHHC4 as a D2R interacting protein. Co-immunoprecipitation analysis revealed that several other PATs, including zDHHC3 and zDHHC8, also interacted with the D2R and that each of the three PATs was capable of affecting the palmitoylation status of the D2R. Finally, biochemical analyses using D2R mutants and the palmitoylation blocker, 2-bromopalmitate indicate that palmitoylation of the receptor plays a role in stability of the D2R.

  19. Subcellular mechanisms in pulmonary arterial hypertension: combinatorial modalities that inhibit anterograde trafficking and cause bone morphogenetic protein receptor type 2 mislocalization.

    Science.gov (United States)

    Yang, Yang-Ming; Lane, Kirk B; Sehgal, Pravin B

    2013-09-01

    Abstract The natural history of familial pulmonary arterial hypertension (PAH) typically involves mutations in and/or haploinsuffciency of BMPR2 (gene for bone morphogenetic protein receptor type 2) but with low penetrance (10%-15%), delayed onset (in the third or fourth decade), and a gender bias (two- to fourfold more prevalent in postpubertal women). Thus, investigators have sought an understanding of "second-hit" modalities that might affect BMPR2 anterograde trafficking and/or function. Indeed, vascular lung lesions in PAH have been reported to contain enlarged "vacuolated" endothelial and smooth muscle cells with dilated endoplasmic reticulum (ER) cisternae, increased ER structural protein reticulon 4 (also called Nogo-B), and enlarged and fragmented Golgi apparatus. We recently replicated this cellular phenotype in primary human pulmonary arterial endothelial cells and human pulmonary arterial smooth muscle cells in culture by acute knockdown of the estradiol 17β (E2)-responsive proteins signal transducer and activator of transcription 5a (STAT5a) and STAT5b using small interfering RNAs (siRNAs). We have now investigated whether functional haploinsufficiences of these molecules, alone or in combination with other modalities, might interfere with anterograde membrane trafficking using (a) the quantitative tsO45VSV-G-GFP trafficking assay and (b) assays for cell-surface localization of Flag-tagged BMPR2 molecules. The G glycoprotein of the vesicular stomatitis virus (VSV-G) trafficking assay was validated in EA.hy926 endothelial cells by showing that cells exposed to monocrotaline pyrrole displayed reduced anterograde trafficking. Thereafter, the combinatorial knockdowns of STAT5a, STAT5b, BMPR2, and/or endothelial nitric oxide synthase as well as exposure to E2 or 2-methoxyestradiol were observed to significantly inhibit VSV-G trafficking. These combinations also led to intracellular trapping of wild-type Flag-tagged BMPR2. Overexpression of the PAH disease

  20. A role of Rab29 in the integrity of the trans-Golgi network and retrograde trafficking of mannose-6-phosphate receptor.

    Directory of Open Access Journals (Sweden)

    Shicong Wang

    Full Text Available Rab29 (also referred as Rab7L1 is a novel Rab protein, and is recently demonstrated to regulate phagocytosis and traffic from the Golgi to the lysosome. However, its roles in membrane trafficking have not been investigated extensively. Our results in this study revealed that Rab29 is associated with the trans-Golgi network (TGN, and is essential for maintaining the integrity of the TGN, because inhibition of the activity of Rab29 or depletion of Rab29 resulted in fragmentation of the TGN marked by TGN46. Expression of the dominant negative form Rab29T21N or shRNA-Rab29 also altered the distribution of mannose-6-phosphate receptor (M6PR, and interrupted the retrograde trafficking of M6PR through monitoring the endocytosis of CD8-tagged calcium dependent M6PR (cdM6PR or calcium independent M6PR (ciM6PR, but without significant effects on the anterograde trafficking of vesicular stomatitis virus G protein (VSV-G. Our results suggest that Rab29 is essential for the integrity of the TGN and participates in the retrograde trafficking of M6PRs.

  1. A Role of Rab29 in the Integrity of the Trans-Golgi Network and Retrograde Trafficking of Mannose-6-Phosphate Receptor

    Science.gov (United States)

    Wang, Shicong; Ma, Zexu; Xu, Xiaohui; Wang, Zhen; Sun, Lixiang; Zhou, Yunhe; Lin, Xiaosi; Hong, Wanjin; Wang, Tuanlao

    2014-01-01

    Rab29 (also referred as Rab7L1) is a novel Rab protein, and is recently demonstrated to regulate phagocytosis and traffic from the Golgi to the lysosome. However, its roles in membrane trafficking have not been investigated extensively. Our results in this study revealed that Rab29 is associated with the trans-Golgi network (TGN), and is essential for maintaining the integrity of the TGN, because inhibition of the activity of Rab29 or depletion of Rab29 resulted in fragmentation of the TGN marked by TGN46. Expression of the dominant negative form Rab29T21N or shRNA-Rab29 also altered the distribution of mannose-6-phosphate receptor (M6PR), and interrupted the retrograde trafficking of M6PR through monitoring the endocytosis of CD8-tagged calcium dependent M6PR (cdM6PR) or calcium independent M6PR (ciM6PR), but without significant effects on the anterograde trafficking of vesicular stomatitis virus G protein (VSV-G). Our results suggest that Rab29 is essential for the integrity of the TGN and participates in the retrograde trafficking of M6PRs. PMID:24788816

  2. Trafficking of α1B-adrenergic receptor mediated by inverse agonist in living cells

    Institute of Scientific and Technical Information of China (English)

    MingXU; Ying-huaGUAN; NingXU; Zhang-yiLIANG; Shu-yiWang; YaoSONG; Chi-deHAN; Xin-shengZHAO; You-yiZHANG

    2005-01-01

    AIM The project is aimed at understanding the action of inverse agonist at single molecule level and capturing the real time picture of molecular behavior of α1B-adrenergic receptor (AR) mediated by inverse agonist in living cells by single molecule detection (SMD). METHODS The location and distribution of α1B-AR was detected by laser confocal and whole cell 3H-prazosin binding assay. Dynamic imaging of BODIPY-FL-labeled prazosin (Praz), specific antagonist of (1-AR, was observed in α1B-AR stably expressed human embryonic kidney 293 (HEK293) living cells. The detection of real-time dynamic behaviors of AR was achieved by using fluorescence-labeled AR and its ligand combined with SMD techniques. RESULTS α1B-AR was predominantly distributed on the cell surface and 8.2% of the total receptors were located in cytosol.

  3. A Naturally Occurring Isoform Inhibits Parathyroid Hormone Receptor Trafficking and Signaling

    OpenAIRE

    Alonso, Ver?nica; Ardura, Juan A; WANG Bin; Sneddon, W Bruce; Peter A Friedman

    2010-01-01

    Parathyroid hormone (PTH) regulates calcium homeostasis and bone remodeling through its cognitive receptor (PTHR). We describe here a PTHR isoform harboring an in-frame 42-bp deletion of exon 14 (?e14-PTHR) that encodes transmembrane domain 7. ?e14-PTHR was detected in human kidney and buccal epithelial cells. We characterized its topology, cellular localization, and signaling, as well as its interactions with PTHR. The C-terminus of the ?e14-PTHR is extracellular, and cell surface expression...

  4. Safety, tumor trafficking and immunogenicity of chimeric antigen receptor (CAR)-T cells specific for TAG-72 in colorectal cancer.

    Science.gov (United States)

    Hege, Kristen M; Bergsland, Emily K; Fisher, George A; Nemunaitis, John J; Warren, Robert S; McArthur, James G; Lin, Andy A; Schlom, Jeffrey; June, Carl H; Sherwin, Stephen A

    2017-01-01

    T cells engineered to express chimeric antigen receptors (CARs) have established efficacy in the treatment of B-cell malignancies, but their relevance in solid tumors remains undefined. Here we report results of the first human trials of CAR-T cells in the treatment of solid tumors performed in the 1990s. Patients with metastatic colorectal cancer (CRC) were treated in two phase 1 trials with first-generation retroviral transduced CAR-T cells targeting tumor-associated glycoprotein (TAG)-72 and including a CD3-zeta intracellular signaling domain (CART72 cells). In trial C-9701 and C-9702, CART72 cells were administered in escalating doses up to 10(10) total cells; in trial C-9701 CART72 cells were administered by intravenous infusion. In trial C-9702, CART72 cells were administered via direct hepatic artery infusion in patients with colorectal liver metastases. In both trials, a brief course of interferon-alpha (IFN-α) was given with each CART72 infusion to upregulate expression of TAG-72. Fourteen patients were enrolled in C-9701 and nine in C-9702. CART72 manufacturing success rate was 100% with an average transduction efficiency of 38%. Ten patients were treated in CC-9701 and 6 in CC-9702. Symptoms consistent with low-grade, cytokine release syndrome were observed in both trials without clear evidence of on target/off tumor toxicity. Detectable, but mostly short-term (≤14 weeks), persistence of CART72 cells was observed in blood; one patient had CART72 cells detectable at 48 weeks. Trafficking to tumor tissues was confirmed in a tumor biopsy from one of three patients. A subset of patients had (111)Indium-labeled CART72 cells injected, and trafficking could be detected to liver, but T cells appeared largely excluded from large metastatic deposits. Tumor biomarkers carcinoembryonic antigen (CEA) and TAG-72 were measured in serum; there was a precipitous decline of TAG-72, but not CEA, in some patients due to induction of an interfering antibody to the TAG

  5. Activity-dependent PI(3,5)P2 synthesis controls AMPA receptor trafficking during synaptic depression.

    Science.gov (United States)

    McCartney, Amber J; Zolov, Sergey N; Kauffman, Emily J; Zhang, Yanling; Strunk, Bethany S; Weisman, Lois S; Sutton, Michael A

    2014-11-11

    Dynamic regulation of phosphoinositide lipids (PIPs) is crucial for diverse cellular functions, and, in neurons, PIPs regulate membrane trafficking events that control synapse function. Neurons are particularly sensitive to the levels of the low abundant PIP, phosphatidylinositol 3,5-bisphosphate [PI(3,5)P2], because mutations in PI(3,5)P2-related genes are implicated in multiple neurological disorders, including epilepsy, severe neuropathy, and neurodegeneration. Despite the importance of PI(3,5)P2 for neural function, surprisingly little is known about this signaling lipid in neurons, or any cell type. Notably, the mammalian homolog of yeast vacuole segregation mutant (Vac14), a scaffold for the PI(3,5)P2 synthesis complex, is concentrated at excitatory synapses, suggesting a potential role for PI(3,5)P2 in controlling synapse function and/or plasticity. PI(3,5)P2 is generated from phosphatidylinositol 3-phosphate (PI3P) by the lipid kinase PI3P 5-kinase (PIKfyve). Here, we present methods to measure and control PI(3,5)P2 synthesis in hippocampal neurons and show that changes in neural activity dynamically regulate the levels of multiple PIPs, with PI(3,5)P2 being among the most dynamic. The levels of PI(3,5)P2 in neurons increased during two distinct forms of synaptic depression, and inhibition of PIKfyve activity prevented or reversed induction of synaptic weakening. Moreover, altering neuronal PI(3,5)P2 levels was sufficient to regulate synaptic strength bidirectionally, with enhanced synaptic function accompanying loss of PI(3,5)P2 and reduced synaptic strength following increased PI(3,5)P2 levels. Finally, inhibiting PI(3,5)P2 synthesis alters endocytosis and recycling of AMPA-type glutamate receptors (AMPARs), implicating PI(3,5)P2 dynamics in AMPAR trafficking. Together, these data identify PI(3,5)P2-dependent signaling as a regulatory pathway that is critical for activity-dependent changes in synapse strength.

  6. Poliovirus trafficking toward central nervous system via human poliovirus receptor-dependent and -independent pathway.

    Directory of Open Access Journals (Sweden)

    Seii eOHKA

    2012-04-01

    Full Text Available In humans, paralytic poliomyelitis results from the invasion of the central nervous system by circulating poliovirus (PV via the blood-brain barrier (BBB. After the virus enters the central nervous system (CNS, it replicates in neurons, especially in motor neurons (MNs, inducing the cell death that causes paralytic poliomyelitis. Along with this route of dissemination, neural pathway has been reported in humans, monkeys, and PV-sensitive human PV receptor (hPVR/CD155-transgenic (Tg mice. We demonstrated that a fast retrograde axonal transport process is required for PV dissemination through the sciatic nerve of hPVR-Tg mice and that intramuscularly inoculated PV causes paralysis in a hPVR-dependent manner. We also showed that hPVR-independent axonal transport of PV exists in hPVR-Tg and non-Tg mice, indicating that several different pathways for PV axonal transport exist in these mice. Circulating PV after intravenous inoculation in mice cross the BBB at a high rate in a hPVR-independent manner. Recently, we identified transferrin receptor 1 (TfR1 of mouse brain capillary endothelial cells as a binding protein to PV, implicating that TfR1 is a possible receptor for PV to permeate the BBB.

  7. Molecular basis of parathyroid hormone receptor signaling and trafficking: a family B GPCR paradigm.

    Science.gov (United States)

    Vilardaga, Jean-Pierre; Romero, Guillermo; Friedman, Peter A; Gardella, Thomas J

    2011-01-01

    The parathyroid hormone (PTH) receptor type 1 (PTHR), a G protein-coupled receptor (GPCR), transmits signals to two hormone systems-PTH, endocrine and homeostatic, and PTH-related peptide (PTHrP), paracrine-to regulate different biological processes. PTHR responds to these hormonal stimuli by activating heterotrimeric G proteins, such as G(S) that stimulates cAMP production. It was thought that the PTHR, as for all other GPCRs, is only active and signals through G proteins on the cell membrane, and internalizes into a cell to be desensitized and eventually degraded or recycled. Recent studies with cultured cell and animal models reveal a new pathway that involves sustained cAMP signaling from intracellular domains. Not only do these studies challenge the paradigm that cAMP production triggered by activated GPCRs originates exclusively at the cell membrane but they also advance a comprehensive model to account for the functional differences between PTH and PTHrP acting through the same receptor.

  8. Dopamine D1-dependent trafficking of striatal N-methyl-D-aspartate glutamate receptors requires Fyn protein tyrosine kinase but not DARPP-32.

    Science.gov (United States)

    Dunah, Anthone W; Sirianni, Ana C; Fienberg, Allen A; Bastia, Elena; Schwarzschild, Michael A; Standaert, David G

    2004-01-01

    Interactions between dopaminergic and glutamatergic systems in the striatum are thought to underlie both the symptoms and adverse effects of treatment of Parkinson's disease. We have previously reported that activation of the dopamine D1 receptor triggers a rapid redistribution of striatal N-methyl-d-aspartate (NMDA) receptors between intracellular and postsynaptic sub-cellular compartments. To unravel the signaling pathways underlying this trafficking, we studied mice with targeted disruptions of either the gene that encodes the dopamine- and cAMP-regulated phosphoprotein (DARPP-32), a potent and selective inhibitor of protein phosphatase-1, or the protein tyrosine kinase Fyn. In striatal tissue from DARPP-32-depleted mice, basal tyrosine and serine phosphorylation of striatal NMDA receptor subunits NR1, NR2A, and NR2B was normal, and activation of dopamine D1 receptors with the agonist SKF-82958 [(+/-)-6-chloro-7,8-dihydroxy-3-allyl-1-phenyl-2,3,4,5-tetra-hydro-1H-benzazepine] produced redistribution of NMDA receptors from vesicular compartments (P3 and LP2) to synaptosomal membranes (LP1). In the Fyn knockout mice, basal tyrosine phosphorylation of NR2A and NR2B was drastically reduced, whereas serine phosphorylation of these NMDA subunits was unchanged. In the Fyn knockout mice, the dopamine D1 receptor agonist failed to induce subcellular redistribution of NMDA receptors. In addition, Fyn-depleted mice lesioned with 6-hydroxydopamine also failed to exhibit l-DOPA-induced behavioral sensitization, but this may be caused, at least in part, by resistance of these mice to the neurotoxic lesion. These findings suggest a novel mechanism for the trafficking of striatal NMDA receptors by signaling pathways that are independent of DARPP-32 but require Fyn protein tyrosine kinase. Strategies that prevent NMDA receptor subcellular redistribution through inhibition of Fyn kinase may prove useful in the treatment of Parkinson's disease.

  9. Fibronectin-induced VEGF receptor and calcium channel transactivation stimulate GLUT-1 synthesis and trafficking through PPARγ and TC10 in mouse embryonic stem cells.

    Science.gov (United States)

    Suh, Han Na; Han, Ho Jae

    2013-05-01

    Extracellular matrix (ECM) mediates interactions between integrin and growth factor receptor (GFR) or ion channel. Although this crosstalk promotes integration of the downstream signal pathways and then regulates cellular function, the effect of ECM on glucose transporter (GLUT) in stem cells has not been elucidated. Therefore, we examined the effect of fibronectin on GLUT-1 expression, trafficking, and its related signal pathways in mouse embryonic stem cells (mESCs). Fibronectin increased 2-deoxyglucose (DG) uptake and GLUT-1 protein expression that were blocked by transcription or translation inhibitors. Integrin α5β1-bound fibronectin increased 2-DG uptake through cluster formation with vascular endothelial growth factor receptor (VEGFR) 2, and then activated Ras and PI3K/Akt. In another pathway, integrin α5β1 displayed structural and functional interactions with calcium channels, and stimulated 2-DG uptake through calcium influx and PKC activation. Akt and PKC-induced PPARγ phosphorylation enhanced the decreased expression of PPARγ protein, and subsequently increased GLUT-1 protein synthesis and 2-DG uptake. Fibronectin stimulated TC10 activity and cytoskeleton (F-actin) rearrangement, followed by GLUT-1 trafficking. In conclusion, integrin-bound fibronectin stimulates GLUT-1 synthesis through VEGFR2/Ras/PI3K/Akt and calcium channel/Ca(2+)/PKC, which are merged at PPARγ and GLUT-1 trafficking through TC10 and F-actin.

  10. Osteoblast regulation via ligand-activated nuclear trafficking of the oxytocin receptor

    Science.gov (United States)

    Di Benedetto, Adriana; Sun, Li; Zambonin, Carlo G.; Tamma, Roberto; Nico, Beatrice; Calvano, Cosima D.; Colaianni, Graziana; Ji, Yaoting; Mori, Giorgio; Grano, Maria; Lu, Ping; Colucci, Silvia; Yuen, Tony; New, Maria I.; Zallone, Alberta; Zaidi, Mone

    2014-01-01

    We report that oxytocin (Oxt) receptors (Oxtrs), on stimulation by the ligand Oxt, translocate into the nucleus of osteoblasts, implicating this process in the action of Oxt on osteoblast maturation. Sequential immunocytochemistry of intact cells or isolated nucleoplasts stripped of the outer nuclear membrane showed progressive nuclear localization of the Oxtr; this nuclear translocation was confirmed by monitoring the movement of Oxtr–EGFP as well as by immunogold labeling. Nuclear Oxtr localization was conclusively shown by Western immunoblotting and MS of nuclear lysate proteins. We found that the passage of Oxtrs into the nucleus was facilitated by successive interactions with β-arrestins (Arrbs), the small GTPase Rab5, importin-β (Kpnb1), and transportin-1 (Tnpo1). siRNA-mediated knockdown of Arrb1, Arrb2, or Tnpo1 abrogated Oxt-induced expression of the osteoblast differentiation genes osterix (Sp7), Atf4, bone sialoprotein (Ibsp), and osteocalcin (Bglap) without affecting Erk phosphorylation. Likewise and again, without affecting pErk, inhibiting Arrb recruitment by mutating Ser rich clusters of the nuclear localization signal to Ala abolished nuclear import and Oxtr-induced gene expression. These studies define a previously unidentified mechanism for Oxtr action on bone and open possibilities for direct transcriptional modulation by nuclear G protein-coupled receptors. PMID:25378700

  11. Calcium-Modulating Cyclophilin Ligand Regulates Membrane Trafficking of Postsynaptic GABAA Receptors

    Science.gov (United States)

    Yuan, Xu; Yao, Jun; Norris, David; Tran, David D.; Bram, Richard J.; Chen, Gong; Luscher, Bernhard

    2008-01-01

    Accumulation of GABAA receptors (GABAARs) at GABAergic synapses requires the cytoplasmic loop region and C-terminal transmembrane domain of the receptor γ2 subunit. We here report a novel interaction of γ2 with Calcium-Modulating cyclophilin Ligand (CAML), an integral membrane protein that regulates this mechanism. Interaction of GABAARs with CAML depends on both the cytoplasmic region and fourth transmembrane domain of the γ2 subunit, CAML immunoprecipitates with GABAARs from transfected cells and brain lysates and colocalizes with γ2 in ER vesicles in soma and dendrites of neurons. CAML shRNA treatment results in reduced expression of postsynaptic GABAARs, along with significant reductions in GABA-evoked whole-cell currents and GABAergic synaptic function, while glutamatergic transmission is unaffected. Reduced surface expression of GABAARs in CAML mutant neurons is associated with selective deficits in recycling of endocytosed GABAARs to the cell surface. Our results indicate a specific role of CAML in functional expression and endocytic recycling of postsynaptic GABAARs. PMID:18424167

  12. Human Trafficking

    Science.gov (United States)

    Wilson, David McKay

    2011-01-01

    The shadowy, criminal nature of human trafficking makes evaluating its nature and scope difficult. The U.S. State Department and anti-trafficking groups estimate that worldwide some 27 million people are caught in a form of forced servitude today. Public awareness of modern-day slavery is gaining momentum thanks to new abolitionist efforts. Among…

  13. Functions of Kinesin Superfamily Proteins in Neuroreceptor Trafficking

    Directory of Open Access Journals (Sweden)

    Na Wang

    2015-01-01

    Full Text Available Synaptic plasticity is widely regarded as the cellular basis of learning and memory. Understanding the molecular mechanism of synaptic plasticity has been one of center pieces of neuroscience research for more than three decades. It has been well known that the trafficking of α-amino-3-hydroxy-5-methylisoxazoloe-4-propionic acid- (AMPA- type, N-methyl-D-aspartate- (NMDA- type glutamate receptors to and from synapses is a key molecular event underlying many forms of synaptic plasticity. Kainate receptors are another type of glutamate receptors playing important roles in synaptic transmission. In addition, GABA receptors also play important roles in modulating the synaptic plasticity. Kinesin superfamily proteins (also known as KIFs transport various cargos in both anterograde and retrograde directions through the interaction with different adaptor proteins. Recent studies indicate that KIFs regulate the trafficking of NMDA receptors, AMPA receptors, kainate receptors, and GABA receptors and thus play important roles in neuronal activity. Here we review the essential functions of KIFs in the trafficking of neuroreceptor and synaptic plasticity.

  14. The G-protein coupled receptor associated sorting protein GASP-1 regulates the signalling and trafficking of the viral chemokine receptor US28.

    Science.gov (United States)

    Tschische, Pia; Moser, Elisabeth; Thompson, Dawn; Vischer, Henry F; Parzmair, Gerald P; Pommer, Veronika; Platzer, Wolfgang; Schwarzbraun, Thomas; Schaider, Helmut; Smit, Martine J; Martini, Lene; Whistler, Jennifer L; Waldhoer, Maria

    2010-05-01

    Human cytomegalovirus (HCMV) encodes the seven transmembrane(7TM)/G-protein coupled receptor (GPCR) US28, which signals and endocytoses in a constitutive, ligand-independent manner. Here we show that, following endocytosis, US28 is targeted to the lysosomes for degradation as a consequence of its interaction with the GPCR-associated sorting protein-1 (GASP-1). We find that GASP-1 binds to US28 in vitro and that disruption of the GASP-1/US28 interaction by either (i) overexpression of dominant negative cGASP-1 or by (ii) shRNA knock-down of endogenous GASP-1 is sufficient to inhibit the lysosomal targeting of US28 and slow its post-endocytic degradation. Furthermore, we found that GASP-1 affects US28-mediated signalling. The knock-down of endogenous GASP-1 impairs the US28-mediated Galphaq/PLC/inositol phosphate (IP) accumulation as well as the activation of the transcription factors Nuclear Factor-kappaB (NF-kappaB) and cyclic AMP responsive element binding protein (CREB). Overexpression of GASP-1 enhances both IP accumulation and transcription factor activity. Thus, GASP-1 is an important cellular determinant that not only regulates the post-endocytic trafficking of US28, but also regulates the signalling capacities of US28.

  15. Rebooting Trafficking

    Directory of Open Access Journals (Sweden)

    Nicholas de Villiers

    2016-09-01

    Full Text Available While popular psychology and appeals to emotion have unfortunately dominated discussions of ‘sex trafficking’, this article suggests that feminist psychoanalytic film theory and theories of affect are still useful for making sense of the appeal of sensational exposés like Lifetime Television’s Human Trafficking (2005. The dynamic of identification with (and impersonation of a human trafficking ‘victim’ by the rescuing Immigration and Customs Enforcement agent (Mira Sorvino is particularly worthy of scrutiny. Film theory about the ‘rebooting’ of film franchises (iconic brands like Batman also helps explain the preponderance of similar programming—Sex Slaves (2005, Selling the Girl Next Door (2011, Trafficked (2016—and the way contemporary discourses of human trafficking have effectively rebranded the myth of ‘white slavery’.

  16. Rebooting Trafficking

    OpenAIRE

    Nicholas de Villiers

    2016-01-01

    While popular psychology and appeals to emotion have unfortunately dominated discussions of ‘sex trafficking’, this article suggests that feminist psychoanalytic film theory and theories of affect are still useful for making sense of the appeal of sensational exposés like Lifetime Television’s Human Trafficking (2005). The dynamic of identification with (and impersonation of) a human trafficking ‘victim’ by the rescuing Immigration and Customs Enforcement agent (Mira Sorvino) is particularly ...

  17. Minireview: aquaporin 2 trafficking.

    Science.gov (United States)

    Valenti, Giovanna; Procino, Giuseppe; Tamma, Grazia; Carmosino, Monica; Svelto, Maria

    2005-12-01

    In the kidney aquaporin-2 (AQP2) provides a target for hormonal regulation of water transport by vasopressin. Short-term control of water permeability occurs via vesicular trafficking of AQP2 and long-term control through changes in the abundance of AQP2 and AQP3 water channels. Defective AQP2 trafficking causes nephrogenic diabetes insipidus, a condition characterized by the kidney inability to produce concentrated urine because of the insensitivity of the distal nephron to vasopressin. AQP2 is redistributed to the apical membrane of collecting duct cells through activation of a cAMP signaling cascade initiated by the binding of vasopressin to its V2-receptor. Protein kinase A-mediated phosphorylation of AQP2 has been proposed to be essential in regulating AQP2-containing vesicle exocytosis. Cessation of the stimulus is followed by endocytosis of the AQP2 proteins exposed on the plasma membrane and their recycling to the original stores, in which they are retained. Soluble N-ethylmaleimide sensitive fusion factor attachment protein receptors (SNARE) and actin cytoskeleton organization regulated by small GTPase of the Rho family were also proved to be essential for AQP2 trafficking. Data for functional involvement of the SNARE vesicle-associated membrane protein 2 in AQP2 targeting has recently been provided. Changes in AQP2 expression/trafficking are of particular importance in pathological conditions characterized by both dilutional and concentrating defects. One of these conditions, hypercalciuria, has shown to be associated with alteration of AQP2 urinary excretion. More precisely, recent data support the hypothesis that, in vivo external calcium, through activation of calcium-sensing receptors, modulates the expression/trafficking of AQP2. Together these findings underscore the importance of AQP2 in kidney pathophysiology.

  18. AP1S3 mutations are associated with pustular psoriasis and impaired Toll-like receptor 3 trafficking

    NARCIS (Netherlands)

    Setta-Kaffetzi, N.; Simpson, M.A.; Navarini, A.A.; Patel, V.M.; Lu, H.C.; Allen, M.H.; Duckworth, M.; Bachelez, H.; Burden, A.D.; Choon, S.E.; Griffiths, C.E.; Kirby, B.; Kolios, A.; Seyger, M.M.B.; Prins, C.; Smahi, A.; Trembath, R.C.; Fraternali, F.; Smith, C.H.; Barker, J.N.; Capon, F.

    2014-01-01

    Adaptor protein complex 1 (AP-1) is an evolutionary conserved heterotetramer that promotes vesicular trafficking between the trans-Golgi network and the endosomes. The knockout of most murine AP-1 complex subunits is embryonically lethal, so the identification of human disease-associated alleles has

  19. Involvement of β3A Subunit of Adaptor Protein-3 in Intracellular Trafficking of Receptor-like Protein Tyrosine Phosphatase PCP-2

    Institute of Scientific and Technical Information of China (English)

    Hui DONG; Hong YUAN; Weirong JIN; Yan SHEN; Xiaojing XU; Hongyang WANG

    2007-01-01

    PCP-2 is a human receptor-like protein tyrosine phosphatase and a member of the MAM domain family cloned in human pancreatic adenocarcinoma cells. Previous studies showed that PCP-2 directly interacted with β-catenin through the juxtamembrane domain, dephosphorylated β-catenin and played an important role in the regulation of cell adhesion. Recent study showed that PCP-2 was also involved in the repression of β-catenin-induced transcriptional activity. Here we describe the interactions of PCP-2 with the β3A subunit of adaptor protein (AP)-3 and sorting nexin (SNX) 3. These protein complexes were detected using the yeast two-hybrid assay with the juxtamembrane and membrane-proximal catalytic domain of PCP-2 as "bait". Both AP-3 and SNX3 are molecules involved in intracellular trafficking of membrane receptors. The association between the β3A subunit of AP-3 and PCP-2 was further confirmed in mammalian cells. Our results suggested a possible mechanism of intracellular trafficking of PCP-2 mediated by AP-3 and SNX3 which might participate in the regulation of PCP-2 functions.

  20. Peripheral inflammation induces tumor necrosis factor dependent AMPA receptor trafficking and Akt phosphorylation in spinal cord in addition to pain behavior.

    Science.gov (United States)

    Choi, Jeong Il; Svensson, Camilla I; Koehrn, Fred J; Bhuskute, Aditi; Sorkin, Linda S

    2010-05-01

    In the present study, intraplantar carrageenan induced increased mechanical allodynia, phosphorylation of PKB/Akt and GluR1 ser 845 (PKA site) as well as GluR1, but not GluR2 movement into neuronal membranes. This change in membrane GluR1/GluR2 ratio is indicative of Ca(2+) permeable AMPA receptor insertion. Pain behavior was reduced and biochemical changes blocked by spinal pretreatment, but not post-treatment, with a tumor necrosis factor (TNF) antagonist, Etanercept (100microg). Pain behavior was also reduced by spinal inhibition of phosphatidylinositol 3-kinase (PI-3K) (wortmannin; 1 and 5microg) and LY294002; 50 and 100microg) and Akt (Akt inhibitor IV; 3microg). Phosphorylated Akt was found exclusively in neurons in grey matter and in oligodendrocytes in white matter. Interestingly, this increase was seen first in superficial dorsal horn and alpha-motor neurons (peak 45min) and later (peak 2h post-injection) in deep dorsal horn neurons. Akt and GluR1 phosphorylation, AMPA receptor trafficking and mechanical allodynia were all TNF dependent. Whether phosphorylation of Akt and of GluR1 are in series or in parallel or upstream of pain behavior remains to be determined. Certainly, TNF-mediated GluR1 trafficking appears to play a major role in inflammatory pain and TNF-mediated effects such as these could represent a path by which glia contribute to neuronal sensitization (spinal LTP) and pathological pain.

  1. Regulation of Monocarboxylic Acid Transporter 1 Trafficking by the Canonical Wnt/β-Catenin Pathway in Rat Brain Endothelial Cells Requires Cross-talk with Notch Signaling.

    Science.gov (United States)

    Liu, Zejian; Sneve, Mary; Haroldson, Thomas A; Smith, Jeffrey P; Drewes, Lester R

    2016-04-01

    The transport of monocarboxylate fuels such as lactate, pyruvate, and ketone bodies across brain endothelial cells is mediated by monocarboxylic acid transporter 1 (MCT1). Although the canonical Wnt/β-catenin pathway is required for rodent blood-brain barrier development and for the expression of associated nutrient transporters, the role of this pathway in the regulation of brain endothelial MCT1 is unknown. Here we report expression of nine members of the frizzled receptor family by the RBE4 rat brain endothelial cell line. Furthermore, activation of the canonical Wnt/β-catenin pathway in RBE4 cells via nuclear β-catenin signaling with LiCl does not alter brain endothelialMct1mRNA but increases the amount of MCT1 transporter protein. Plasma membrane biotinylation studies and confocal microscopic examination of mCherry-tagged MCT1 indicate that increased transporter results from reduced MCT1 trafficking from the plasma membrane via the endosomal/lysosomal pathway and is facilitated by decreased MCT1 ubiquitination following LiCl treatment. Inhibition of the Notch pathway by the γ-secretase inhibitorN-[N-(3,5-difluorophenacetyl)-l-alanyl]-S-phenylglycinet-butyl ester negated the up-regulation of MCT1 by LiCl, demonstrating a cross-talk between the canonical Wnt/β-catenin and Notch pathways. Our results are important because they show, for the first time, the regulation of MCT1 in cerebrovascular endothelial cells by the multifunctional canonical Wnt/β-catenin and Notch signaling pathways.

  2. Cyclic GMP signaling in cardiomyocytes modulates fatty acid trafficking and prevents triglyceride accumulation

    Science.gov (United States)

    While the balance between carbohydrates and fatty acids for energy production appears to be crucial for cardiac homeostasis, much remains to be learned about the molecular mechanisms underlying this relationship. Given the reported benefits of cGMP signaling on the myocardium, we investigated the im...

  3. Stress differentially alters mu opioid receptor density and trafficking in parvalbumin-containing interneurons in the female and male rat hippocampus.

    Science.gov (United States)

    Milner, Teresa A; Burstein, Suzanne R; Marrone, Gina F; Khalid, Sana; Gonzalez, Andreina D; Williams, Tanya J; Schierberl, Kathryn C; Torres-Reveron, Annelyn; Gonzales, Keith L; McEwen, Bruce S; Waters, Elizabeth M

    2013-11-01

    Stress differentially affects hippocampal-dependent learning relevant to addiction and morphology in male and female rats. Mu opioid receptors (MORs), which are located in parvalbumin (PARV)-containing GABAergic interneurons and are trafficked in response to changes in the hormonal environment, play a critical role in promoting principal cell excitability and long-term potentiation. Here, we compared the effects of acute and chronic immobilization stress (AIS and CIS) on MOR trafficking in PARV-containing neurons in the hilus of the dentate gyrus in female and male rats using dual label immunoelectron microscopy. Following AIS, the density of MOR silver-intensified gold particles (SIGs) in the cytoplasm of PARV-labeled dendrites was significantly reduced in females (estrus stage). Conversely, AIS significantly increased the proportion of cytoplasmic MOR SIGs in PARV-labeled dendrites in male rats. CIS significantly reduced the number of PARV-labeled neurons in the dentate hilus of males but not females. However, MOR/PARV-labeled dendrites and terminals were significantly smaller in CIS females, but not males, compared with controls. Following CIS, the density of cytoplasmic MOR SIGs increased in PARV-labeled dendrites and terminals in females. Moreover, the proportion of near-plasmalemmal MOR SIGs relative to total decreased in large PARV-labeled dendrites in females. After CIS, no changes in the density or trafficking of MOR SIGs were seen in PARV-labeled dendrites or terminals in males. These data show that AIS and CIS differentially affect available MOR pools in PARV-containing interneurons in female and male rats. Furthermore, they suggest that CIS could affect principal cell excitability in a manner that maintains learning processes in females but not males.

  4. The Viral G Protein-Coupled Receptor ORF74 Hijacks β-Arrestins for Endocytic Trafficking in Response to Human Chemokines.

    Science.gov (United States)

    de Munnik, Sabrina M; Kooistra, Albert J; van Offenbeek, Jody; Nijmeijer, Saskia; de Graaf, Chris; Smit, Martine J; Leurs, Rob; Vischer, Henry F

    2015-01-01

    Kaposi's sarcoma-associated herpesvirus-infected cells express the virally encoded G protein-coupled receptor ORF74. Although ORF74 is constitutively active, it binds human CXC chemokines that modulate this basal activity. ORF74-induced signaling has been demonstrated to underlie the development of the angioproliferative tumor Kaposi's sarcoma. Whereas G protein-dependent signaling of ORF74 has been the subject of several studies, the interaction of this viral GPCR with β-arrestins has hitherto not been investigated. Bioluminescence resonance energy transfer experiments demonstrate that ORF74 recruits β-arrestins and subsequently internalizes in response to human CXCL1 and CXCL8, but not CXCL10. Internalized ORF74 traffics via early endosomes to recycling and late endosomes. Site-directed mutagenesis and homology modeling identified four serine and threonine residues at the distal end of the intracellular carboxyl-terminal of ORF74 that are required for β-arrestin recruitment and subsequent endocytic trafficking. Hijacking of the human endocytic trafficking machinery is a previously unrecognized action of ORF74.

  5. Molecular pharmacology of 4-substituted glutamic acid analogues at ionotropic and metabotropic excitatory amino acid receptors

    DEFF Research Database (Denmark)

    Bräuner-Osborne, Hans; Nielsen, B; Stensbøl, T B;

    1997-01-01

    using rat brain ionotropic glutamate receptors, and in functional assays using cloned metabotropic glutamate (mGlu) receptors. As a notable result of these studies, (2S,4R)-4-methylglutamic acid and (2S,4S)-4-methylglutamic acid were shown to be selective for kainic acid receptors and mGlu receptors......The pharmacology of (2S,4R)-4-methylglutamic acid, (2S,4S)-4-methylglutamic acid and (S)- and (R)-4-methyleneglutamic acids (obtained in high chemical and enantiomeric purity from racemic 4-methyleneglutamic acid by chiral HPLC using a Crownpak CR(+) column), was examined in binding experiments...... (subtypes 1alpha and 2), respectively, whereas (S)-4-methyleneglutamic acid showed high but rather non-selective affinity for the (RS)-2-amino-3-(3-hydroxy-5-methylisoxazol-4-yl)propionic acid (AMPA), kainic acid, NMDA and mGlu receptors (subtypes 1alpha and 2). Although none of the compounds were specific...

  6. Promiscuous Seven Transmembrane Receptors Sensing L-α-amino Acids

    DEFF Research Database (Denmark)

    Smajilovic, Sanela; Wellendorph, Petrine; Bräuner-Osborne, Hans

    2014-01-01

    A number of nutrient sensing seven trans-membrane (7TM) receptors have been identified and characterized over the past few years. While the sensing mechanisms to carbohydrates and free fatty acids are well understood, the molecular basis of amino acid sensing has recently come to the limelight. T....... The present review describes the current status of promiscuous L-α-amino acid sensors, the calcium sensing receptor (CaSR), the GPRC6A receptor, the T1R1/T1R3 receptor and also their molecular pharmacology, expression pattern and physiological significance....

  7. Quinoline based receptor in fluorometric discrimination of carboxylic acids

    Directory of Open Access Journals (Sweden)

    2008-12-01

    Full Text Available Quinoline and naphthalene-based fluororeceptors 1 and 2 have been designed and synthesized for detection of hydroxy carboxylic acids in less polar solvents. The receptor 1 shows monomer emission quenching followed by excimer emission upon hydrogen bond-mediated complexation of carboxylic acids. The excimer emission distinguishes aromatic dicarboxylic acids from aliphatic dicarboxylic acids and even long chain aliphatic dicarboxylic acids from short chain aliphatic dicarboxylic acids. The receptor 1 is found to be selective for citric acid with a strong excimer emission in CHCl3. On the contrary, the receptor 2 exhibited less binding constant value and did not form any excimer upon complexation with the same acids under similar conditions. This established the role of quinoline ring nitrogen in binding with the acids.

  8. Trafficking of calcium-permeable and calcium-impermeable AMPA receptors in nucleus accumbens medium spiny neurons co-cultured with prefrontal cortex neurons.

    Science.gov (United States)

    Werner, Craig T; Murray, Conor H; Reimers, Jeremy M; Chauhan, Niravkumar M; Woo, Kenneth K Y; Molla, Hanna M; Loweth, Jessica A; Wolf, Marina E

    2017-04-01

    AMPA receptor (AMPAR) transmission onto medium spiny neurons (MSNs) of the adult rat nucleus accumbens (NAc) is normally dominated by GluA2-containing, Ca(2+)-impermeable AMPAR (CI-AMPARs). However, GluA2-lacking, Ca(2+)-permeable AMPA receptors (CP-AMPARs) accumulate after prolonged withdrawal from extended-access cocaine self-administration and thereafter their activation is required for the intensified (incubated) cue-induced cocaine craving that characterizes prolonged withdrawal from such regimens. These findings suggest the existence of mechanisms in NAc MSNs that differentially regulate CI-AMPARs and CP-AMPARs. Here, we compared trafficking of GluA1A2 CI-AMPARs and homomeric GluA1 CP-AMPARs using immunocytochemical assays in cultured NAc MSNs plated with prefrontal cortical neurons to restore excitatory inputs. We began by evaluating constitutive internalization of surface receptors and found that this occurs more rapidly for CP-AMPARs. Next, we studied receptor insertion into the membrane; combined with past results, the present findings suggest that activation of protein kinase A accelerates insertion of both CP-AMPARs and CI-AMPARs. We also studied constitutive cycling (net loss of receptors from the membrane under conditions where internalization and recycling are both occurring). Interestingly, although CP-AMPARs exhibit faster constitutive internalization, they cycle at similar rates as CI-AMPARs, suggesting faster reinsertion of CP-AMPARs. In studies of synaptic scaling, long-term (24 h) activity blockade increased surface expression and cycling rates of CI-AMPARs but not CP-AMPARs, whereas long-term increases in activity produced more pronounced scaling down of CI-AMPARs than CP-AMPARs but did not alter receptor cycling. These findings can be used to evaluate and generate hypotheses regarding AMPAR plasticity in the rat NAc following cocaine exposure. Copyright © 2016 Elsevier Ltd. All rights reserved.

  9. GABAB receptor trafficking and interacting proteins: targets for the development of highly specific therapeutic strategies to treat neurological disorders?

    Science.gov (United States)

    Benke, Dietmar

    2013-12-01

    GABAB receptors mediate slow inhibitory neurotransmission throughout the central nervous system thereby controlling the excitability of neurons. They have been implicated in numerous neurological disorders making them an attractive drug target. However, due to considerable side effects, the agonist baclofen is so far the only drug on the market targeting GABAB receptors, primarily for the treatment of spasticity. Because GABAB receptors are involved in a variety of brain functions it is rather unlikely to avoid unwanted effects with systemically administered drugs directly addressing ligand binding sites of the receptor. To minimize side effects, it would be desirable to target only those receptors involved in a given pathological state. This commentary discusses the idea that restoring impaired GABAB receptor function in diseased neurons by interfering with receptor-protein interactions may be an approach to specifically target only those receptors involved in the pathological state. Two recently discovered mechanisms that down-regulate the level of functional GABAB receptors most likely contribute to cerebral ischemia and neuropathic pain, respectively. In both mechanisms, small interfering peptides disrupting protein-protein interactions may offer a highly specific means to restore normal receptor function selectively at the site of malfunction. If restored functional GABAB receptor expression in these diseases has beneficial effects, this may serve as a starting point for the development of a highly specific therapeutic interventions. Such an approach is expected to minimize side effects because it promises to leave those GABAB receptors unaffected which are not involved in the dysfunction.

  10. TIM-1 glycoprotein binds the adhesion receptor P-selectin and mediates T cell trafficking during inflammation and autoimmunity.

    Science.gov (United States)

    Angiari, Stefano; Donnarumma, Tiziano; Rossi, Barbara; Dusi, Silvia; Pietronigro, Enrica; Zenaro, Elena; Della Bianca, Vittorina; Toffali, Lara; Piacentino, Gennj; Budui, Simona; Rennert, Paul; Xiao, Sheng; Laudanna, Carlo; Casasnovas, Jose M; Kuchroo, Vijay K; Constantin, Gabriela

    2014-04-17

    Selectins play a central role in leukocyte trafficking by mediating tethering and rolling on vascular surfaces. Here we have reported that T cell immunoglobulin and mucin domain 1 (TIM-1) is a P-selectin ligand. We have shown that human and murine TIM-1 binds to P-selectin, and that TIM-1 mediates tethering and rolling of T helper 1 (Th1) and Th17, but not Th2 and regulatory T cells on P-selectin. Th1 and Th17 cells lacking the TIM-1 mucin domain showed reduced rolling in thrombin-activated mesenteric venules and inflamed brain microcirculation. Inhibition of TIM-1 had no effect on naive T cell homing, but it reduced T cell recruitment in a skin hypersensitivity model and blocked experimental autoimmune encephalomyelitis. Uniquely, the TIM-1 immunoglobulin variable domain was also required for P-selectin binding. Our data demonstrate that TIM-1 is a major P-selectin ligand with a specialized role in T cell trafficking during inflammatory responses and the induction of autoimmune disease.

  11. Overexpression of human fatty acid transport protein 2/very long chain acyl-CoA synthetase 1 (FATP2/Acsvl1) reveals distinct patterns of trafficking of exogenous fatty acids

    Energy Technology Data Exchange (ETDEWEB)

    Melton, Elaina M. [Department of Biochemistry, University of Nebraska, Lincoln, NE (United States); Center for Cardiovascular Sciences, Albany Medical College, Albany, NY (United States); Cerny, Ronald L. [Department of Chemistry, University of Nebraska, Lincoln, NE (United States); DiRusso, Concetta C. [Department of Biochemistry, University of Nebraska, Lincoln, NE (United States); Black, Paul N., E-mail: pblack2@unl.edu [Department of Biochemistry, University of Nebraska, Lincoln, NE (United States)

    2013-11-01

    Highlights: •Roles of FATP2 in fatty acid transport/activation contribute to lipid homeostasis. •Use of 13C- and D-labeled fatty acids provide novel insights into FATP2 function. •FATP2-dependent trafficking of FA into phospholipids results in distinctive profiles. •FATP2 functions in the transport and activation pathways for exogenous fatty acids. -- Abstract: In mammals, the fatty acid transport proteins (FATP1 through FATP6) are members of a highly conserved family of proteins, which function in fatty acid transport proceeding through vectorial acylation and in the activation of very long chain fatty acids, branched chain fatty acids and secondary bile acids. FATP1, 2 and 4, for example directly function in fatty acid transport and very long chain fatty acids activation while FATP5 does not function in fatty acid transport but activates secondary bile acids. In the present work, we have used stable isotopically labeled fatty acids differing in carbon length and saturation in cells expressing FATP2 to gain further insights into how this protein functions in fatty acid transport and intracellular fatty acid trafficking. Our previous studies showed the expression of FATP2 modestly increased C16:0-CoA and C20:4-CoA and significantly increased C18:3-CoA and C22:6-CoA after 4 h. The increases in C16:0-CoA and C18:3-CoA suggest FATP2 must necessarily partner with a long chain acyl CoA synthetase (Acsl) to generate C16:0-CoA and C18:3-CoA through vectorial acylation. The very long chain acyl CoA synthetase activity of FATP2 is consistent in the generation of C20:4-CoA and C22:6-CoA coincident with transport from their respective exogenous fatty acids. The trafficking of exogenous fatty acids into phosphatidic acid (PA) and into the major classes of phospholipids (phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylinositol (PI), and phosphatidyserine (PS)) resulted in distinctive profiles, which changed with the expression of FATP2. The

  12. Ionotropic excitatory amino acid receptor ligands. Synthesis and pharmacology of a new amino acid AMPA antagonist

    DEFF Research Database (Denmark)

    Madsen, U; Sløk, F A; Stensbøl, T B;

    2000-01-01

    We have previously described the potent and selective (RS)-2-amino-3-(3-hydroxy-5-methyl-4-isoxazolyl)propionic acid (AMPA) receptor agonist, (RS)-2-amino-3-(3-carboxy-5-methyl-4-isoxazolyl)propionic acid (ACPA), and the AMPA receptor antagonist (RS)-2-amino-3-[3-(carboxymethoxy)-5-methyl-4......-isoxazolyl]propionic acid (AMOA). Using these AMPA receptor ligands as leads, a series of compounds have been developed as tools for further elucidation of the structural requirements for activation and blockade of AMPA receptors. The synthesized compounds have been tested for activity at ionotropic...... excitatory amino acid (EAA) receptors using receptor binding and electrophysiological techniques, and for activity at metabotropic EAA receptors using second messenger assays. Compounds 1 and 4 were essentially inactive. (RS)-2-Amino-3-[3-(2-carboxyethyl)-5-methyl-4-isoxazolyl]propionic acid (ACMP, 2...

  13. Slow-pressor angiotensin II hypertension and concomitant dendritic NMDA receptor trafficking in estrogen receptor β-containing neurons of the mouse hypothalamic paraventricular nucleus are sex and age dependent.

    Science.gov (United States)

    Marques-Lopes, Jose; Van Kempen, Tracey; Waters, Elizabeth M; Pickel, Virginia M; Iadecola, Costantino; Milner, Teresa A

    2014-09-01

    The incidence of hypertension increases after menopause. Similar to humans, "slow-pressor" doses of angiotensin II (AngII) increase blood pressure in young males, but not in young female mice. However, AngII increases blood pressure in aged female mice, paralleling reproductive hormonal changes. These changes could influence receptor trafficking in central cardiovascular circuits and contribute to hypertension. Increased postsynaptic N-methyl-D-aspartate (NMDA) receptor activity in the hypothalamic paraventricular nucleus (PVN) is crucial for the sympathoexcitation driving AngII hypertension. Estrogen receptors β (ERβs) are present in PVN neurons. We tested the hypothesis that changes in ovarian hormones with age promote susceptibility to AngII hypertension, and influence NMDA receptor NR1 subunit trafficking in ERβ-containing PVN neurons. Transgenic mice expressing enhanced green fluorescent protein (EGFP) in ERβ-containing cells were implanted with osmotic minipumps delivering AngII (600 ng/kg/min) or saline for 2 weeks. AngII increased blood pressure in 2-month-old males and 18-month-old females, but not in 2-month-old females. By electron microscopy, NR1-silver-intensified immunogold (SIG) was mainly in ERβ-EGFP dendrites. At baseline, NR1-SIG density was greater in 2-month-old females than in 2-month-old males or 18-month-old females. After AngII infusion, NR1-SIG density was decreased in 2-month-old females, but increased in 2-month-old males and 18-month-old females. These findings suggest that, in young female mice, NR1 density is decreased in ERβ-PVN dendrites thus reducing NMDA receptor activity and preventing hypertension. Conversely, in young males and aged females, NR1 density is upregulated in ERβ-PVN dendrites and ultimately leads to the neurohumoral dysfunction driving hypertension.

  14. Excitatory synapses are stronger in the hippocampus of Rett syndrome mice due to altered synaptic trafficking of AMPA-type glutamate receptors.

    Science.gov (United States)

    Li, Wei; Xu, Xin; Pozzo-Miller, Lucas

    2016-03-15

    Deficits in long-term potentiation (LTP) at central excitatory synapses are thought to contribute to cognitive impairments in neurodevelopmental disorders associated with intellectual disability and autism. Using the methyl-CpG-binding protein 2 (Mecp2) knockout (KO) mouse model of Rett syndrome, we show that naïve excitatory synapses onto hippocampal pyramidal neurons of symptomatic mice have all of the hallmarks of potentiated synapses. Stronger Mecp2 KO synapses failed to undergo LTP after either theta-burst afferent stimulation or pairing afferent stimulation with postsynaptic depolarization. On the other hand, basal synaptic strength and LTP were not affected in slices from younger presymptomatic Mecp2 KO mice. Furthermore, spine synapses in pyramidal neurons from symptomatic Mecp2 KO are larger and do not grow in size or incorporate GluA1 subunits after electrical or chemical LTP. Our data suggest that LTP is occluded in Mecp2 KO mice by already potentiated synapses. The higher surface levels of GluA1-containing receptors are consistent with altered expression levels of proteins involved in AMPA receptor trafficking, suggesting previously unidentified targets for therapeutic intervention for Rett syndrome and other MECP2-related disorders.

  15. ARF6 and GASP-1 are post-endocytic sorting proteins selectively involved in the intracellular trafficking of dopamine D₂ receptors mediated by GRK and PKC in transfected cells.

    Science.gov (United States)

    Cho, D I; Zheng, M; Min, C; Kwon, K J; Shin, C Y; Choi, H K; Kim, K M

    2013-03-01

    GPCRs undergo both homologous and heterologous regulatory processes in which receptor phosphorylation plays a critical role. The protein kinases responsible for each pathway are well established; however, other molecular details that characterize each pathway remain unclear. In this study, the molecular mechanisms that determine the differences in the functional roles and intracellular trafficking between homologous and PKC-mediated heterologous internalization pathways for the dopamine D₂ receptor were investigated. All of the S/T residues located within the intracellular loops of D₂ receptor were mutated, and the residues responsible for GRK- and PKC-mediated internalization were determined in HEK-293 cells and SH-SY5Y cells. The functional role of receptor internalization and the cellular components that determine the post-endocytic fate of internalized D₂ receptors were investigated in the transfected cells. T134, T225/S228/S229 and S325 were involved in PKC-mediated D₂ receptor desensitization. S229 and adjacent S/T residues mediated the PKC-dependent internalization of D₂ receptors, which induced down-regulation and desensitization. S/T residues within the second intracellular loop and T225 were the major residues involved in GRK-mediated internalization of D₂ receptors, which induced receptor resensitization. ARF6 mediated the recycling of D₂ receptors internalized in response to agonist stimulation. In contrast, GASP-1 mediated the down-regulation of D₂ receptors internalized in a PKC-dependent manner. GRK- and PKC-mediated internalizations of D₂ receptors occur through different intracellular trafficking pathways and mediate distinct functional roles. Distinct S/T residues within D₂ receptors and different sorting proteins are involved in the dissimilar regulation of D₂ receptors by GRK2 and PKC. © 2012 The Authors. British Journal of Pharmacology © 2012 The British Pharmacological Society.

  16. ARF6 and GASP-1 are post-endocytic sorting proteins selectively involved in the intracellular trafficking of dopamine D2 receptors mediated by GRK and PKC in transfected cells

    Science.gov (United States)

    Cho, DI; Zheng, M; Min, C; Kwon, KJ; Shin, CY; Choi, HK; Kim, KM

    2013-01-01

    Background and Purpose GPCRs undergo both homologous and heterologous regulatory processes in which receptor phosphorylation plays a critical role. The protein kinases responsible for each pathway are well established; however, other molecular details that characterize each pathway remain unclear. In this study, the molecular mechanisms that determine the differences in the functional roles and intracellular trafficking between homologous and PKC-mediated heterologous internalization pathways for the dopamine D2 receptor were investigated. Experimental Approach All of the S/T residues located within the intracellular loops of D2 receptor were mutated, and the residues responsible for GRK- and PKC-mediated internalization were determined in HEK-293 cells and SH-SY5Y cells. The functional role of receptor internalization and the cellular components that determine the post-endocytic fate of internalized D2 receptors were investigated in the transfected cells. Key Results T134, T225/S228/S229 and S325 were involved in PKC-mediated D2 receptor desensitization. S229 and adjacent S/T residues mediated the PKC-dependent internalization of D2 receptors, which induced down-regulation and desensitization. S/T residues within the second intracellular loop and T225 were the major residues involved in GRK-mediated internalization of D2 receptors, which induced receptor resensitization. ARF6 mediated the recycling of D2 receptors internalized in response to agonist stimulation. In contrast, GASP-1 mediated the down-regulation of D2 receptors internalized in a PKC-dependent manner. Conclusions and Implications GRK- and PKC-mediated internalizations of D2 receptors occur through different intracellular trafficking pathways and mediate distinct functional roles. Distinct S/T residues within D2 receptors and different sorting proteins are involved in the dissimilar regulation of D2 receptors by GRK2 and PKC. PMID:23082996

  17. Complex pharmacology of free fatty acid receptors

    OpenAIRE

    Milligan, Graeme; Shimpukade, Bharat; Ulven, Trond; Hudson, Brian D.

    2017-01-01

    G protein-coupled receptors (GPCRs) are historically the most successful family of drug targets. In recent times it has become clear that the pharmacology of these receptors is far more complex than previously imagined. Understanding of the pharmacological regulation of GPCRs now extends beyond simple competitive agonism or antagonism by ligands interacting with the orthosteric binding site of the receptor to incorporate concepts of allosteric agonism, allosteric modulation, signaling bias, c...

  18. Economics of human trafficking.

    Science.gov (United States)

    Wheaton, Elizabeth M; Schauer, Edward J; Galli, Thomas V

    2010-01-01

    Because freedom of choice and economic gain are at the heart of productivity, human trafficking impedes national and international economic growth. Within the next 10 years, crime experts expect human trafficking to surpass drug and arms trafficking in its incidence, cost to human well-being, and profitability to criminals (Schauer and Wheaton, 2006: 164-165). The loss of agency from human trafficking as well as from modern slavery is the result of human vulnerability (Bales, 2000: 15). As people become vulnerable to exploitation and businesses continually seek the lowest-cost labour sources, trafficking human beings generates profit and a market for human trafficking is created. This paper presents an economic model of human trafficking that encompasses all known economic factors that affect human trafficking both across and within national borders. We envision human trafficking as a monopolistically competitive industry in which traffickers act as intermediaries between vulnerable individuals and employers by supplying differentiated products to employers. In the human trafficking market, the consumers are employers of trafficked labour and the products are human beings. Using a rational-choice framework of human trafficking we explain the social situations that shape relocation and working decisions of vulnerable populations leading to human trafficking, the impetus for being a trafficker, and the decisions by employers of trafficked individuals. The goal of this paper is to provide a common ground upon which policymakers and researchers can collaborate to decrease the incidence of trafficking in humans.

  19. Overexpression of human fatty acid transport protein 2/very long chain acyl-CoA synthetase 1 (FATP2/Acsvl1) reveals distinct patterns of trafficking of exogenous fatty acids.

    Science.gov (United States)

    Melton, Elaina M; Cerny, Ronald L; DiRusso, Concetta C; Black, Paul N

    2013-11-01

    In mammals, the fatty acid transport proteins (FATP1 through FATP6) are members of a highly conserved family of proteins, which function in fatty acid transport proceeding through vectorial acylation and in the activation of very long chain fatty acids, branched chain fatty acids and secondary bile acids. FATP1, 2 and 4, for example directly function in fatty acid transport and very long chain fatty acids activation while FATP5 does not function in fatty acid transport but activates secondary bile acids. In the present work, we have used stable isotopically labeled fatty acids differing in carbon length and saturation in cells expressing FATP2 to gain further insights into how this protein functions in fatty acid transport and intracellular fatty acid trafficking. Our previous studies showed the expression of FATP2 modestly increased C16:0-CoA and C20:4-CoA and significantly increased C18:3-CoA and C22:6-CoA after 4h. The increases in C16:0-CoA and C18:3-CoA suggest FATP2 must necessarily partner with a long chain acyl CoA synthetase (Acsl) to generate C16:0-CoA and C18:3-CoA through vectorial acylation. The very long chain acyl CoA synthetase activity of FATP2 is consistent in the generation of C20:4-CoA and C22:6-CoA coincident with transport from their respective exogenous fatty acids. The trafficking of exogenous fatty acids into phosphatidic acid (PA) and into the major classes of phospholipids (phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylinositol (PI), and phosphatidyserine (PS)) resulted in distinctive profiles, which changed with the expression of FATP2. The trafficking of exogenous C16:0 and C22:6 into PA was significant where there was 6.9- and 5.3-fold increased incorporation, respectively, over the control; C18:3 and C20:4 also trended to increase in the PA pool while there were no changes for C18:1 and C18:2. The trafficking of C18:3 into PC and PI trended higher and approached significance. In the case of C20:4, expression of

  20. Anterograde trafficking of G protein-coupled receptors: function of the C-terminal F(X)6LL motif in export from the endoplasmic reticulum.

    Science.gov (United States)

    Duvernay, Matthew T; Dong, Chunmin; Zhang, Xiaoping; Zhou, Fuguo; Nichols, Charles D; Wu, Guangyu

    2009-04-01

    We have reported previously that the F(X)(6)LL motif in the C termini is essential for export of alpha(2B)-adrenergic (alpha(2B)-AR) and angiotensin II type 1 receptors (AT1Rs) from the endoplasmic reticulum (ER). Here, we further demonstrate that mutation of the F(X)(6)LL motif similarly abolished the cell-surface expression of alpha(2B)-AR, AT1R, alpha(1B)-AR, and beta(2)-AR, suggesting that the F(X)(6)LL motif plays a general role in ER export of G protein-coupled receptors (GPCRs). Mutation of Phe to Val, Leu, Trp, and Tyr, and mutation of LL to FF and VV, markedly inhibited alpha(2B)-AR transport, indicating that the F(X)(6)LL function cannot be fully substituted by other hydrophobic residues. The structural analysis revealed that the Phe residue in the F(X)(6)LL motif is buried in the transmembrane domains and possibly interacts with Ile58 in beta(2)-AR and Val42 in alpha(2B)-AR, whereas the LL motif is exposed to the cytosolic space. Indeed, mutation of Ile58 in beta(2)-AR and Val42 in alpha(2B)-AR markedly disrupted cell surface transport of the receptors. It is noteworthy that the Val and Ile residues are highly conserved among the GPCRs carrying the F(X)(6)LL motif. Furthermore, the Phe mutant exhibited a stronger interaction with ER chaperones and was more potently rescued by physical and chemical treatments than the LL mutant. These data suggest that the Phe residue is probably involved in folding of alpha(2B)-AR and beta(2)-AR, possibly through interaction with other hydrophobic residues in neighboring domains. These data also provide the first evidence implying crucial roles of the C termini possibly through modulating multiple events in anterograde trafficking of GPCRs.

  1. Oligomeric amyloid-{beta} inhibits the proteolytic conversion of brain-derived neurotrophic factor (BDNF), AMPA receptor trafficking, and classical conditioning.

    Science.gov (United States)

    Zheng, Zhaoqing; Sabirzhanov, Boris; Keifer, Joyce

    2010-11-01

    Amyloid-β (Aβ) peptide is thought to have a significant role in the progressive memory loss observed in patients with Alzheimer disease and inhibits synaptic plasticity in animal models of learning. We previously demonstrated that brain-derived neurotrophic factor (BDNF) is critical for synaptic AMPA receptor delivery in an in vitro model of eyeblink classical conditioning. Here, we report that acquisition of conditioned responses was significantly attenuated by bath application of oligomeric (200 nm), but not fibrillar, Aβ peptide. Western blotting revealed that BDNF protein expression during conditioning is significantly reduced by treatment with oligomeric Aβ, as were phosphorylation levels of cAMP-response element-binding protein (CREB), Ca(2+)/calmodulin-dependent protein kinase II (CaMKII), Ca(2+)/calmodulin-dependent protein kinase IV (CaMKIV), and ERK. However, levels of PKA and PKCζ/λ were unaffected, as was PDK-1. Protein localization studies using confocal imaging indicate that oligomeric Aβ, but not fibrillar or scrambled forms, suppresses colocalization of GluR1 and GluR4 AMPA receptor subunits with synaptophysin, indicating that trafficking of these subunits to synapses during the conditioning procedure is blocked. In contrast, coapplication of BDNF with oligomeric Aβ significantly reversed these findings. Interestingly, a tolloid-like metalloproteinase in turtle, tTLLs (turtle tolloid-like protein), which normally processes the precursor proBDNF into mature BDNF, was found to degrade oligomeric Aβ into small fragments. These data suggest that an Aβ-induced reduction in BDNF, perhaps due to interference in the proteolytic conversion of proBDNF to BDNF, results in inhibition of synaptic AMPA receptor delivery and suppression of the acquisition of conditioning.

  2. Exendin-4 promotes the membrane trafficking of the AMPA receptor GluR1 subunit and ADAM10 in the mouse neocortex.

    Science.gov (United States)

    Ohtake, Nobuaki; Saito, Mieko; Eto, Masaaki; Seki, Kenjiro

    2014-05-01

    Glucagon-like peptide-1 (GLP-1) is a novel treatment modality for type 2 diabetes mellitus. However, GLP-1 has been suggested as a therapeutic target for Alzheimer's disease (AD). In rodent studies, GLP-1 reduces amyloid beta (Aβ) and facilitates synaptic plasticity. Therefore, in the present study, we investigated how GLP-1 facilitates synaptic plasticity and reduces the Aβ in vivo. Exendin-4, a GLP-1 receptor agonist that can cross the blood brain barrier, was subcutaneously administered to adult mice. We then extracted the total and the plasma membrane proteins from the mouse neocortex. Exendin-4 significantly increased the phosphorylation level of cAMP response element-binding protein (CREB). Consistently, the expression level of brain-derived neurotrophic factor (BDNF), a transcriptional target of CREB, was increased. Furthermore, exendin-4 increased the membrane protein level of the AMPA receptor GluR1 subunit and postsynaptic density protein-95 (PSD-95), whereas GluR2 was unaffected. These exendin-4-dependent increases in membrane GluR1, total PSD-95 and BDNF were abrogated by pretreatment with temozolomide (TMZ), a DNA-alkylating agent, indicating that these alterations were dependent on exendin-4-induced transcriptional activity. In addition, we found that exendin-4 increased the level of the α-C terminal fragment (α-CTF) of amyloid precursor protein (APP). Furthermore, protein levels of both mature and immature ADAM10, the α-secretase of APP in the plasma membrane, were increased, whereas the total mature and immature ADAM10 levels were unchanged. These exendin-4-dependent increases in α-CTF and ADAM10 were not affected by TMZ. These findings suggested that GLP-1 facilitates the GluR1 membrane insertion through CREB activation and increases α-secretase activity through ADAM10 membrane trafficking. Upregulation of GluR1 and ADAM10 at the plasma membrane were also observed in mice with intracerebroventricular administration of Aβ oligomer

  3. Identifying the receptor subtype selectivity of retinoid X and retinoic acid receptors via quantum mechanics.

    Science.gov (United States)

    Tsuji, Motonori; Shudo, Koichi; Kagechika, Hiroyuki

    2017-03-01

    Understanding and identifying the receptor subtype selectivity of a ligand is an important issue in the field of drug discovery. Using a combination of classical molecular mechanics and quantum mechanical calculations, this report assesses the receptor subtype selectivity for the human retinoid X receptor (hRXR) and retinoic acid receptor (hRAR) ligand-binding domains (LBDs) complexed with retinoid ligands. The calculated energies show good correlation with the experimentally reported binding affinities. The technique proposed here is a promising method as it reveals the origin of the receptor subtype selectivity of selective ligands.

  4. Fission yeast arrestin-related trafficking adaptor, Arn1/Any1, is ubiquitinated by Pub1 E3 ligase and regulates endocytosis of Cat1 amino acid transporter

    Directory of Open Access Journals (Sweden)

    Akio Nakashima

    2014-05-01

    Full Text Available The Tsc1–Tsc2 complex homologous to human tuberous sclerosis complex proteins governs amino acid uptake by regulating the expression and intracellular distribution of amino acid transporters in Schizosaccharomyces pombe. Here, we performed a genetic screening for molecules that are involved in amino acid uptake and found Arn1 (also known as Any1. Arn1 is homologous to ART1, an arrestin-related trafficking adaptor (ART in Saccharomyces cerevisiae, and contains a conserved arrestin motif, a ubiquitination site, and two PY motifs. Overexpression of arn1+ confers canavanine resistance on cells, whereas its disruption causes hypersensitivity to canavanine. We also show that Arn1 regulates endocytosis of the Cat1 amino acid transporter. Furthermore, deletion of arn1+ suppresses a defect of amino acid uptake and the aberrant Cat1 localization in tsc2Δ. Arn1 interacts with and is ubiquitinated by the Pub1 ubiquitin ligase, which is necessary to regulate Cat1 endocytosis. Cat1 undergoes ubiquitinations on lysine residues within the N-terminus, which are mediated, in part, by Arn1 to determine Cat1 localization. Correctively, Arn1 is an ART in S. pombe and contributes to amino acid uptake through regulating Cat1 endocytosis in which Tsc2 is involved.

  5. Two-stage AMPA receptor trafficking in classical conditioning and selective role for glutamate receptor subunit 4 (tGluA4) flop splice variant.

    Science.gov (United States)

    Zheng, Zhaoqing; Sabirzhanov, Boris; Keifer, Joyce

    2012-07-01

    Previously, we proposed a two-stage model for an in vitro neural correlate of eyeblink classical conditioning involving the initial synaptic incorporation of glutamate receptor A1 (GluA1)-containing α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid type receptors (AMPARs) followed by delivery of GluA4-containing AMPARs that support acquisition of conditioned responses. To test specific elements of our model for conditioning, selective knockdown of GluA4 AMPAR subunits was used using small-interfering RNAs (siRNAs). Recently, we sequenced and characterized the GluA4 subunit and its splice variants from pond turtles, Trachemys scripta elegans (tGluA4). Analysis of the relative abundance of mRNA expression by real-time RT-PCR showed that the flip/flop variants of tGluA4, tGluA4c, and a novel truncated variant tGluA4trc1 are major isoforms in the turtle brain. Here, transfection of in vitro brain stem preparations with anti-tGluA4 siRNA suppressed conditioning, tGluA4 mRNA and protein expression, and synaptic delivery of tGluA4-containing AMPARs but not tGluA1 subunits. Significantly, transfection of abducens motor neurons by nerve injections of tGluA4 flop rescue plasmid prior to anti-tGluA4 siRNA application restored conditioning and synaptic incorporation of tGluA4-containing AMPARs. In contrast, treatment with rescue plasmids for tGluA4 flip or tGluA4trc1 failed to rescue conditioning. Finally, treatment with a siRNA directed against GluA1 subunits inhibited conditioning and synaptic delivery of tGluA1-containing AMPARs and importantly, those containing tGluA4. These data strongly support our two-stage model of conditioning and our hypothesis that synaptic incorporation of tGluA4-containing AMPARs underlies the acquisition of in vitro classical conditioning. Furthermore, they suggest that tGluA4 flop may have a critical role in conditioning mechanisms compared with the other tGluA4 splice variants.

  6. N-methyl-D-aspartic acid receptor agonists

    DEFF Research Database (Denmark)

    Madsen, U; Frydenvang, Karla Andrea; Ebert, B

    1996-01-01

    (R,S)-2-Amino-2-(3-hydroxy-5-methyl-4-isoxazolyl)acetic acid [(R,S)-AMAA, 4] is a potent and selective agonist at the N-methyl-D-aspartic acid (NMDA) subtype of excitatory amino acid receptors. Using the Ugi "four-component condensation" method, the two diastereomers (2R)- and (2S)-2-[3-(benzyloxy......) showed peak affinity for [3H]AMPA receptor sites (IC50 = 72 +/- 13 microM) and was shown to be a more potent inhibitor of [3H]CPP binding (IC50 = 3.7 +/- 1.5 microM) than (S)-AMAA (9) (IC50 = 61 +/- 6.4 microM). Neither enantiomer of AMAA affected [3H]kainic acid receptor binding significantly...

  7. Multiple roles of the extracellular vestibule amino acid residues in the function of the rat P2X4 receptor.

    Directory of Open Access Journals (Sweden)

    Milos B Rokic

    Full Text Available The binding of ATP to trimeric P2X receptors (P2XR causes an enlargement of the receptor extracellular vestibule, leading to opening of the cation-selective transmembrane pore, but specific roles of vestibule amino acid residues in receptor activation have not been evaluated systematically. In this study, alanine or cysteine scanning mutagenesis of V47-V61 and F324-N338 sequences of rat P2X4R revealed that V49, Y54, Q55, F324, and G325 mutants were poorly responsive to ATP and trafficking was only affected by the V49 mutation. The Y54F and Y54W mutations, but not the Y54L mutation, rescued receptor function, suggesting that an aromatic residue is important at this position. Furthermore, the Y54A and Y54C receptor function was partially rescued by ivermectin, a positive allosteric modulator of P2X4R, suggesting a rightward shift in the potency of ATP to activate P2X4R. The Q55T, Q55N, Q55E, and Q55K mutations resulted in non-responsive receptors and only the Q55E mutant was ivermectin-sensitive. The F324L, F324Y, and F324W mutations also rescued receptor function partially or completely, ivermectin action on channel gating was preserved in all mutants, and changes in ATP responsiveness correlated with the hydrophobicity and side chain volume of the substituent. The G325P mutant had a normal response to ATP, suggesting that G325 is a flexible hinge. A topological analysis revealed that the G325 and F324 residues disrupt a β-sheet upon ATP binding. These results indicate multiple roles of the extracellular vestibule amino acid residues in the P2X4R function: the V49 residue is important for receptor trafficking to plasma membrane, the Y54 and Q55 residues play a critical role in channel gating and the F324 and G325 residues are critical for vestibule widening.

  8. Regulation of amino acid transporter trafficking by mTORC1 in primary human trophoblast cells is mediated by the ubiquitin ligase Nedd4-2.

    Science.gov (United States)

    Rosario, Fredrick J; Dimasuay, Kris Genelyn; Kanai, Yoshikatsu; Powell, Theresa L; Jansson, Thomas

    2016-04-01

    Changes in placental amino acid transfer directly contribute to altered fetal growth, which increases the risk for perinatal complications and predisposes for the development of obesity, diabetes and cardiovascular disease later in life. Placental amino acid transfer is critically dependent on the expression of specific transporters in the plasma membrane of the trophoblast, the transporting epithelium of the human placenta. However, the molecular mechanisms regulating this process are largely unknown. Nedd4-2 is an ubiquitin ligase that catalyses the ubiquitination of proteins, resulting in proteasomal degradation. We hypothesized that inhibition of mechanistic target of rapamycin complex 1 (mTORC1) decreases amino acid uptake in primary human trophoblast (PHT) cells by activation of Nedd4-2, which increases transporter ubiquitination resulting in decreased transporter expression in the plasma membrane. mTORC 1 inhibition increased the expression of Nedd4-2, promoted ubiquitination and decreased the plasma membrane expression of SNAT2 (an isoform of the System A amino acid transporter) and LAT1 (a System L amino acid transporter isoform), resulting in decreased cellular amino acid uptake. Nedd4-2 silencing markedly increased the trafficking of SNAT2 and LAT1 to the plasma membrane, which stimulated cellular amino acid uptake. mTORC1 inhibition by silencing of raptor failed to decrease amino acid transport following Nedd4-2 silencing. In conclusion, we have identified a novel link between mTORC1 signalling and ubiquitination, a common posttranslational modification. Because placental mTORC1 is inhibited in fetal growth restriction and activated in fetal overgrowth, we propose that regulation of placental amino acid transporter ubiquitination by mTORC1 and Nedd4-2 constitutes a molecular mechanisms underlying abnormal fetal growth.

  9. The C-terminal domains of the GABA(b) receptor subunits mediate intracellular trafficking but are not required for receptor signaling.

    Science.gov (United States)

    Calver, A R; Robbins, M J; Cosio, C; Rice, S Q; Babbs, A J; Hirst, W D; Boyfield, I; Wood, M D; Russell, R B; Price, G W; Couve, A; Moss, S J; Pangalos, M N

    2001-02-15

    GABA(B) receptors are G-protein-coupled receptors that mediate slow synaptic inhibition in the brain and spinal cord. These receptors are heterodimers assembled from GABA(B1) and GABA(B2) subunits, neither of which is capable of producing functional GABA(B) receptors on homomeric expression. GABA(B1,) although able to bind GABA, is retained within the endoplasmic reticulum (ER) when expressed alone. In contrast, GABA(B2) is able to access the cell surface when expressed alone but does not couple efficiently to the appropriate effector systems or produce any detectable GABA-binding sites. In the present study, we have constructed chimeric and truncated GABA(B1) and GABA(B2) subunits to explore further GABA(B) receptor signaling and assembly. Removal of the entire C-terminal intracellular domain of GABA(B1) results in plasma membrane expression without the production of a functional GABA(B) receptor. However, coexpression of this truncated GABA(B1) subunit with either GABA(B2) or a truncated GABA(B2) subunit in which the C terminal has also been removed is capable of functional signaling via G-proteins. In contrast, transferring the entire C-terminal tail of GABA(B1) to GABA(B2) leads to the ER retention of the GABA(B2) subunit when expressed alone. These results indicate that the C terminal of GABA(B1) mediates the ER retention of this protein and that neither of the C-terminal tails of GABA(B1) or GABA(B2) is an absolute requirement for functional coupling of heteromeric receptors. Furthermore although GABA(B1) is capable of producing GABA-binding sites, GABA(B2) is of central importance in the functional coupling of heteromeric GABA(B) receptors to G-proteins and the subsequent activation of effector systems.

  10. Mechanisms Underlying Activation of α1-Adrenergic Receptor-Induced Trafficking of AQP5 in Rat Parotid Acinar Cells under Isotonic or Hypotonic Conditions

    Science.gov (United States)

    Bragiel, Aneta M.; Wang, Di; Pieczonka, Tomasz D.; Shono, Masayuki; Ishikawa, Yasuko

    2016-01-01

    Defective cellular trafficking of aquaporin-5 (AQP5) to the apical plasma membrane (APM) in salivary glands is associated with the loss of salivary fluid secretion. To examine mechanisms of α1-adrenoceptor (AR)-induced trafficking of AQP5, immunoconfocal microscopy and Western blot analysis were used to analyze AQP5 localization in parotid tissues stimulated with phenylephrine under different osmolality. Phenylephrine-induced trafficking of AQP5 to the APM and lateral plasma membrane (LPM) was mediated via the α1A-AR subtype, but not the α1B- and α1D-AR subtypes. Phenylephrine-induced trafficking of AQP5 was inhibited by ODQ and KT5823, inhibitors of nitric oxide (NO)-stimulated guanylcyclase (GC) and protein kinase (PK) G, respectively, indicating the involvement of the NO/ soluble (c) GC/PKG signaling pathway. Under isotonic conditions, phenylephrine-induced trafficking was inhibited by La3+, implying the participation of store-operated Ca2+ channel. Under hypotonic conditions, phenylephrine-induced trafficking of AQP5 to the APM was higher than that under isotonic conditions. Under non-stimulated conditions, hypotonicity-induced trafficking of AQP5 to the APM was inhibited by ruthenium red and La3+, suggesting the involvement of extracellular Ca2+ entry. Thus, α1A-AR activation induced the trafficking of AQP5 to the APM and LPM via the Ca2+/ cyclic guanosine monophosphate (cGMP)/PKG signaling pathway, which is associated with store-operated Ca2+ entry. PMID:27367668

  11. Truncation of merozoite surface protein 3 disrupts its trafficking and that of acidic-basic repeat protein to the surface of Plasmodium falciparum merozoites.

    Science.gov (United States)

    Mills, Kerry E; Pearce, J Andrew; Crabb, Brendan S; Cowman, Alan F

    2002-03-01

    Merozoite surface protein 3 (MSP3), an important vaccine candidate, is a soluble polymorphic antigen associated with the surface of Plasmodium falciparum merozoites. The MSP3 sequence contains three blocks of heptad repeats that are consistent with the formation of an intramolecular coiled-coil. MSP3 also contains a glutamic acid-rich region and a putative leucine zipper sequence at the C-terminus. We have disrupted the msp3 gene by homologous recombination, resulting in the expression of a truncated form of MSP3 that lacks the putative leucine zipper sequence but retains the glutamic acid-rich region and the heptad repeats. Here, we show that truncated MSP3, lacking the putative leucine zipper region, does not localize to the parasitophorous vacuole or interact with the merozoite surface. Furthermore, the acidic-basic repeat antigen (ABRA), which is present on the merozoite surface, also was not localized to the merozoite surface in parasites expressing the truncated form of MSP3. The P. falciparum merozoites lacking MSP3 and ABRA on the surface show reduced invasion into erythrocytes. These results suggest that MSP3 is not absolutely essential for blood stage growth and that the putative leucine zipper region is required for the trafficking of both MSP3 and ABRA to the parasitophorous vacuole.

  12. Activation of muscarinic receptors in rat parotid acinar cells induces AQP5 trafficking to nuclei and apical plasma membrane.

    Science.gov (United States)

    Cho, Gota; Bragiel, Aneta M; Wang, Di; Pieczonka, Tomasz D; Skowronski, Mariusz T; Shono, Masayuki; Nielsen, Søren; Ishikawa, Yasuko

    2015-04-01

    The subcellular distribution of aquaporin-5 (AQP5) in rat parotid acinar cells in response to muscarinic acetylcholine receptor (mAChR) activation remains unclear. Immunoconfocal and immunoelectron microscopy were used to visualize the distribution of AQP5 in parotid acinar cells. Western blotting was used to analyze AQP5 levels in membranes. To clarify the characteristics of membrane domains associated with AQP5, detergent solubility and sucrose-density flotation experiments were performed. Under control conditions, AQP5 was diffusely distributed on the apical plasma membrane (APM) and apical plasmalemmal region and throughout the cytoplasm. Upon mAChR activation, AQP5 was predominantly located in the nucleus, APM and lateral plasma membrane (LPM). Subsequently, localization of AQP5 in the nucleus, APM and LPM was decreased. Prolonged atropine treatment inhibited mAChR agonist-induced translocation of AQP5 to the nucleus, APM and LPM. AQP5 levels were enhanced in isolated nuclei and nuclear membranes prepared from parotid tissues incubated with mAChR agonist. mAChR agonist induced AQP5 levels in both soluble and insoluble nuclear fractions solubilized with Triton X-100 or Lubrol WX. Small amounts of AQP5 in nuclei were detected using low-density sucrose gradient. When AQP5 was present in the nuclear membrane, nuclear size decreased. The activation of mAChR induced AQP5 translocation to the nucleus, APM and LPM, and AQP5 may trigger water transport across the nuclear membrane and plasma membrane in rat parotid acinar cells. AQP5 translocates to the nuclear membrane and may trigger the movement of water, inducing shrinkage of the nucleus and the start of nuclear functions. Copyright © 2015 Elsevier B.V. All rights reserved.

  13. High affinity retinoic acid receptor antagonists: analogs of AGN 193109.

    Science.gov (United States)

    Johnson, A T; Wang, L; Gillett, S J; Chandraratna, R A

    1999-02-22

    A series of high affinity retinoic acid receptor (RAR) antagonists were prepared based upon the known antagonist AGN 193109 (2). Introduction of various phenyl groups revealed a preference for substitution at the para-position relative to the meta-site. Antagonists with the highest affinities for the RARs possessed hydrophobic groups, however, the presence of polar functionality was also well tolerated.

  14. Bile acid receptors and nonalcoholic fatty liver disease

    Institute of Scientific and Technical Information of China (English)

    2015-01-01

    With the high prevalence of obesity, diabetes, and otherfeatures of the metabolic syndrome in United States,nonalcoholic fatty liver disease (NAFLD) has inevitablybecome a very prevalent chronic liver disease and isnow emerging as one of the leading indications for livertransplantation. Insulin resistance and derangementof lipid metabolism, accompanied by activation ofthe pro-inflammatory response and fibrogenesis, areessential pathways in the development of the moreclinically significant form of NAFLD, known as nonalcoholicsteatohepatitis (NASH). Recent advances inthe functional characterization of bile acid receptors,such as farnesoid X receptor (FXR) and transmembraneG protein-coupled receptor (TGR) 5, have providedfurther insight in the pathophysiology of NASH andhave led to the development of potential therapeutictargets for NAFLD and NASH. Beyond maintaining bileacid metabolism, FXR and TGR5 also regulate lipidmetabolism, maintain glucose homeostasis, increaseenergy expenditure, and ameliorate hepatic inflammation.These intriguing features have been exploitedto develop bile acid analogues to target pathways inNAFLD and NASH pathogenesis. This review providesa brief overview of the pathogenesis of NAFLD andNASH, and then delves into the biological functions ofbile acid receptors, particularly with respect to NASHpathogenesis, with a description of the associatedexperimental data, and, finally, we discuss the prospectsof bile acid analogues in the treatment of NAFLD andNASH.

  15. Study of binding glycyrrhetic acid to AT1 receptor

    Institute of Scientific and Technical Information of China (English)

    ZHANG; Fengyun; (张凤云); YUE; Baozhen; (岳保珍); HE; Shipeng; (贺师鹏)

    2003-01-01

    To analyze the binding of glycyrrhetic acid (GA) to angiotensin II type I (AT1) receptor and to explore the mechanisms underlying the binding, primary cell culture of rat vascular smooth muscle cell (VSMC), radioactive ligand-receptor binding assay, lascer confocal scanning microscope (LCSM), Northern blot, 3H-TdR incorporation DNA assay were used in this study. The results suggest that specific binding of GA to AT1 receptor (IC50 value was 35.0 μmol/L) increases intracellular [Ca2+]i of VSMC, activates transcription factor c-myc and promotes the proliferation of VSMC, therefore GA was probably an agonist of AT1 receptor, providing a new target for GA's pharmaceutical effects.

  16. The Arabidopsis Vacuolar Sorting Receptor1 Is Required for Osmotic Stress-Induced Abscisic Acid Biosynthesis

    KAUST Repository

    Wang, Zhen-Yu

    2014-11-21

    Osmotic stress activates the biosynthesis of the phytohormone abscisic acid (ABA) through a pathway that is rate limited by the carotenoid cleavage enzyme 9-cis-epoxycarotenoid dioxygenase (NCED). To understand the signal transduction mechanism underlying the activation of ABA biosynthesis, we performed a forward genetic screen to isolate mutants defective in osmotic stress regulation of the NCED3 gene. Here, we identified the Arabidopsis (Arabidopsis thaliana) Vacuolar Sorting Receptor1 (VSR1) as a unique regulator of ABA biosynthesis. The vsr1 mutant not only shows increased sensitivity to osmotic stress, but also is defective in the feedback regulation of ABA biosynthesis by ABA. Further analysis revealed that vacuolar trafficking mediated by VSR1 is required for osmotic stress-responsive ABA biosynthesis and osmotic stress tolerance. Moreover, under osmotic stress conditions, the membrane potential, calcium flux, and vacuolar pH changes in the vsr1 mutant differ from those in the wild type. Given that manipulation of the intracellular pH is sufficient to modulate the expression of ABA biosynthesis genes, including NCED3, and ABA accumulation, we propose that intracellular pH changes caused by osmotic stress may play a signaling role in regulating ABA biosynthesis and that this regulation is dependent on functional VSR1.

  17. Molecular pharmacology of homologues of ibotenic acid at cloned metabotropic glutamic acid receptors

    DEFF Research Database (Denmark)

    Bräuner-Osborne, Hans; Nielsen, B; Krogsgaard-Larsen, P

    1998-01-01

    We have studied the effects of the enantiomers of 2-amino-3-(3-hydroxyisoxazol-5-yl)propionic acid (homoibotenic acid, HIBO) and analogues substituted with a methyl, bromo or butyl group in the four position of the ring at cloned metabotropic glutamate (mGlu) receptors expressed in Chinese hamste...

  18. Role of Bile Acids and Bile Acid Receptors in Metabolic Regulation

    NARCIS (Netherlands)

    Lefebvre, Philippe; Cariou, Bertrand; Lien, Fleur; Kuipers, Folkert; Staels, Bart

    2009-01-01

    Lefebvre P, Cariou B, Lien F, Kuipers F, Staels B. Role of Bile Acids and Bile Acid Receptors in Metabolic Regulation. Physiol Rev 89: 147-191,2009; doi: 10.1152/physrev.00010.2008. - The incidence of the metabolic syndrome has taken epidemic proportions in the past decades, contributing to an incre

  19. Bile acid nuclear receptor FXR and digestive system diseases

    Directory of Open Access Journals (Sweden)

    Lili Ding

    2015-03-01

    Full Text Available Bile acids (BAs are not only digestive surfactants but also important cell signaling molecules, which stimulate several signaling pathways to regulate some important biological processes. The bile-acid-activated nuclear receptor, farnesoid X receptor (FXR, plays a pivotal role in regulating bile acid, lipid and glucose homeostasis as well as in regulating the inflammatory responses, barrier function and prevention of bacterial translocation in the intestinal tract. As expected, FXR is involved in the pathophysiology of a wide range of diseases of gastrointestinal tract, including inflammatory bowel disease, colorectal cancer and type 2 diabetes. In this review, we discuss current knowledge of the roles of FXR in physiology of the digestive system and the related diseases. Better understanding of the roles of FXR in digestive system will accelerate the development of FXR ligands/modulators for the treatment of digestive system diseases.

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

    DEFF Research Database (Denmark)

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

    2016-01-01

    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. He...

  1. 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......-2) and T1R2-3 receptor, all receptors are either activated or positively modulated by amino acids. In this review, we outline mutational, biophysical and structural studies which have elucidated the interaction of the amino acids with the Venus flytrap domains, molecular mechanisms of receptor selectivity...

  2. Soman- or kainic acid-induced convulsions decrease muscarinic receptors but not benzodiazepine receptors

    Energy Technology Data Exchange (ETDEWEB)

    Churchill, L.; Pazdernik, T.L.; Cross, R.S.; Nelson, S.R.; Samson, F.E. (Univ. of Kansas Medical Center, Kansas City (USA))

    (3H)Quinuclidinyl benzilate (QNB) binding to muscarinic receptors decreased in the rat forebrain after convulsions induced by a single dose of either soman, a potent inhibitor of acetylcholinesterase, or kainic acid, an excitotoxin. A Rosenthal plot revealed that the receptors decreased in number rather than affinity. When the soman-induced convulsions were blocked, the decrease in muscarinic receptors at 3 days was less extensive than when convulsions occurred and at 10 days they approached control levels in most of the brain areas. The most prominent decrements in QNB binding were in the piriform cortex where the decline in QNB binding is probably related to the extensive convulsion-associated neuropathology. The decrements in QNB binding after convulsions suggest that the convulsive state leads to a down-regulation of muscarinic receptors in some brain areas. In contrast to the decrease in QNB binding after convulsions, (3H)flunitrazepam binding to benzodiazepine receptors did not change even in the piriform cortex where the loss in muscarinic receptors was most prominent. Thus, it appears that those neuronal processes that bear muscarinic receptors are more vulnerable to convulsion-induced change than those with benzodiazepine receptors.

  3. Hydroxylated analogues of 5-aminovaleric acid as 4-aminobutyric acidB receptor antagonists

    DEFF Research Database (Denmark)

    Kristiansen, U; Hedegaard, A; Herdeis, C;

    1992-01-01

    The (R) and (S) forms of 5-amino-2-hydroxyvaleric acid (2-OH-DAVA) and 5-amino-4-hydroxyvaleric acid (4-OH-DAVA) were designed as structural hybrids of the 4-aminobutyric acidB (GABAB) agonist (R)-(-)-4-amino-3-hydroxybutyric acid [(R)-(-)-3-OH-GABA] and the GABAB antagonist 5-aminovaleric acid....... All four compounds were weak inhibitors of GABAA receptor binding in rat brain, and none of them significantly affected synaptosomal GABA uptake. Based on molecular modeling studies it has been demonstrated that low-energy conformations of (R)-(-)-3-OH-GABA, (S)-(-)-2-OH-DAVA, and (R)-(-)-4-OH...

  4. Ursolic acid, a natural pentacyclic triterpenoid, inhibits intracellular trafficking of proteins and induces accumulation of intercellular adhesion molecule-1 linked to high-mannose-type glycans in the endoplasmic reticulum

    Directory of Open Access Journals (Sweden)

    Satoshi Mitsuda

    2014-01-01

    Full Text Available Ursolic acid (3β-hydroxy-urs-12-en-28-oic acid is a natural pentacyclic triterpenoid that is present in many plants, including medicinal herbs, and foods. Ursolic acid was initially identified as an inhibitor of the expression of intercellular adhesion molecule-1 (ICAM-1 in response to interleukin-1α (IL-1α. We report here a novel biological activity: ursolic acid inhibits intracellular trafficking of proteins. Ursolic acid markedly inhibited the IL-1α-induced cell-surface ICAM-1 expression in human cancer cell lines and human umbilical vein endothelial cells. By contrast, ursolic acid exerted weak inhibitory effects on the IL-1α-induced ICAM-1 expression at the protein level. Surprisingly, we found that ursolic acid decreased the apparent molecular weight of ICAM-1 and altered the structures of N-linked oligosaccharides bound to ICAM-1. Ursolic acid induced the accumulation of ICAM-1 in the endoplasmic reticulum, which was linked mainly to high-mannose-type glycans. Moreover, in ursolic-acid-treated cells, the Golgi apparatus was fragmented into pieces and distributed over the cells. Thus, our results reveal that ursolic acid inhibits intracellular trafficking of proteins and induces the accumulation of ICAM-1 linked to high-mannose-type glycans in the endoplasmic reticulum.

  5. Selective Orthosteric Free Fatty Acid Receptor 2 (FFA2) Agonists

    Science.gov (United States)

    Schmidt, Johannes; Smith, Nicola J.; Christiansen, Elisabeth; Tikhonova, Irina G.; Grundmann, Manuel; Hudson, Brian D.; Ward, Richard J.; Drewke, Christel; Milligan, Graeme; Kostenis, Evi; Ulven, Trond

    2011-01-01

    Free fatty acid receptor 2 (FFA2; GPR43) is a G protein-coupled seven-transmembrane receptor for short-chain fatty acids (SCFAs) that is implicated in inflammatory and metabolic disorders. The SCFA propionate has close to optimal ligand efficiency for FFA2 and can hence be considered as highly potent given its size. Propionate, however, does not discriminate between FFA2 and the closely related receptor FFA3 (GPR41). To identify FFA2-selective ligands and understand the molecular basis for FFA2 selectivity, a targeted library of small carboxylic acids was examined using holistic, label-free dynamic mass redistribution technology for primary screening and the receptor-proximal G protein [35S]guanosine 5′-(3-O-thio)triphosphate activation, inositol phosphate, and cAMP accumulation assays for hit confirmation. Structure-activity relationship analysis allowed formulation of a general rule to predict selectivity for small carboxylic acids at the orthosteric binding site where ligands with substituted sp3-hybridized α-carbons preferentially activate FFA3, whereas ligands with sp2- or sp-hybridized α-carbons prefer FFA2. The orthosteric binding mode was verified by site-directed mutagenesis: replacement of orthosteric site arginine residues by alanine in FFA2 prevented ligand binding, and molecular modeling predicted the detailed mode of binding. Based on this, selective mutation of three residues to their non-conserved counterparts in FFA3 was sufficient to transfer FFA3 selectivity to FFA2. Thus, selective activation of FFA2 via the orthosteric site is achievable with rather small ligands, a finding with significant implications for the rational design of therapeutic compounds selectively targeting the SCFA receptors. PMID:21220428

  6. Complement receptor 1 is a sialic acid-independent erythrocyte receptor of Plasmodium falciparum.

    Directory of Open Access Journals (Sweden)

    Carmenza Spadafora

    Full Text Available Plasmodium falciparum is a highly lethal malaria parasite of humans. A major portion of its life cycle is dedicated to invading and multiplying inside erythrocytes. The molecular mechanisms of erythrocyte invasion are incompletely understood. P. falciparum depends heavily on sialic acid present on glycophorins to invade erythrocytes. However, a significant proportion of laboratory and field isolates are also able to invade erythrocytes in a sialic acid-independent manner. The identity of the erythrocyte sialic acid-independent receptor has been a mystery for decades. We report here that the complement receptor 1 (CR1 is a sialic acid-independent receptor for the invasion of erythrocytes by P. falciparum. We show that soluble CR1 (sCR1 as well as polyclonal and monoclonal antibodies against CR1 inhibit sialic acid-independent invasion in a variety of laboratory strains and wild isolates, and that merozoites interact directly with CR1 on the erythrocyte surface and with sCR1-coated microspheres. Also, the invasion of neuraminidase-treated erythrocytes correlates with the level of CR1 expression. Finally, both sialic acid-independent and dependent strains invade CR1 transgenic mouse erythrocytes preferentially over wild-type erythrocytes but invasion by the latter is more sensitive to neuraminidase. These results suggest that both sialic acid-dependent and independent strains interact with CR1 in the normal red cell during the invasion process. However, only sialic acid-independent strains can do so without the presence of glycophorin sialic acid. Our results close a longstanding and important gap in the understanding of the mechanism of erythrocyte invasion by P. falciparum that will eventually make possible the development of an effective blood stage vaccine.

  7. In silico discovery of novel Retinoic Acid Receptor agonist structures

    Directory of Open Access Journals (Sweden)

    Samuels Herbert H

    2001-06-01

    Full Text Available Abstract Background Several Retinoic Acid Receptors (RAR agonists have therapeutic activity against a variety of cancer types; however, unacceptable toxicity profiles have hindered the development of drugs. RAR agonists presenting novel structural and chemical features could therefore open new avenues for the discovery of leads against breast, lung and prostate cancer or leukemia. Results We have analysed the induced fit of the active site residues upon binding of a known ligand. The derived binding site models were used to dock over 150,000 molecules in silico (or virtually to the structure of the receptor with the Internal Coordinates Mechanics (ICM program. Thirty ligand candidates were tested in vitro. Conclusions Two novel agonists resulting from the predicted receptor model were active at 50 nM. One of them displays novel structural features which may translate into the development of new ligands for cancer therapy.

  8. mGlu5 Receptors and Relapse to Cocaine-Seeking: The Role of Receptor Trafficking in Postrelapse Extinction Learning Deficits

    Directory of Open Access Journals (Sweden)

    Lori A. Knackstedt

    2016-01-01

    Full Text Available We have previously demonstrated that MTEP, an allosteric antagonist of mGlu5, infused into the nucleus accumbens attenuates relapse after abstinence from cocaine self-administration. MTEP infused into the dorsolateral striatum (dlSTR does not alter relapse but has long-lasting effects on subsequent extinction learning. Here we tested whether systemic MTEP would prevent relapse after abstinence or alter extinction learning. We also investigated the mechanism of action by which intra-dlSTR MTEP on test day alters extinction on subsequent days. Animals self-administered cocaine for 12 days followed by abstinence for 20-21 days. MTEP (0.5–5 mg/kg IP was administered prior to placement into the operant chamber for a context-primed relapse test. A separate group of animals received intra-dlSTR MTEP prior to the relapse test and were sacrificed day later. Systemic administration of MTEP attenuated abstinent-relapse without significantly affecting extinction learning. Surface biotinylation analysis of protein expression in the dlSTR revealed that, in cocaine animals, intra-dlSTR MTEP administration decreased mGlu5 surface expression and prevented changes in Arc and GluA1/GluA2 observed in their vehicle counterparts. Thus, blockade of mGlu5 receptors may be utilized in future treatment strategies for relapse prevention in humans, although the effects of chronic blockade on extinction learning should be further evaluated.

  9. 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...... anion channels activated by depolarization. In addition to locally produced lactate, lactate produced by exercising muscle as well as exogenous HCAR1 agonists, e.g., from fruits and berries, might activate the receptor on cerebral blood vessels and brain cells....

  10. Smuggled or trafficked?

    Directory of Open Access Journals (Sweden)

    Jacqueline Bhabha

    2006-05-01

    Full Text Available The UN Convention Against Transnational Organized Crime (TNC and its two Protocols on Trafficking and Smuggling, adopted in 2000, seek to distinguish between trafficking and smuggling. In reality these distinctions are often blurred. A more nuanced approach is needed to ensure protection for all those at risk.

  11. Development of novel silicon-containing inverse agonists of retinoic acid receptor-related orphan receptors.

    Science.gov (United States)

    Toyama, Hirozumi; Nakamura, Masaharu; Nakamura, Masahiko; Matsumoto, Yotaro; Nakagomi, Madoka; Hashimoto, Yuichi

    2014-03-15

    Retinoic acid receptor (RAR)-related orphan receptors (RORs) regulate a variety of physiological processes, including hepatic gluconeogenesis, lipid metabolism, circadian rhythm and immune function. The RAR agonist: all-trans retinoic acid was reported to be an RORβ inverse agonist, but no information is available regarding ROR activity of its synthetic analogue Am580. Therefore, we screened Am580 and some related tetramethyltetrahydronaphthalene derivatives and carried out structural development studies, including substitution of carbon atoms with silicon, with the aim of creating a potent ROR transcriptional inhibitor. The phenyl amide disila compound 22 showed the most potent ROR-inhibitory activity among the compounds examined. Its activity towards RORα, RORβ and RORγ was increased compared to that of Am580. The IC₅₀ values for RORα, RORβ and RORγ are 1.3, >10 and 4.5 μM, respectively.

  12. Hyaluronic acid induces activation of the κ-opioid receptor.

    Directory of Open Access Journals (Sweden)

    Barbara Zavan

    Full Text Available INTRODUCTION: Nociceptive pain is one of the most common types of pain that originates from an injury involving nociceptors. Approximately 60% of the knee joint innervations are classified as nociceptive. The specific biological mechanism underlying the regulation of nociceptors is relevant for the treatment of symptoms affecting the knee joint. Intra-articular administration of exogenous hyaluronic acid (HA in patients with osteoarthritis (OA appears to be particularly effective in reducing pain and improving patient function. METHODS: We performed an in vitro study conducted in CHO cells that expressed a panel of opioid receptors and in primary rat dorsal root ganglion (DRG neurons to determine if HA induces the activation of opioid peptide receptors (OPr using both aequorin and the fluorescent dye Fura-2/AM. RESULTS: Selective agonists and antagonists for each OPr expressed on CHO cells were used to test the efficacy of our in vitro model followed by stimulation with HA. The results showed that HA induces stimulatory effects on the κ receptor (KOP. These effects of HA were also confirmed in rat DRG neurons, which express endogenously the OPr. CONCLUSIONS: HA activates the KOP receptor in a concentration dependent manner, with a pEC(50 value of 7.57.

  13. Receptor for protons: First observations on Acid Sensing Ion Channels.

    Science.gov (United States)

    Krishtal, Oleg

    2015-07-01

    The history of ASICs began in 1980 with unexpected observation. The concept of highly selective Na(+) current gated by specific receptors for protons was not easily accepted. It took 16 years to get these receptor/channels cloned and start a new stage in their investigation. "The receptor for protons" became ASIC comprising under this name a family of receptor/channels ubiquitous for mammalian nervous system, both peripheral and central. The role of ASICs as putative nociceptors was suggested almost immediately after their discovery. This role subsequently was proven in many forms of pain-related phenomena. Many other functions of ASICs have been also found or primed for speculations both in physiology and in disease. Despite the width of field and strength of efforts, numerous basic questions are to be answered before we understand how the local changes in pH in the nervous tissue transform into electric and messenger signaling via ASICs as transducers. This article is part of the Special Issue entitled 'Acid-Sensing Ion Channels in the Nervous System'.

  14. Investigation on bile acid receptor regulators. Discovery of cholanoic acid derivatives with dual G-protein coupled bile acid receptor 1 (GPBAR1) antagonistic and farnesoid X receptor (FXR) modulatory activity.

    Science.gov (United States)

    Sepe, Valentina; Renga, Barbara; Festa, Carmen; Finamore, Claudia; Masullo, Dario; Carino, Adriana; Cipriani, Sabrina; Distrutti, Eleonora; Fiorucci, Stefano; Zampella, Angela

    2016-01-01

    Bile acids, the end products of cholesterol metabolism, activate multiple mechanisms through the interaction with membrane G-protein coupled receptors including the bile acid receptor GPBAR1 and nuclear receptors such as the bile acid sensor, farnesoid X receptor (FXR). Even if dual FXR/GPBAR1 agonists are largely considered a novel opportunity in the treatment of several liver and metabolic diseases, selective targeting of one of these receptors represents an attractive therapeutic approach for a wide range of disorders in which dual modulation is associated to severe side effects. In the present study we have investigated around the structure of LCA generating a small library of cholane derivatives, endowed with dual FXR agonism/GPBAR1 antagonism. To the best of our knowledge, this is the first report of bile acid derivatives able to antagonize GPBAR1.

  15. Tetrazolyl isoxazole amino acids as ionotropic glutamate receptor antagonists: synthesis, modelling and molecular pharmacology

    DEFF Research Database (Denmark)

    Frølund, Bente; Greenwood, Jeremy R; Holm, Mai Marie

    2005-01-01

    and 1b were pharmacologically characterized in receptor binding assays, and electrophysiologically on homomeric AMPA receptors (GluR1-4), homomeric (GluR5 and GluR6) and heteromeric (GluR6/KA2) kainic acid receptors, using two-electrode voltage-clamped Xenopus laevis oocytes expressing these receptors...

  16. Non-Acidic Free Fatty Acid Receptor 4 Agonists with Antidiabetic Activity

    DEFF Research Database (Denmark)

    Goncalves de Azavedo, Carlos M. B. P.; Watterson, Kenneth R; Wargent, Ed T

    2016-01-01

    The free fatty acid receptor 4 (FFA4 or GPR120) has appeared as an interesting potential target for the treatment of metabolic disorders. At present, most FFA4 ligands are carboxylic acids that are assumed to mimic the endogenous long-chain fatty acid agonists. Here, we report preliminary structure......-activity relationship studies of a previously disclosed non-acidic sulfonamide FFA4 agonist. Mutagenesis studies indicate that the compounds are orthosteric agonists despite the absence of a carboxylate function. The preferred compounds showed full agonist activity on FFA4 and complete selectivity over FFA1, although...... a significant fraction of these non-carboxylic acids also showed partial antagonistic activity on FFA1. Studies in normal and diet-induced obese (DIO) mice with the preferred compound 34 showed improved glucose tolerance after oral dosing in an oral glucose tolerance test. Chronic dosing of 34 in DIO mice...

  17. TGD4 involved in endoplasmic reticulum-to-chloroplast lipid trafficking is a phosphatidic acid binding protein

    Energy Technology Data Exchange (ETDEWEB)

    Wang Z.; Xu C.; Benning, C.

    2012-05-01

    The synthesis of galactoglycerolipids, which are prevalent in photosynthetic membranes, involves enzymes at the endoplasmic reticulum (ER) and the chloroplast envelope membranes. Genetic analysis of trigalactosyldiacylglycerol (TGD) proteins in Arabidopsis has demonstrated their role in polar lipid transfer from the ER to the chloroplast. The TGD1, 2, and 3 proteins resemble components of a bacterial-type ATP-binding cassette (ABC) transporter, with TGD1 representing the permease, TGD2 the substrate binding protein, and TGD3 the ATPase. However, the function of the TGD4 protein in this process is less clear and its location in plant cells remains to be firmly determined. The predicted C-terminal {beta}-barrel structure of TGD4 is weakly similar to proteins of the outer cell membrane of Gram-negative bacteria. Here, we show that, like TGD2, the TGD4 protein when fused to DsRED specifically binds phosphatidic acid (PtdOH). As previously shown for tgd1 mutants, tgd4 mutants have elevated PtdOH content, probably in extraplastidic membranes. Using highly purified and specific antibodies to probe different cell fractions, we demonstrated that the TGD4 protein was present in the outer envelope membrane of chloroplasts, where it appeared to be deeply buried within the membrane except for the N-terminus, which was found to be exposed to the cytosol. It is proposed that TGD4 is either directly involved in the transfer of polar lipids, possibly PtdOH, from the ER to the outer chloroplast envelope membrane or in the transfer of PtdOH through the outer envelope membrane.

  18. GPCR Signaling and Trafficking: The Long and Short of It.

    Science.gov (United States)

    Pavlos, Nathan J; Friedman, Peter A

    2017-03-01

    Emerging findings disclose unexpected components of G protein-coupled receptor (GPCR) signaling and cell biology. Select GPCRs exhibit classical signaling, that is restricted to cell membranes, as well as newly described persistent signaling that depends on internalization of the GPCR bound to β-arrestins. Termination of non-canonical endosomal signaling requires intraluminal acidification and sophisticated protein trafficking machineries. Recent studies reveal the structural determinants of the trafficking chaperones. This review summarizes advances in GPCR signaling and trafficking with a focus on the parathyroid hormone receptor (PTHR) as a prototype, and on the actin-sorting nexin 27 (SNX27)-retromer tubule (ASRT) complex, an endosomal sorting hub responsible for recycling and preservation of cell surface receptors. The findings are integrated into a model of PTHR trafficking with implications for signal transduction, bone growth, and mineral ion metabolism. Copyright © 2016 Elsevier Ltd. All rights reserved.

  19. Concomitant action of structural elements and receptor phosphorylation determines arrestin-3 interaction with the free fatty acid receptor FFA4

    DEFF Research Database (Denmark)

    Butcher, Adrian J; Hudson, Brian D; Shimpukade, Bharat;

    2014-01-01

    In addition to being nutrients, free fatty acids act as signaling molecules by activating a family of G protein-coupled receptors. Among these is FFA4, previously called GPR120, which responds to medium and long chain fatty acids, including health-promoting ω-3 fatty acids, which have been implic...

  20. Treatment of Type 2 Diabetes by Free Fatty Acid Receptor Agonists

    OpenAIRE

    Kenneth R. Watterson; Hudson, Brian D.; Ulven, Trond; Milligan, Graeme

    2014-01-01

    Dietary free fatty acids (FFAs), such as ω-3 fatty acids, regulate metabolic and anti-inflammatory processes, with many of these effects attributed to FFAs interacting with a family of G protein-coupled receptors. Selective synthetic ligands for Free Fatty Acid receptors (FFA1-4) have consequently been developed as potential treatments for type 2 diabetes (T2D). In particular, clinical studies show that Fasiglifam, an agonist of the long chain FFA receptor, FFA1, improved glycaemic control an...

  1. UK victims of trafficking

    Directory of Open Access Journals (Sweden)

    Bob Burgoyne

    2006-05-01

    Full Text Available Analysis of court cases shows how hard it is forvictims of trafficking to win the right to remain in the UK. Case law is inconsistent and more research and data collection are urgently needed.

  2. Biological roles and therapeutic potential of hydroxy-carboxylic acid receptors

    Directory of Open Access Journals (Sweden)

    Kashan eAhmed

    2011-10-01

    Full Text Available In the recent past, deorphanization studies have described intermediates of energy metabolism to activate G protein-coupled receptors (GPCRs and to thereby regulate metabolic functions. GPR81, GPR109A and GPR109B, formerly known as the nicotinic acid receptor family, are encoded by clustered genes and share a high degree of sequence homology. Recently, hydroxy-carboxylic acids were identified as endogenous ligands of GPR81, GPR109A and GPR109B, and therefore these receptors have been placed into a novel receptor family of hydroxy-carboxylic acid (HCA receptors. The HCA1 receptor (GPR81 is activated by the glycolytic metabolite 2-hydroxy-propionic acid (lactate, the HCA2 receptor is activated by the ketone body 3-hydroxy-butyric acid and the HCA3 receptor (GPR109B is a receptor for the β-oxidation intermediate 3-hydroxy-octanoic acid. While HCA1 and HCA2 receptors are present in most mammalian species, the HCA3 receptor is exclusively found in humans and higher primates. HCA receptors are expressed in adipose tissue and mediate anti-lipolytic effects in adipocytes through Gi-type G-protein-dependent inhibition of adenylyl cyclase. HCA2 and HCA3 inhibit lipolysis during conditions of increased β-oxidation such as prolonged fasting, whereas HCA1 mediates the anti-lipolytic effects of insulin in the fed state. As HCA2 is a receptor for the established anti-dyslipidemic drug nicotinic acid, HCA1 and HCA3 also represent promising drug targets and several synthetic ligands for HCA receptors have been developed. In this article, we will summarize the deorphanization and pharmacological characterization of HCA receptors. Moreover, we will discuss recent progress in elucidating the physiological and pathophysiological role to further evaluate the therapeutic potential of the HCA receptor family for the treatment of metabolic disease.

  3. Oral Administration of a Retinoic Acid Receptor Antagonist Reversibly Inhibits Spermatogenesis in Mice

    OpenAIRE

    Chung, Sanny S. W.; Wang, Xiangyuan; Roberts, Shelby S.; Stephen M Griffey; Reczek, Peter R.; Wolgemuth, Debra J.

    2011-01-01

    Meeting men's contraceptive needs, orally administered retinoic acid receptor antagonists represent new lead molecules in developing non-hormonal, reversible male contraceptives without adverse side effects.

  4. FATTY ACIDS MODULATE TOLL-LIKE RECEPTOR 4 ACTIVATION THROUGH REGULATION OF RECEPTOR DIMERIZATION AND RECRUITMENT INTO LIPID RAFTS

    Science.gov (United States)

    The saturated fatty acids acylated on Lipid A of lipopolysaccharide (LPS) or bacterial lipoproteins play critical roles in ligand recognition and receptor activation for Toll-like Receptor 4 (TLR4) and TLR2. The results from our previous studies (J Biol Chem 2003, 2004) demonstrated that saturated ...

  5. Tannic Acid Preferentially Targets Estrogen Receptor-Positive Breast Cancer

    Directory of Open Access Journals (Sweden)

    Brian W. Booth

    2013-01-01

    Full Text Available Research efforts investigating the potential of natural compounds in the fight against cancer are growing. Tannic acid (TA belongs to the class of hydrolysable tannins and is found in numerous plants and foods. TA is a potent collagen cross-linking agent; the purpose of this study was to generate TA-cross-linked beads and assess the effects on breast cancer cell growth. Collagen beads were stable at body temperature following crosslinking. Exposure to collagen beads with higher levels of TA inhibited proliferation and induced apoptosis in normal and cancer cells. TA-induced apoptosis involved activation of caspase 3/7 and caspase 9 but not caspase 8. Breast cancer cells expressing the estrogen receptor were more susceptible to the effects of TA. Taken together the results suggest that TA has the potential to become an anti-ER+ breast cancer treatment or preventative agent.

  6. Rigid nonproteinogenic cyclic amino acids as ligands for glutamate receptors: trans-tris(homoglutamic) acids

    DEFF Research Database (Denmark)

    Meyer, Udo; Bisel, Philippe; Bräuner-Osborne, Hans

    2005-01-01

    ) yielded diastereomeric mixtures of secondary alpha-amino amido-esters, which after separation were hydrogenolyzed and hydrolyzed each to the enantiomeric trans-1-amino-2-carboxymethylcyclopentanecarboxylic acids. Their configuration was completely established by NMR methods, CD spectra, and X-ray analysis...... of the trans-1S,2R-configured secondary alpha-amino amido-ester. In receptor binding assays and functional tests, trans-1S,2R-1-amino-2-carboxymethylcyclopentanecarboxylic acid hydrochloride was found to behave as a selective mGluR(2)-antagonist without relevant binding properties at iGluRs.......The second-generation asymmetric synthesis of the trans-tris(homoglutamic) acids reported herein proceeds via Strecker reaction of chiral ketimines, obtained from condensation of racemic 2-ethoxycarbonylmethylcyclopentanone and commercially available (S)- and (R)-1-phenylethylamine, respectively...

  7. 1,2,3-triazolyl amino acids as AMPA receptor ligands

    DEFF Research Database (Denmark)

    Stanley, Nathan J.; Pedersen, Daniel Sejer; Nielsen, Birgitte

    2010-01-01

    The central nervous system glutamate receptors are an important target for drug discovery. Herein we report initial investigations into the synthesis and glutamate receptor activity of 1,2,3-triazolyl amino acids. Two compounds were found to be selective AMPA receptor ligands, which warrant further...

  8. Chemical labelling for visualizing native AMPA receptors in live neurons

    Science.gov (United States)

    Wakayama, Sho; Kiyonaka, Shigeki; Arai, Itaru; Kakegawa, Wataru; Matsuda, Shinji; Ibata, Keiji; Nemoto, Yuri L.; Kusumi, Akihiro; Yuzaki, Michisuke; Hamachi, Itaru

    2017-04-01

    The location and number of neurotransmitter receptors are dynamically regulated at postsynaptic sites. However, currently available methods for visualizing receptor trafficking require the introduction of genetically engineered receptors into neurons, which can disrupt the normal functioning and processing of the original receptor. Here we report a powerful method for visualizing native α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-type glutamate receptors (AMPARs) which are essential for cognitive functions without any genetic manipulation. This is based on a covalent chemical labelling strategy driven by selective ligand-protein recognition to tether small fluorophores to AMPARs using chemical AMPAR modification (CAM) reagents. The high penetrability of CAM reagents enables visualization of native AMPARs deep in brain tissues without affecting receptor function. Moreover, CAM reagents are used to characterize the diffusion dynamics of endogenous AMPARs in both cultured neurons and hippocampal slices. This method will help clarify the involvement of AMPAR trafficking in various neuropsychiatric and neurodevelopmental disorders.

  9. Pharmacological Tool Compounds for the Free Fatty Acid Receptor 4 (FFA4/GPR120)

    DEFF Research Database (Denmark)

    Hansen, Steffen V F; Ulven, Trond

    2017-01-01

    The free fatty acid receptor 4 (FFA4), also known as GPR120, is a G protein-coupled receptor that is activated by long-chain fatty acids and that has been associated with regulation of appetite, release of insulin controlling hormones, insulin sensitization, anti-inflammatory and potentially anti...

  10. 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

    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...... 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....

  11. Ist2 in the yeast cortical endoplasmic reticulum promotes trafficking of the amino acid transporter Bap2 to the plasma membrane.

    Directory of Open Access Journals (Sweden)

    Wendelin Wolf

    Full Text Available The equipment of the plasma membrane in Saccharomyces cerevisiae with specific nutrient transporters is highly regulated by transcription, translation and protein trafficking allowing growth in changing environments. The activity of these transporters depends on a H(+ gradient across the plasma membrane generated by the H(+-ATPase Pma1. We found that the polytopic membrane protein Ist2 in the cortical endoplasmic reticulum (ER is required for efficient leucine uptake during the transition from fermentation to respiration. Experiments employing tandem fluorescence timer protein tag showed that Ist2 was necessary for efficient trafficking of newly synthesized leucine transporter Bap2 from the ER to the plasma membrane. This finding explains the growth defect of ist2Δ mutants during nutritional challenges and illustrates the important role of physical coupling between cortical ER and plasma membrane.

  12. Data for amino acid alignment of Japanese stingray melanocortin receptors with other gnathostome melanocortin receptor sequences, and the ligand selectivity of Japanese stingray melanocortin receptors

    OpenAIRE

    Akiyoshi Takahashi; Perry Davis; Christina Reinick; Kanta Mizusawa; Tatsuya Sakamoto; Dores, Robert M.

    2016-01-01

    This article contains structure and pharmacological characteristics of melanocortin receptors (MCRs) related to research published in “Characterization of melanocortin receptors from stingray Dasyatis akajei, a cartilaginous fish” (Takahashi et al., 2016) [1]. The amino acid sequences of the stingray, D. akajei, MC1R, MC2R, MC3R, MC4R, and MC5R were aligned with the corresponding melanocortin receptor sequences from the elephant shark, Callorhinchus milii, the dogfish, Squalus acanthias, the ...

  13. Activity of L-alpha-amino acids at the promiscuous goldfish odorant receptor 5.24

    DEFF Research Database (Denmark)

    Christiansen, Bolette; Wellendorph, Petrine; Bräuner-Osborne, Hans

    2006-01-01

    The goldfish odorant receptor 5.24 is a member of family C of G protein-coupled receptors and is closely related to the human receptor GPRC6A. Receptor 5.24 has previously been shown to have binding affinity for L-alpha-amino acids, especially the basic amino acids arginine and lysine. Here we...... a preference for basic amino acids....... report the agonist activities of the 20 proteinogenic L-alpha-amino acids, and L-ornithine and L-citrulline, measured in an intracellular calcium release assay in mammalian tsA cells. The results show that receptor 5.24 is broadly activated by 19 of the tested L-alpha-amino acids and displays...

  14. Synaptic plasticity, AMPA-R trafficking, and Ras-MAPK signaling

    Institute of Scientific and Technical Information of China (English)

    Yun GU; Ruth L STORNETTA

    2007-01-01

    Synaptic modification of transmission is a general phenomenon expressed at al-most every excitatory synapse in the mammalian brain. Over the last three decades,much has been discovered about the cellular, synaptic, molecular, and signalingmechanisms responsible for controlling synaptic transmission and plasticity. Here,we present a brief review of these mechanisms with emphasis on the currentunderstanding of α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid recep-tor (AMPA-R) trafficking and Ras-mitogen-activated protein kinase (MAPK)signaling events involved in controlling synaptic transmission.

  15. Sex Trafficking of Minors.

    Science.gov (United States)

    Moore, Jessica L; Kaplan, Dana M; Barron, Christine E

    2017-04-01

    Sex trafficking is an increasingly recognized global health crisis affecting every country and region in the world. Domestic minor sex trafficking is a subset of commercial sexual exploitation of children, defined as engagement of minors (<18 years of age) in sexual acts for items of value (eg, food, shelter, drugs, money) involving children victimized within US borders. These involved youth are at risk for serious immediate and long-term physical and mental health consequences. Continued efforts are needed to improve preventive efforts, identification, screening, appropriate interventions, and subsequent resource provision for victimized and high-risk youth.

  16. Trafficking in women in China

    OpenAIRE

    Jiang, Quanbao; Sánchez-Barricarte, Jesús

    2011-01-01

    Based on existing data and prior research, this paper reviews studies of and investigations into the trafficking of women in China. First, the “industrial chain” of human trafficking is analyzed. Second, the analysis indicates that the “buyers market” exhibits a strong demand for trafficking in women. The scale of trafficking is escalating: originally the crime was mainly committed in a few provinces, but has now spread to nearly every province in China. Furthermore, human traf...

  17. LRP-1 and LRP-2 receptors function in the membrane neuron. Trafficking mechanisms and proteolytic processing in Alzheimer’s disease

    Directory of Open Access Journals (Sweden)

    Carlos eSpuch

    2012-07-01

    Full Text Available Low density lipoprotein receptor-related protein (LRP belongs to the low-density lipoprotein receptor family, generally recognized as cell surface endocytic receptors, which bind and internalize extracellular ligands for degradation in lysosomes. Neurons require cholesterol to function and keep the membrane rafts stable. Cholesterol uptake into the neuron is carried out by ApoE via LRPs receptors on the cell surface. In neurons the most important are LRP-1 and LRP-2, even it is thought that a causal factor in Alzheimer’s disease (AD is the malfunction of this process which cause impairment intracellular signalling as well as storage and/or release of nutrients and toxic compounds. Both receptors are multifunctional cell surface receptors that are widely expressed in several tissues including neurons and astrocytes. LRPs are constituted by an intracellular (ICD and extracellular domain (ECD. Through its ECD, LRPs bind at least 40 different ligands ranging from lipoprotein and protease inhibitor complex to growth factors and extracellular matrix proteins. These receptors has also been shown to interact with scaffolding and signalling proteins via its ICD in a phosphorylation-dependent manner and to function as a co-receptor partnering with other cell surface or integral membrane proteins. Thus, LRPs are implicated in two major physiological processes: endocytosis and regulation of signalling pathways, which are both involved in diverse biological roles including lipid metabolism, cell growth processes, degradation of proteases, and tissue invasion. Interestingly, LRPs were also localized in neurons in different stages, suggesting that both receptors could be implicated in signal transduction during embryonic development, neuronal outgrowth or in the pathogenesis of AD

  18. Tetrazolyl isoxazole amino acids as ionotropic glutamate receptor antagonists: synthesis, modelling and molecular pharmacology.

    Science.gov (United States)

    Frølund, Bente; Greenwood, Jeremy R; Holm, Mai M; Egebjerg, Jan; Madsen, Ulf; Nielsen, Birgitte; Bräuner-Osborne, Hans; Stensbøl, Tine B; Krogsgaard-Larsen, Povl

    2005-09-15

    Two 3-(5-tetrazolylmethoxy) analogues, 1a and 1b, of (RS)-2-amino-3-(3-hydroxy-5-methyl-4-isoxazolyl)propionic acid (AMPA), a selective AMPA receptor agonist, and (RS)-2-amino-3-(5-tert-butyl-3-hydroxy-4-isoxazolyl)propionic acid (ATPA), a GluR5-preferring agonist, were synthesized. Compounds 1a and 1b were pharmacologically characterized in receptor binding assays, and electrophysiologically on homomeric AMPA receptors (GluR1-4), homomeric (GluR5 and GluR6) and heteromeric (GluR6/KA2) kainic acid receptors, using two-electrode voltage-clamped Xenopus laevis oocytes expressing these receptors. Both analogues proved to be antagonists at all AMPA receptor subtypes, showing potencies (Kb=38-161 microM) similar to that of the AMPA receptor antagonist (RS)-2-amino-3-[3-(carboxymethoxy)-5-methyl-4-isoxazolyl]propionic acid (AMOA) (Kb=43-76 microM). Furthermore, the AMOA analogue, 1a, blocked two kainic acid receptor subtypes (GluR5 and GluR6/KA2), showing sevenfold preference for GluR6/KA2 (Kb=19 microM). Unlike the iGluR antagonist (S)-2-amino-3-[5-tert-butyl-3-(phosphonomethoxy)-4-isoxazolyl]propionic acid [(S)-ATPO], the corresponding tetrazolyl analogue, 1b, lacks kainic acid receptor effects. On the basis of docking to a crystal structure of the isolated extracellular ligand-binding core of the AMPA receptor subunit GluR2 and a homology model of the kainic acid receptor subunit GluR5, we were able to rationalize the observed structure-activity relationships.

  19. Farnesoid X receptor, the bile acid sensing nuclear receptor, in liver regeneration

    Directory of Open Access Journals (Sweden)

    Guodong Li

    2015-03-01

    Full Text Available The liver is unique in regenerative potential, which could recover the lost mass and function after injury from ischemia and resection. The underlying molecular mechanisms of liver regeneration have been extensively studied in the past using the partial hepatectomy (PH model in rodents, where 2/3 PH is carried out by removing two lobes. The whole process of liver regeneration is complicated, orchestrated event involving a network of connected interactions, which still remain fully elusive. Bile acids (BAs are ligands of farnesoid X receptor (FXR, a nuclear receptor of ligand-activated transcription factor. FXR has been shown to be highly involved in liver regeneration. BAs and FXR not only interact with each other but also regulate various downstream targets independently during liver regeneration. Moreover, recent findings suggest that tissue-specific FXR also contributes to liver regeneration significantly. These novel findings suggest that FXR has much broader role than regulating BA, cholesterol, lipid and glucose metabolism. Therefore, these researches highlight FXR as an important pharmaceutical target for potential use of FXR ligands to regulate liver regeneration in clinic. This review focuses on the roles of BAs and FXR in liver regeneration and the current underlying molecular mechanisms which contribute to liver regeneration.

  20. Intracellular delivery and trafficking dynamics of a lymphoma-targeting antibody-polymer conjugate.

    Science.gov (United States)

    Berguig, Geoffrey Y; Convertine, Anthony J; Shi, Julie; Palanca-Wessels, Maria Corinna; Duvall, Craig L; Pun, Suzie H; Press, Oliver W; Stayton, Patrick S

    2012-12-03

    Ratiometric fluorescence and cellular fractionation studies were employed to characterize the intracellular trafficking dynamics of antibody-poly(propylacrylic acid) (PPAA) conjugates in CD22+ RAMOS-AW cells. The HD39 monoclonal antibody (mAb) directs CD22-dependent, receptor-mediated uptake in human B-cell lymphoma cells, where it is rapidly trafficked to the lysosomal compartment. To characterize the intracellular-release dynamics of the polymer-mAb conjugates, HD39-streptavidin (HD39/SA) was dual-labeled with pH-insensitive Alexa Fluor 488 and pH-sensitive pHrodo fluorophores. The subcellular pH distribution of the HD39/SA-polymer conjugates was quantified as a function of time by live-cell fluorescence microscopy, and the average intracellular pH value experienced by the conjugates was also characterized as a function of time by flow cytometry. PPAA was shown to alter the intracellular trafficking kinetics strongly relative to HD39/SA alone or HD39/SA conjugates with a control polymer, poly(methacryclic acid) (PMAA). Subcellular trafficking studies revealed that after 6 h, only 11% of the HD39/SA-PPAA conjugates had been trafficked to acidic lysosomal compartments with values at or below pH 5.6. In contrast, the average intracellular pH of HD39/SA alone dropped from 6.7 ± 0.2 at 1 h to 5.6 ± 0.5 after 3 h and 4.7 ± 0.6 after 6 h. Conjugation of the control polymer PMAA to HD39/SA showed an average pH drop similar to that of HD39/SA. Subcellular fractionation studies with tritium-labeled HD39/SA demonstrated that after 6 h, 89% of HD39/SA was associated with endosomes (Rab5+) and lysosomes (Lamp2+), while 45% of HD39/SA-PPAA was translocated to the cytosol (lactate dehydrogenase+). These results demonstrate the endosomal-releasing properties of PPAA with antibody-polymer conjugates and detail their intracellular trafficking dynamics and subcellular compartmental distributions over time.

  1. Characterization of bicuculline/baclofen-insensitive (rho-like) gamma-aminobutyric acid receptors expressed in Xenopus oocytes. II. Pharmacology of gamma-aminobutyric acidA and gamma-aminobutyric acidB receptor agonists and antagonists.

    Science.gov (United States)

    Woodward, R M; Polenzani, L; Miledi, R

    1993-04-01

    Poly(A)+ RNA from mammalian retina expresses bicuculline/baclofen-insensitive gamma-aminobutyric acid (GABA) receptors in Xenopus oocytes with properties similar to those of homooligomeric GABA rho 1 receptors. The pharmacological profile of these rho-like receptors was extended by measuring sensitivities to various GABAA and GABAB receptor ligands. For direct comparison the same compounds were also assayed with GABAA receptors expressed by rat brain RNA. The potency sequence for heterocyclic GABA analogues at the GABA rho-like receptors was GABA (1.3) > muscimol (2.3) > isoguvacine (100) (approximate EC50 in parentheses; all EC50 and Kb values given in microM). Both muscimol and isoguvacine were partial agonists at the rho-like receptors. 4,5,6,7-Tetrahydroisoxazolo[5,4-c]pyridin-3-ol (Kb congruent to 32), piperidine-4-sulfonic acid (Kb congruent to 85), and isonipecotic acid (Kb congruent to 1000) acted primarily as competitive antagonists, showing little or no activity as agonists. The sulfonic acid GABA analogue 3-aminopropanesulfonic acid was also a competitive antagonist (Kb congruent to 20). Conformationally restricted GABA analogues trans- and cis-4-aminocrotonic acid (TACA and CACA) were agonists at the rho-like receptors. TACA (EC50 congruent to 0.6) had twice the potency of GABA and was 125 times more potent than CACA (EC50 congruent to 75). Z-3-(Amidinothio)propenoic acid, an isothiouronium analogue of GABA, had little activity as an agonist but instead acted as a competitive antagonist (Kb congruent to 20). At concentrations of > 100 microM, bicuculline did have some weak competitive inhibitory effects on the GABA rho-like receptors (Kb congruent to 6000), but it was at least 5000 times more potent at GABAA receptors. Strychnine (Kb congruent to 70) and SR-95531 (Kb congruent to 35) also were competitive inhibitors of the rho-like receptors but were, respectively, 20 and 240 times more potent at GABAA receptors. The GABAB receptor ligands baclofen

  2. Nuclear trafficking of secreted factors and cell-surface receptors: new pathways to regulate cell proliferation and differentiation, and involvement in cancers

    Directory of Open Access Journals (Sweden)

    Planque Nathalie

    2006-10-01

    Full Text Available Abstract Secreted factors and cell surface receptors can be internalized by endocytosis and translocated to the cytoplasm. Instead of being recycled or proteolysed, they sometimes translocate to the nucleus. Nuclear import generally involves a nuclear localization signal contained either in the secreted factor or its transmembrane receptor, that is recognized by the importins machinery. In the nucleus, these molecules regulate transcription of specific target genes by direct binding to transcription factors or general coregulators. In addition to the transcription regulation, nuclear secreted proteins and receptors seem to be involved in other important processes for cell life and cellular integrity such as DNA replication, DNA repair and RNA metabolism. Nuclear secreted proteins and transmembrane receptors now appear to induce new signaling pathways to regulate cell proliferation and differentiation. Their nuclear localization is often transient, appearing only during certain phases of the cell cycle. Nuclear secreted and transmembrane molecules regulate the proliferation and differentiation of a large panel of cell types during embryogenesis and adulthood and are also potentially involved in wound healing. Secreted factors such as CCN proteins, EGF, FGFs and their receptors are often detected in the nucleus of cancer cells. Nuclear localization of these molecules has been correlated with tumor progression and poor prognosis for patient survival. Nuclear growth factors and receptors may be responsible for resistance to radiotherapy.

  3. Expression of retinoic acid receptors in human endometrial carcinoma.

    Science.gov (United States)

    Tanabe, Kojiro; Utsunomiya, Hiroki; Tamura, Mitsutoshi; Niikura, Hitoshi; Takano, Tadao; Yoshinaga, Kohsuke; Nagase, Satoru; Suzuki, Takashi; Ito, Kiyoshi; Matsumoto, Mitsuyo; Hayashi, Shin-ichi; Yaegashi, Nobuo

    2008-02-01

    The retinoids (vitamin A and its biologically active derivatives) are essential for the health and survival of the individual. Several studies have reported a strong rationale for the use of retinoids in cancer treatment and chemoprevention. It has been discovered that expression of retinoic acid receptor (RAR) beta is frequently silenced in epithelial carcinogenesis, which has led to the hypothesis that RAR beta could act as a tumor suppressor. However, the status of RAR beta in human endometrial carcinoma has not been examined. In the present study, we initially studied the effects of retinoic acid on cell proliferation and the expression of RAR alpha, RAR beta, and RAR gamma using AM580 (a RAR-specific agonist) in the Ishikawa endometrial cancer cell line. We also examined the expression of RAR in human eutopic endometrium (30 cases), endometrial hyperplasia (28 cases), and endometrial carcinoma (103 cases) using immunohistochemistry. Finally, we correlated these findings with the clinicopathological parameters. In vitro, cell growth was inhibited and RAR beta and RAR gamma mRNA was significantly induced by AM580, compared with vehicle controls, whereas RAR alpha mRNA was significantly attenuated by AM580, compared with vehicle. RAR beta was detected predominantly in endometrial hyperplasia, compared with endometrial carcinoma. No statistically significant correlation was obtained between the expression of any other RAR subtypes and clinicopathological parameters in human endometrial carcinoma. The results of our study demonstrate that AM580 inhibits cell growth and induces RAR beta mRNA expression in the Ishikawa cell line, and the expression level of RAR beta in endometrial carcinoma is significantly lower than that in endometrial hyperplasia. AM580 might therefore be considered as a potential treatment for endometrial carcinoma.

  4. ErbB-2 blockade and prenyltransferase inhibition alter epidermal growth factor and epidermal growth factor receptor trafficking and enhance (111)In-DTPA-hEGF Auger electron radiation therapy.

    Science.gov (United States)

    Cornelissen, Bart; Darbar, Sonali; Hernandez, Rebecca; Kersemans, Veerle; Tullis, Iain; Barber, Paul R; Smart, Sean; Vojnovic, Borivoj; Reilly, Raymond; Vallis, Katherine A

    2011-05-01

    The intracellular distribution of Auger electron-emitting radiopharmaceuticals is a determinant of cytotoxicity. However, the mechanisms by which these agents are routed through the cell are ill understood. The aim of this study was to investigate how trafficking of (111)In-labeled human epidermal growth factor ((111)In-DTPA-hEGF) relates to that of the EGF receptor (EGFR) and whether coadministration of agents that modulate EGFR signaling alters the efficacy of (111)In-DTPA-hEGF. The spatiotemporal interaction between AlexaFluor488-EGF (AF488-EGF) and Cy3-conjugated anti-EGFR antibody (Cy3-anti-EGFR) was studied in the breast cancer cell line MDA-MB-468 using fluorescence resonance energy transfer and 2-photon fluorescence lifetime imaging. (111)In internalization and nuclear fractionation assays were performed to investigate the effect of the ErbB-2-blocking antibody trastuzumab and a prenyltransferase inhibitor, L-778,123, on the subcellular localization of (111)In-DTPA-hEGF in MDA-MB-468 (1.3 × 10(6) EGFR per cell; ErbB-2 negative) and 231-H2N (0.2 × 10(6) EGFR per cell; 0.4 × 10(5) ErbB-2 per cell) cell lines. The cytotoxicity of (111)In-DTPA-hEGF (0-64 nM) plus trastuzumab (0-50 μg/mL) or L-778,123 (0-22.5 μM) was measured using clonogenic assays in a panel of breast cancer cell lines that express different levels of EGFR and ErB-2. Clonogenic survival data were used to calculate combination indices. Tumor growth inhibition was measured in vivo in 231-H2N xenograft-bearing mice treated with (111)In-DTPA-hEGF plus trastuzumab or L-788,123. Using fluorescence resonance energy transfer, we showed that EGF interacts with EGFR in the cytoplasm and nucleus after internalization of the ligand-receptor complex in MDA-MB-468 cells. Nuclear localization of (111)In-DTPA-hEGF is enhanced by trastuzumab and L-788,123. Trastuzumab and L-788,123 sensitized 231-H2N cells to (111)In-DTPA-hEGF. Nuclear localization and cytotoxicity of (111)In-DTPA-hEGF were significantly

  5. Molecular identification of high and low affinity receptors for nicotinic acid.

    Science.gov (United States)

    Wise, Alan; Foord, Steven M; Fraser, Neil J; Barnes, Ashley A; Elshourbagy, Nabil; Eilert, Michelle; Ignar, Diane M; Murdock, Paul R; Steplewski, Klaudia; Green, Andrew; Brown, Andrew J; Dowell, Simon J; Szekeres, Philip G; Hassall, David G; Marshall, Fiona H; Wilson, Shelagh; Pike, Nicholas B

    2003-03-14

    Nicotinic acid has been used clinically for over 40 years in the treatment of dyslipidemia producing a desirable normalization of a range of cardiovascular risk factors, including a marked elevation of high density lipoprotein and a reduction in mortality. The precise mechanism of action of nicotinic acid is unknown, although it is believed that activation of a G(i)-G protein-coupled receptor may contribute. Utilizing available information on the tissue distribution of nicotinic acid receptors, we identified candidate orphan receptors. The selected orphan receptors were screened for responses to nicotinic acid, in an assay for activation of G(i)-G proteins. Here we describe the identification of the G protein-coupled receptor HM74 as a low affinity receptor for nicotinic acid. We then describe the subsequent identification of HM74A in follow-up bioinformatics searches and demonstrate that it acts as a high affinity receptor for nicotinic acid and other compounds with related pharmacology. The discovery of HM74A as a molecular target for nicotinic acid may facilitate the discovery of superior drug molecules to treat dyslipidemia.

  6. Altered food consumption in mice lacking lysophosphatidic acid receptor-1.

    Science.gov (United States)

    Dusaulcy, R; Daviaud, D; Pradère, J P; Grès, S; Valet, Ph; Saulnier-Blache, J S

    2009-12-01

    The release of lysophosphatidic acid (LPA) by adipocytes has previously been proposed to play a role in obesity and associated pathologies such as insulin resistance and diabetes. In the present work, the sensitivity to diet-induced obesity was studied in mice lacking one of the LPA receptor subtype (LPA1R). Conversely to what was observed in wild type (WT) mice, LPA1R-KO-mice fed a high fat diet (HFD) showed no significant increase in body weight or fat mass when compared to low fat diet (LFD). In addition, in contrast to what was observed in WT mice, LPA1R-KO mice did not exhibit over-consumption of food associated with HFD. Surprisingly, when fed a LFD, LPA1R-KO mice exhibited significant higher plasma leptin concentration and higher level of adipocyte leptin mRNA than WT mice. In conclusion, LPA1R-KO mice were found to be resistant to diet-induced obesity consecutive to a resistance to fat-induced over-consumption of food that may result at least in part from alterations in leptin expression and production.

  7. Structural basis and functions of abscisic acid receptors PYLs

    Science.gov (United States)

    Zhang, Xing L.; Jiang, Lun; Xin, Qi; Liu, Yang; Tan, Jian X.; Chen, Zhong Z.

    2015-01-01

    Abscisic acid (ABA) plays a key role in many developmental processes and responses to adaptive stresses in plants. Recently, a new family of nucleocytoplasmic PYR/PYL/RCAR (PYLs) has been identified as bona fide ABA receptors. PYLs together with protein phosphatases type-2C (PP2Cs), Snf1 (Sucrose-non-fermentation 1)-related kinases subfamily 2 (SnRK2s) and downstream substrates constitute the core ABA signaling network. Generally, PP2Cs inactivate SnRK2s kinases by physical interaction and direct dephosphorylation. Upon ABA binding, PYLs change their conformations and then contact and inhibit PP2Cs, thus activating SnRK2s. Here, we reviewed the recent progress in research regarding the structures of the core signaling pathways of ABA, including the (+)-ABA, (−)-ABA and ABA analogs pyrabactin as well as 6AS perception by PYLs, SnRK2s mimicking PYLs in binding PP2Cs. PYLs inhibited PP2Cs in both the presence and absence of ABA and activated SnRK2s. The present review elucidates multiple ABA signal perception and transduction by PYLs, which might shed light on how to design small chemical compounds for improving plant performance in the future. PMID:25745428

  8. The anticonvulsant gabapentin (neurontin) does not act through gamma-aminobutyric acid-B receptors

    DEFF Research Database (Denmark)

    Jensen, Anders A.; Mosbacher, Johannes; Elg, Susanne;

    2002-01-01

    The actions of the anticonvulsant gabapentin [1-(aminomethyl)cyclohexaneacetic acid, Neurontin] have been somewhat enigmatic until recently, when it was claimed to be a gamma-aminobutyric acid-B (GABA(B)) receptor agonist acting exclusively at a heterodimeric complex containing the GABA(B(1a...... in vitro assays. In light of these results, we find it highly questionable that gabapentin is a GABA(B) receptor agonist. Hence, the anticonvulsive effects of the compound have to arise from GABA(B) receptor-independent mechanisms. This also implies that the first GABA(B) receptor splice variant...

  9. Effects of gamma-aminobutyric acid receptors on muscarinic receptor-mediated free calcium ion levels in the facial nucleus following facial nerve injury

    Institute of Scientific and Technical Information of China (English)

    Guangfeng Jiang; Dawei Sun; Rui Zhou; Fugao Zhu; Yanqing Wang; Xiuming Wan; Banghua Liu

    2011-01-01

    Muscarinic receptors and nicotine receptors can increase free calcium ion levels in the facial nucleus via different channels following facial nerve injury. In addition, γ-aminobutyric acid A (GABAA) receptors have been shown to negatively regulate free calcium ion levels in the facial nucleus by inhibiting nicotine receptors. The present study investigated the influence of GABAA, γ-aminobutyric acid B (GABAB) and C (GABAC) receptors on muscarinic receptors in rats with facial nerve injury by confocal laser microscopy. GABAA and GABAB receptors exhibited significant dose-dependent inhibitory effects on increased muscarinic receptor-mediated free calcium ion levels following facial nerve injury. Results showed that GABAA and GABAB receptors negatively regulate muscarinic receptor effects and interplay with cholinergic receptors to regulate free calcium ion levels for facial neural regeneration.

  10. Trafficking of ThermoTRP Channels

    Directory of Open Access Journals (Sweden)

    Clotilde Ferrandiz-Huertas

    2014-08-01

    Full Text Available ThermoTRP channels (thermoTRPs define a subfamily of the transient receptor potential (TRP channels that are activated by changes in the environmental temperature, from noxious cold to injurious heat. Acting as integrators of several stimuli and signalling pathways, dysfunction of these channels contributes to several pathological states. The surface expression of thermoTRPs is controlled by both, the constitutive and regulated vesicular trafficking. Modulation of receptor surface density during pathological processes is nowadays considered as an interesting therapeutic approach for management of diseases, such as chronic pain, in which an increased trafficking is associated with the pathological state. This review will focus on the recent advances trafficking of the thermoTRP channels, TRPV1, TRPV2, TRPV4, TRPM3, TRPM8 and TRPA1, into/from the plasma membrane. Particularly, regulated membrane insertion of thermoTRPs channels contributes to a fine tuning of final channel activity, and indeed, it has resulted in the development of novel therapeutic approaches with successful clinical results such as disruption of SNARE-dependent exocytosis by botulinum toxin or botulinomimetic peptides.

  11. Rigid nonproteinogenic cyclic amino acids as ligands for glutamate receptors: trans-tris(homoglutamic) acids.

    Science.gov (United States)

    Meyer, Udo; Bisel, Philippe; Bräuner-Osborne, Hans; Madsen, Ulf; Höfner, Georg; Wanner, Klaus Th; Frahm, August Wilhelm

    2005-02-01

    The second-generation asymmetric synthesis of the trans-tris(homoglutamic) acids reported herein proceeds via Strecker reaction of chiral ketimines, obtained from condensation of racemic 2-ethoxycarbonylmethylcyclopentanone and commercially available (S)- and (R)-1-phenylethylamine, respectively. In the key stereodifferentiating step, the cyanide addition leads to mixtures of diastereomeric alpha-amino nitrile-esters, the composition of which is independent of the reaction temperature and the type of the solvent, respectively. Hydrolysis of the alpha-amino nitrile-esters with concentrated H(2)SO(4) yielded diastereomeric mixtures of secondary alpha-amino amido-esters, which after separation were hydrogenolyzed and hydrolyzed each to the enantiomeric trans-1-amino-2-carboxymethylcyclopentanecarboxylic acids. Their configuration was completely established by NMR methods, CD spectra, and X-ray analysis of the trans-1S,2R-configured secondary alpha-amino amido-ester. In receptor binding assays and functional tests, trans-1S,2R-1-amino-2-carboxymethylcyclopentanecarboxylic acid hydrochloride was found to behave as a selective mGluR(2)-antagonist without relevant binding properties at iGluRs. Copyright 2005 Wiley-Liss, Inc.

  12. Interaction between retinoid acid receptor-related orphan receptor alpha (RORA and neuropeptide S receptor 1 (NPSR1 in asthma.

    Directory of Open Access Journals (Sweden)

    Nathalie Acevedo

    Full Text Available Retinoid acid receptor-related Orphan Receptor Alpha (RORA was recently identified as a susceptibility gene for asthma in a genome-wide association study. To investigate the impact of RORA on asthma susceptibility, we performed a genetic association study between RORA single nucleotide polymorphisms (SNPs in the vicinity of the asthma-associated SNP (rs11071559 and asthma-related traits. Because the regulatory region of a previously implicated asthma susceptibility gene, Neuropeptide S receptor 1 (NPSR1, has predicted elements for RORA binding, we hypothesized that RORA may interact biologically and genetically with NPSR1. 37 RORA SNPs and eight NPSR1 SNPs were genotyped in the Swedish birth cohort BAMSE (2033 children and the European cross-sectional PARSIFAL study (1120 children. Seven RORA SNPs confined into a 49 kb region were significantly associated with physician-diagnosed childhood asthma. The most significant association with rs7164773 (T/C was driven by the CC genotype in asthma cases (OR = 2.0, 95%CI 1.36-2.93, p = 0.0003 in BAMSE; and 1.61, 1.18-2.19, p = 0.002 in the combined BAMSE-PARSIFAL datasets, respectively, and strikingly, the risk effect was dependent on the Gln344Arg mutation in NPSR1. In cell models, stimulation of NPSR1 activated a pathway including RORA and other circadian clock genes. Over-expression of RORA decreased NPSR1 promoter activity further suggesting a regulatory loop between these genes. In addition, Rora mRNA expression was lower in the lung tissue of Npsr1 deficient mice compared to wildtype littermates during the early hours of the light period. We conclude that RORA SNPs are associated with childhood asthma and show epistasis with NPSR1, and the interaction between RORA and NPSR1 may be of biological relevance. Combinations of common susceptibility alleles and less common functional polymorphisms may modify the joint risk effects on asthma susceptibility.

  13. Regulation of retinoic acid receptor beta expression by peroxisome proliferator-activated receptor gamma ligands in cancer cells.

    Science.gov (United States)

    James, Sharon Y; Lin, Feng; Kolluri, Siva Kumar; Dawson, Marcia I; Zhang, Xiao-kun

    2003-07-01

    The peroxisome proliferator-activated receptor gamma (PPAR gamma) is a nuclear receptor family member that can form a heterodimeric complex with retinoid X receptor (RXR) and initiate transcription of target genes. In this study, we have examined the effects of the PPAR gamma ligand ciglitazone and the RXR ligand SR11237 on growth and induction of retinoic acid receptor (RAR) beta expression in breast and lung cancer cells. Our results demonstrated that ciglitazone and SR11237 cooperatively inhibited the growth of ZR-75-1 and T-47D breast cancer and Calu-6 lung cancer cells. Gel shift analysis indicated that PPAR gamma, in the presence of RXR, formed a strong complex with a retinoic acid response element (beta retinoic acid response element) in the RAR beta promoter. In reporter gene assays, RXR ligands and ciglitazone, but not the PPAR gamma ligand 15d-PGJ(2), cooperatively promoted the transcriptional activity of the beta retinoic acid response element. Ciglitazone, but not 15d-PGJ(2), strongly induced RAR beta expression in human breast and lung cancer cell lines when used together with SR11237. The induction of RAR beta expression by the ciglitazone and SR11237 combination was diminished by a PPAR gamma-selective antagonist, bisphenol A diglycidyl ether. All-trans-retinoic acid or the combination of ciglitazone and SR11237 was able to induce RAR beta in all-trans-retinoic acid-resistant MDA-MB-231 breast cancer cells only when the orphan receptor chick ovalbumin upstream promoter transcription factor was expressed, or in the presence of the histone deacetylase inhibitor trichostatin A. These studies indicate the existence of a novel RAR beta-mediated signaling pathway of PPAR gamma action, which may provide a molecular basis for developing novel therapies involving RXR and PPAR gamma ligands in potentiating antitumor responses.

  14. Role of retinoic acid receptors in squamous-cell carcinoma in human esophagus

    DEFF Research Database (Denmark)

    Bergheim, I.; Wolfgarten, E.; Bollschweiler, E.

    2005-01-01

    BACKGROUND: Worldwide, cancer in the esophagus ranks among the 10 most common cancers. Alterations of retinoic acid receptors (e.g. RARalpha, beta, gamma, and RXRalpha, beta, gamma) expression is considered to play an important role in development of squamous-cell carcinoma (SCC), which is the most...... common esophageal cancer. Alcohol consumption and smoking, which can alter retinoic acid receptor levels, have been identified as key risk factors in the development of carcinoma in the aero-digestive tract. Therefore, the aim of the present study was to evaluate protein levels of retinoic acid receptors...... were found for RARalpha, beta, and RXRbeta protein levels between normal esophageal tissue of patients and that of controls. CONCLUSION: In conclusion, results of the present study suggest that alterations of retinoic acid receptors protein may contribute in the development of SCC in esophagus...

  15. Synthesis and pharmacological characterization at glutamate receptors of the four enantiopure isomers of tricholomic acid

    DEFF Research Database (Denmark)

    Pinto, Andrea; Conti, Paola; De Amici, Marco

    2008-01-01

    of the studied amino acids reflect the relationship between the activity/selectivity and the stereochemistry of the two stereogenic centers: while the (2 S,5' S) stereoisomer is an agonist at the AMPA and KA receptors, its (2 R,5' R) enantiomer interacts selectively with the NMDA receptors; the (2 S,5' R...

  16. Development and Characterization of a Potent Free Fatty Acid Receptor 1 (FFA1) Fluorescent Tracer

    DEFF Research Database (Denmark)

    Christiansen, Elisabeth; Hudson, Brian D; Hansen, Anders Højgaard;

    2016-01-01

    The free fatty acid receptor 1 (FFA1/GPR40) is a potential target for treatment of type 2 diabetes. Although several potent agonists have been described, there remains a strong need for suitable tracers to interrogate ligand binding to this receptor. We address this by exploring fluorophore-tethe...

  17. Novel 3-carboxy- and 3-phosphonopyrazoline amino acids as potent and selective NMDA receptor antagonists

    DEFF Research Database (Denmark)

    Conti, Paola; Pinto, Andrea; Tamborini, Lucia

    2010-01-01

    The design and synthesis of new N1-substituted 3-carboxy- and 3-phosphonopyrazoline and pyrazole amino acids that target the glutamate binding site of NMDA receptors are described. An analysis of the stereochemical requirements for high-affinity interaction with these receptors was performed. We...

  18. Scavenger receptor class B type I (SR-BI) in pig enterocytes: trafficking from the brush border to lipid droplets during fat absorption

    DEFF Research Database (Denmark)

    Hansen, Gert Helge; Niels-Christiansen, Lise-Lotte W; Immerdal, Lissi

    2003-01-01

    BACKGROUND: Scavenger receptor class B type I (SR-BI) is known to mediate cellular uptake of cholesterol from high density lipoprotein particles and is particularly abundant in liver and steroidogenic tissues. In addition, SR-BI expression in the enterocyte brush border has also been reported...... but its role in the small intestine remains unclear. AIM AND METHODS: To gain insight into the possible function of pig SR-BI during uptake of dietary fat, its localisation in enterocytes was studied in the fasting state and during fat absorption by immunogold electron microscopy and subcellular...... fat, SR-BI is endocytosed from the enterocyte brush border and accumulates in cytoplasmic lipid droplets. Internalisation of the receptor occurs mainly by clathrin coated pits rather than by a caveolae/lipid raft based mechanism....

  19. Aryl Hydrocarbon Receptor Activation in Hematopoietic Stem/Progenitor Cells Alters Cell Function and Pathway-Specific Gene Modulation Reflecting Changes in Cellular Trafficking and MigrationS⃞

    OpenAIRE

    Casado, Fanny L.; Singh, Kameshwar P.; Gasiewicz, Thomas A.

    2011-01-01

    The aryl hydrocarbon receptor (AhR) is a transcription factor belonging to the Per-ARNT-Sim family of proteins. These proteins sense molecules and stimuli from the cellular/tissue environment and initiate signaling cascades to elicit appropriate cellular responses. Recent literature reports suggest an important function of AhR in hematopoietic stem cell (HSC) biology. However, the molecular mechanisms by which AhR signaling regulates HSC functions are unknown. In previous studies, we and othe...

  20. The molecular basis of ligand interaction at free fatty acid receptor 4 (FFA4/GPR120)

    OpenAIRE

    Hudson, Brian D.; Shimpukade, Bharat; Milligan, Graeme; Ulven, Trond

    2014-01-01

    The long-chain fatty acid receptor FFA4(previously GPR120) is receiving substantial interest as a novel target for the treatment of metabolic and inflammatory disease. The current study examines for the first time the detailed mode of binding of both a long-chain fatty acid and synthetic agonist ligands at FFA4 by integrating molecular modeling, receptor mutagenesis, and ligand structure-activity relationship approaches in an iterative format. In doing so, residues required for binding of fat...

  1. Synthesis and pharmacology of glutamate receptor ligands: new isothiazole analogues of ibotenic acid.

    Science.gov (United States)

    Jørgensen, Charlotte G; Clausen, Rasmus P; Hansen, Kasper B; Bräuner-Osborne, Hans; Nielsen, Birgitte; Metzler, Bjørn; Metzler, Birgitte Bjørn; Kehler, Jan; Krogsgaard-Larsen, Povl; Madsen, Ulf

    2007-02-07

    The naturally occurring heterocyclic amino acid ibotenic acid (Ibo) and the synthetic analogue thioibotenic acid (Thio-Ibo) possess interesting but dissimilar pharmacological activity at ionotropic and metabotropic glutamate receptors (iGluRs and mGluRs). Therefore, a series of Thio-Ibo analogues was synthesized. The synthesis included introduction of substituents by Suzuki and Grignard reactions on 4-halogenated 3-benzyloxyisothiazolols, reduction of the obtained alcohols, followed by introduction of the amino acid moiety by use of 2-(N-tert-butoxycarbonylimino)malonic acid diethyl ester. The obtained Thio-Ibo analogues (1, 2a-g) were characterized in functional assays on recombinant mGluRs and in receptor binding assays on native iGluRs. At mGluRs, the activity at Group II was retained for compounds with small substituents (2a-2d), whereas the Group I and Group III receptor activities for all new compounds were lost. Detection of NMDA receptor affinity prompted further characterization, and two-electrode voltage-clamp recordings at recombinant NMDA receptor subtypes NR1/NR2A-D expressed in Xenopus oocytes were carried out for compounds with small substituents (chloro, bromo, methyl or ethyl, compounds 2a-d). This series of Thio-Ibo analogues defines a structural threshold for NMDA receptor activation and reveals that the individual subtypes have different steric requirements for receptor activation. The compounds 2a and 2c are the first examples of agonists discriminating individual NMDA subtypes.

  2. Nicotinic acid receptor abnormalities in human skin cancer: implications for a role in epidermal differentiation.

    Directory of Open Access Journals (Sweden)

    Yira Bermudez

    Full Text Available BACKGROUND: Chronic UV skin exposure leads to epidermal differentiation defects in humans that can be largely restored by pharmacological doses of nicotinic acid. Nicotinic acid has been identified as a ligand for the human G-protein-coupled receptors GPR109A and GPR109B that signal through G(i-mediated inhibition of adenylyl cyclase. We have examined the expression, cellular distribution, and functionality of GPR109A/B in human skin and skin derived epidermal cells. RESULTS: Nicotinic acid increases epidermal differentiation in photodamaged human skin as judged by the terminal differentiation markers caspase 14 and filaggrin. Both GPR109A and GPR109B genes are transcribed in human skin and in epidermal keratinocytes, but expression in dermal fibroblasts is below limits of detection. Receptor transcripts are greatly over-expressed in squamous cell cancers. Receptor protein in normal skin is prominent from the basal through granular layers of the epidermis, with cellular localization more dispersive in the basal layer but predominantly localized at the plasma membrane in more differentiated epidermal layers. In normal human primary and immortalized keratinocytes, nicotinic acid receptors show plasma membrane localization and functional G(i-mediated signaling. In contrast, in a squamous cell carcinoma derived cell line, receptor protein shows a more diffuse cellular localization and the receptors are nearly non-functional. CONCLUSIONS: The results of these studies justify future genetic and pharmacological intervention studies to define possible specific role(s of nicotinic acid receptors in human skin homeostasis.

  3. Potentiation of gamma aminobutyric acid receptors (GABAAR by Ethanol: How are inhibitory receptors affected?

    Directory of Open Access Journals (Sweden)

    Benjamin eFörstera

    2016-05-01

    Full Text Available In recent years there has been an increase in the understanding of ethanol actions on the type A -aminobutyric acid chloride channel (GABAAR, a member of the pentameric ligand gated ion channels (pLGICs. However, the mechanism by which ethanol potentiates the complex is still not fully understood and a number of publications have shown contradictory results. Thus many questions still remain unresolved requiring further studies for a better comprehension of this effect. The present review concentrates on the involvement of GABAAR in the acute actions of ethanol and specifically focuses on the immediate, direct or indirect, synaptic and extra-synaptic modulatory effects. To elaborate on the immediate, direct modulation of GABAAR by acute ethanol exposure, electrophysiological studies investigating the importance of different subunits, and data from receptor mutants will be examined. We will also discuss the nature of the putative binding sites for ethanol based on structural data obtained from other members of the pLGICs family. Finally, we will briefly highlight the glycine gated chloride channel (GlyR, another member of the pLGIC family, as a suitable target for the development of new pharmacological tools.

  4. Epidermal growth factor receptor transactivation by intracellular prostaglandin E2-activated prostaglandin E2 receptors. Role in retinoic acid receptor-β up-regulation.

    Science.gov (United States)

    Fernández-Martínez, Ana B; Lucio Cazaña, Francisco J

    2013-09-01

    The pharmacological modulation of renoprotective factor vascular endothelial growth factor-A (VEGF-A) in the proximal tubule has therapeutic interest. In human proximal tubular HK-2 cells, treatment with all-trans retinoic acid or prostaglandin E2 (PGE2) triggers the production of VEGF-A. The pathway involves an initial increase in intracellular PGE2, followed by activation of EP receptors (PGE2 receptors, most likely an intracellular subset) and increase in retinoic acid receptor-β (RARβ) expression. RARβ then up-regulates transcription factor hypoxia-inducible factor-1α (HIF-1α), which increases the transcription and production of VEGF-A. Here we studied the role in this pathway of epidermal growth factor receptor (EGFR) transactivation by EP receptors. We found that EGFR inhibitor AG1478 prevented the increase in VEGF-A production induced by PGE2- and all-trans retinoic acid. This effect was due to the inhibition of the transcriptional up-regulation of RARβ, which resulted in loss of the RARβ-dependent transcriptional up-regulation of HIF-1α. PGE2 and all-trans retinoic acid also increased EGFR phosphorylation and this effect was sensitive to antagonists of EP receptors. The role of intracellular PGE2 was indicated by two facts; i) PGE2-induced EGFR phosphorylation was substantially prevented by inhibitor of prostaglandin uptake transporter bromocresol green and ii) all-trans retinoic acid treatment, which enhanced intracellular but not extracellular PGE2, had lower effect on EGFR phosphorylation upon pre-treatment with cyclooxygenase inhibitor diclofenac. Thus, EGFR transactivation by intracellular PGE2-activated EP receptors results in the sequential activation of RARβ and HIF-1α leading to increased production of VEGF-A and it may be a target for the therapeutic modulation of HIF-1α/VEGF-A. Copyright © 2013 Elsevier B.V. All rights reserved.

  5. Tranexamic acid induces kaolin intake stimulating a pathway involving tachykinin neurokinin 1 receptors in rats.

    Science.gov (United States)

    Kakiuchi, Hitoshi; Kawarai-Shimamura, Asako; Kuwagata, Makiko; Orito, Kensuke

    2014-01-15

    Tranexamic acid suppresses post-partum haemorrhage and idiopathic menorrhagia through its anti-fibrinolytic action. Although it is clinically useful, it is associated with high risks of side effects such as emesis. Understanding the mechanisms underlying tranexamic acid-induced emesis is very important to explore appropriate anti-emetic drugs for the prevention and/or suppression of emesis. In this study, we examined the receptors involved in tranexamic acid-induced kaolin intake in rats, which reflects the drug's clinical emetogenic potential in humans. Further, we examined the brain regions activated by administration of tranexamic acid and elucidated pivotal pathways of tranexamic acid-induced kaolin intake. We examined the effects of ondansetron, a 5-hydroxytryptamine 3 receptor antagonist, domperidone, a dopamine 2 receptor antagonist, and aprepitant, a tachykinin neurokinin 1 (NK1) receptor antagonist, on tranexamic acid-induced kaolin intake in rats. Then, we determined the brain regions that showed increased numbers of c-Fos immunoreactive cells. Finally, we examined the effects of an antagonist(s) that reduced tranexamic acid-induced kaolin intake on the increase in c-Fos immunoreactive cells. Aprepitant significantly decreased tranexamic acid-induced kaolin intake. However, neither ondansetron nor domperidone decreased kaolin intake. Tranexamic acid significantly increased c-Fos immunoreactive cells by approximately 5.5-fold and 22-fold in the area postrema and nucleus of solitary tract, respectively. Aprepitant decreased the number of c-Fos immunoreactive cells in both areas. Tranexamic acid induced kaolin intake possibly via stimulation of tachykinin NK1 receptors in rats. The tachykinin NK1 receptor could be targeted to prevent and/or suppress emesis in patients receiving tranexamic acid. © 2013 Published by Elsevier B.V.

  6. Stoichiometry of expressed alpha(4)beta(2)delta gamma-aminobutyric acid type A receptors depends on the ratio of subunit cDNA transfected.

    Science.gov (United States)

    Wagoner, Kelly R; Czajkowski, Cynthia

    2010-05-07

    The gamma-aminobutyric acid type A receptor (GABA(A)R) is the target of many depressants, including benzodiazepines, anesthetics, and alcohol. Although the highly prevalent alphabetagamma GABA(A)R subtype mediates the majority of fast synaptic inhibition in the brain, receptors containing delta subunits also play a key role, mediating tonic inhibition and the actions of endogenous neurosteroids and alcohol. However, the fundamental properties of delta-containing GABA(A)Rs, such as subunit stoichiometry, are not well established. To determine subunit stoichiometry of expressed delta-containing GABA(A)Rs, we inserted the alpha-bungarotoxin binding site tag in the alpha(4), beta(2), and delta subunit N termini. An enhanced green fluorescent protein tag was also inserted into the beta(2) subunit to shift its molecular weight, allowing us to separate subunits using SDS-PAGE. Tagged alpha(4)beta(2)delta GABA(A)Rs were expressed in HEK293T cells using various ratios of subunit cDNA, and receptor subunit stoichiometry was determined by quantitating fluorescent alpha-bungarotoxin bound to each subunit on Western blots of surface immunopurified tagged GABA(A)Rs. The results demonstrate that the subunit stoichiometry of alpha(4)beta(2)delta GABA(A)Rs is regulated by the ratio of subunit cDNAs transfected. Increasing the ratio of delta subunit cDNA transfected increased delta subunit incorporation into surface receptors with a concomitant decrease in beta(2) subunit incorporation. Because receptor subunit stoichiometry can directly influence GABA(A)R pharmacological and functional properties, considering how the transfection protocols used affect subunit stoichiometry is essential when studying heterologously expressed alpha(4)beta(2)delta GABA(A)Rs. Successful bungarotoxin binding site tagging of GABA(A)R subunits is a novel tool with which to accurately quantitate subunit stoichiometry and will be useful for monitoring GABA(A)R trafficking in live cells.

  7. Data for amino acid alignment of Japanese stingray melanocortin receptors with other gnathostome melanocortin receptor sequences, and the ligand selectivity of Japanese stingray melanocortin receptors.

    Science.gov (United States)

    Takahashi, Akiyoshi; Davis, Perry; Reinick, Christina; Mizusawa, Kanta; Sakamoto, Tatsuya; Dores, Robert M

    2016-06-01

    This article contains structure and pharmacological characteristics of melanocortin receptors (MCRs) related to research published in "Characterization of melanocortin receptors from stingray Dasyatis akajei, a cartilaginous fish" (Takahashi et al., 2016) [1]. The amino acid sequences of the stingray, D. akajei, MC1R, MC2R, MC3R, MC4R, and MC5R were aligned with the corresponding melanocortin receptor sequences from the elephant shark, Callorhinchus milii, the dogfish, Squalus acanthias, the goldfish, Carassius auratus, and the mouse, Mus musculus. These alignments provide the basis for phylogenetic analysis of these gnathostome melanocortin receptor sequences. In addition, the Japanese stingray melanocortin receptors were separately expressed in Chinese Hamster Ovary cells, and stimulated with stingray ACTH, α-MSH, β-MSH, γ-MSH, δ-MSH, and β-endorphin. The dose response curves reveal the order of ligand selectivity for each stingray MCR.

  8. Data for amino acid alignment of Japanese stingray melanocortin receptors with other gnathostome melanocortin receptor sequences, and the ligand selectivity of Japanese stingray melanocortin receptors

    Directory of Open Access Journals (Sweden)

    Akiyoshi Takahashi

    2016-06-01

    Full Text Available This article contains structure and pharmacological characteristics of melanocortin receptors (MCRs related to research published in “Characterization of melanocortin receptors from stingray Dasyatis akajei, a cartilaginous fish” (Takahashi et al., 2016 [1]. The amino acid sequences of the stingray, D. akajei, MC1R, MC2R, MC3R, MC4R, and MC5R were aligned with the corresponding melanocortin receptor sequences from the elephant shark, Callorhinchus milii, the dogfish, Squalus acanthias, the goldfish, Carassius auratus, and the mouse, Mus musculus. These alignments provide the basis for phylogenetic analysis of these gnathostome melanocortin receptor sequences. In addition, the Japanese stingray melanocortin receptors were separately expressed in Chinese Hamster Ovary cells, and stimulated with stingray ACTH, α-MSH, β-MSH, γ-MSH, δ-MSH, and β-endorphin. The dose response curves reveal the order of ligand selectivity for each stingray MCR.

  9. The relationship between the IL-1 receptor structure and its trafficking function%I型IL-1受体的结构与其转运功能相关的研究

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    AIM:To analyze the relationship between structure and traffickingfunction of IL-1 receptor.METHODS: Wild type and mutant (W514A) IL-1 receptor cDNA containing mouse extracellular domain and human intracellular domain, IL-1 receptor cDNA only containing mouse extracellular domain, were inserted into pEGFP-N2 C-terminal protein fusion vector, respectively. All constructs were transfected respectively into human fibroblasts using calcium phosphate precipitation method. The location of expressing fusion protein at single cell level and translocation after binding with IL-1 were determined under confocal microscopy. RESULTS: The wild type IL-1 receptor located in cell membrane, extended processes and accumulated in areas suggestive of sites of focal adhesions. Further, the fusion proteins moved into cytoplasm and finally concentrate in nuclear after IL-1 stimulation. In contrast, mutant and extracellular domain IL-1 receptors did not translocate to sites of cell adhesion and were unaffected by IL-1 stimulation. CONCLUSION: Fibronectin can increase the expression of IL-1 receptor. The trafficking of IL-1 receptor is dependant on structurally intact, specific structure (tryptophan at 514) of C-terminal end of the cytoplasmic tail of the receptor is necessary.%目的:为进一步了解IL-1受体的结构与IL-1受体在细胞内表达后的转运的关系,以及该结构改变对IL-1受体功能的影响。方法:将重组型、突变型和仅含细胞外段的I型IL-1受体(IL-1RI)的质粒构建入含有绿色荧光蛋白的表达载体中,用磷酸钙沉淀法转染入成纤维细胞中,通过激光扫描共聚焦显微镜,观察其表达的融合蛋白在单个活细胞中的定位,以及与IL-1结合后的运动变化。结果:重组型IL-1RI的融合蛋白表达后主要位于细胞膜和细胞伸出的突起与细胞基质相接触的焦点附着斑处,加入IL-1后,逐渐从细胞膜上移至细胞质内,最后聚集

  10. Castor oil induces laxation and uterus contraction via ricinoleic acid activating prostaglandin EP3 receptors.

    Science.gov (United States)

    Tunaru, Sorin; Althoff, Till F; Nüsing, Rolf M; Diener, Martin; Offermanns, Stefan

    2012-06-01

    Castor oil is one of the oldest drugs. When given orally, it has a laxative effect and induces labor in pregnant females. The effects of castor oil are mediated by ricinoleic acid, a hydroxylated fatty acid released from castor oil by intestinal lipases. Despite the wide-spread use of castor oil in conventional and folk medicine, the molecular mechanism by which ricinoleic acid acts remains unknown. Here we show that the EP(3) prostanoid receptor is specifically activated by ricinoleic acid and that it mediates the pharmacological effects of castor oil. In mice lacking EP(3) receptors, the laxative effect and the uterus contraction induced via ricinoleic acid are absent. Although a conditional deletion of the EP(3) receptor gene in intestinal epithelial cells did not affect castor oil-induced diarrhea, mice lacking EP(3) receptors only in smooth-muscle cells were unresponsive to this drug. Thus, the castor oil metabolite ricinoleic acid activates intestinal and uterine smooth-muscle cells via EP(3) prostanoid receptors. These findings identify the cellular and molecular mechanism underlying the pharmacological effects of castor oil and indicate a role of the EP(3) receptor as a target to induce laxative effects.

  11. Synthesis and structure-activity studies on acidic amino acids and related diacids as NMDA receptor ligands

    DEFF Research Database (Denmark)

    Johansen, T N; Frydenvang, Karla Andrea; Ebert, B

    1994-01-01

    , of (S)-glutamic acid (1) receptors. A number of amino acids and diacids structurally related to AMAA were synthesized and tested electrophysiologically and in receptor-binding assays. The hydroxymethyl analogue 7c of AMAA was an NMDA agonist approximately equipotent with AMAA in the [3H......]CPP-binding assay (IC50 = 7 +/- 3 microM) and electropharmacologically in the rat cortical wedge model (EC50 = 8 +/- 2 microM). In contrast to this, the tertbutyl analogue 7a of AMAA turned out to be an antagonist at NMDA and AMPA receptors. The conformational characteristics of AMAA and 7a, c were studied...... by molecular mechanics calculations. Compound 7a possesses extra steric bulk and shows significant restriction of conformational flexibility compared to AMAA and 7c, which may be determining factors for the observed differences in biological activity. Although the nitrogen atom of quinolinic acid (6) has very...

  12. Eicosopentaneoic Acid and Other Free Fatty Acid Receptor Agonists Inhibit Lysophosphatidic Acid- and Epidermal Growth Factor-Induced Proliferation of Human Breast Cancer Cells

    Directory of Open Access Journals (Sweden)

    Mandi M. Hopkins

    2016-01-01

    Full Text Available Many key actions of ω-3 (n-3 fatty acids have recently been shown to be mediated by two G protein-coupled receptors (GPCRs in the free fatty acid receptor (FFAR family, FFA1 (GPR40 and FFA4 (GPR120. n-3 Fatty acids inhibit proliferation of human breast cancer cells in culture and in animals. In the current study, the roles of FFA1 and FFA4 were investigated. In addition, the role of cross-talk between GPCRs activated by lysophosphatidic acid (LPA, and the tyrosine kinase receptor activated by epidermal growth factor (EGF, was examined. In MCF-7 and MDA-MB-231 human breast cancer cell lines, both LPA and EGF stimulated proliferation, Erk activation, Akt activation, and CCN1 induction. LPA antagonists blocked effects of LPA and EGF on proliferation in MCF-7 and MDA-MB-231, and on cell migration in MCF-7. The n-3 fatty acid eicosopentaneoic acid inhibited LPA- and EGF-induced proliferation in both cell lines. Two synthetic FFAR agonists, GW9508 and TUG-891, likewise inhibited LPA- and EGF-induced proliferation. The data suggest a major role for FFA1, which was expressed by both cell lines. The results indicate that n-3 fatty acids inhibit breast cancer cell proliferation via FFARs, and suggest a mechanism involving negative cross-talk between FFARS, LPA receptors, and EGF receptor.

  13. Health implications of human trafficking.

    Science.gov (United States)

    Richards, Tiffany A

    2014-01-01

    Freedom is arguably the most cherished right in the United States. But each year, approximately 14,500 to 17,500 women, men and children are trafficked into the United States for the purposes of forced labor or sexual exploitation. Human trafficking has significant effects on both physical and mental health. This article describes the features of human trafficking, its physical and mental health effects and the vital role nurses can play in providing care to this vulnerable population.

  14. Women trafficking: causes, concerns, care!

    Science.gov (United States)

    Khowaja, Shaneela Sadaruddin; Tharani, Ambreen Jawed; Agha, Ajmal; Karamaliani, Rozina Sherali

    2012-08-01

    Pakistan is both a country of origin and destination as far as women trafficking is concerned. Poverty, gender discrimination, lack of education, and ignorance about legal rights are some of the underlying causes. Available data suggest several areas of concern, like, for instance: direct health effects, maladaptive coping leading to the use of illicit drugs, and inaccessibility to healthcare facilities. Therefore, numerous interventions would be required at three levels: the prevention of trafficking, the protection of victims and the prosecution of the traffickers.

  15. High affinity receptor labeling based on basic leucine zipper domain peptides conjugated with pH-sensitive fluorescent dye: Visualization of AMPA-type glutamate receptor endocytosis in living neurons.

    Science.gov (United States)

    Hayashi, Ayako; Asanuma, Daisuke; Kamiya, Mako; Urano, Yasuteru; Okabe, Shigeo

    2016-01-01

    Techniques to visualize receptor trafficking in living neurons are important, but currently available methods are limited in their labeling efficiency, specificity and reliability. Here we report a method for receptor labeling with a basic leucine zipper domain peptide (ZIP) and a binding cassette specific to ZIP. Receptors are tagged with a ZIP-binding cassette at their extracellular domain. Tagged receptors expressed in cultured cells were labeled with exogenously applied fluorescently labeled ZIP with low background and high affinity. To test if ZIP labeling is useful in monitoring endocytosis and intracellular trafficking, we next conjugated ZIP with a pH-sensitive dye RhP-M (ZIP-RhP-M). ZIP binding to its binding cassette was pH-resistant and RhP-M fluorescence dramatically increased in acidic environment. Thus AMPA-type glutamate receptors (AMPARs) labeled by ZIP-RhP-M can report receptor endocytosis and subsequent intracellular trafficking. Application of ZIP-RhP-M to cultured hippocampal neurons expressing AMPARs tagged with a ZIP-binding cassette resulted in appearance of fluorescent puncta in PSD-95-positive large spines, suggesting local endocytosis and acidification of AMPARs in individual mature spines. This spine pool of AMPARs in acidic environment was distinct from the early endosomes labeled by transferrin uptake. These results suggest that receptor labeling by ZIP-RhP-M is a useful technique for monitoring endocytosis and intracellular trafficking. This article is part of the Special Issue entitled 'Synaptopathy--from Biology to Therapy'.

  16. The effect pathway of retinoic acid through regulation of retinoic acid receptor in gastric cancer cells

    Institute of Scientific and Technical Information of China (English)

    Su Liu; Qiao Wu; Zheng-Ming Chen; Wen-Jin Su

    2001-01-01

    AIM To evaluate the role of RARa gene in mediating the growth inhibitory effect of ail-trans retinoic acid (ATRA)on gastric cancer cells.``METHODS The expression levels of retinoic acid receptors (RARs) in gastric cancer cells were detected by Northern blot. Transient transfection and chlorophenicol acetyl transferase (CAT) assay were used to show the transcriptional activity of β retinoic acid response element (βRARE) and AP-l activity. Cell growth inhibition was determined by MTT assay and anchorage-independent growth assay, respectively. Stable transfection was performed by the method of Lipofectamine, and the cells were screened by G418.``RESULTS ATRA could induce expression level of RARα in MGC80-3, BGCC8823 and SGC-7901 cells obviously,resulting in growth inhibition of these cell lines. After sense RARa gene was transfected into MKN-45 cells that expressed rather Iow level of RARα and could not be induced by ATRA, the cell growth was inhibited by ATRA markedly. In contrast, when antisense RARα gene was transfected into BGC-825 cells, a little inhibitory effect by ATRA was seen, compared with the parallel BGC-823cells. In transient transfection assay, ATRA effectively induced transcriptional activity of βRARE in MGC80-3,BGC.823, SGC-7902 and MKN/RARa cell lines, but not in MKN-45 and BGC/aRARa cell lines. Similar results were observed in measuring anti-AP-l activity by ATRA in these cancer cell lines.``CONCLUSION ATRA inhibits the growth of gastric cancer cells by up-regulating the level of RARa; RARa is the major mediator of ATRA action in gastric cancer cells; and adequate level of RAPa is required for ATRA effect on gastric cancer cells.``

  17. Human Trafficking and National Morality

    Directory of Open Access Journals (Sweden)

    William R. DI PIETRO

    2015-12-01

    Full Text Available The paper proposes that national morality is an important variable for explaining national anti-trafficking policy. It uses cross country regression analysis to see whether or not empirically national morality is a determinant of anti-trafficking policy. The findings of the paper are consistent with the notion that improved levels of national morality lead to better national anti-trafficking policy. National morality is found to be statistically relevant for national anti-trafficking policy when controlling for the extent of democracy, the share of the private sector in the economy, and the degree of globalization.

  18. Deorphanization of GPRC6A: a promiscuous L-alpha-amino acid receptor with preference for basic amino acids

    DEFF Research Database (Denmark)

    Wellendorph, Petrine; Hansen, Kasper B; Balsgaard, Anders

    2005-01-01

    . To identify agonists at this orphan receptor, we faced the challenges of achieving surface expression in mammalian cell lines and establishing an appropriate functional assay. Generating a chimeric receptor construct, h6A/5.24, containing the ligand binding amino-terminal domain (ATD) of hGPRC6A...... and was employed to rationally select potential ligands. Measurement of Ca2+-dependent chloride currents in Xenopus laevis oocytes facilitated the deorphanization of h6A/5.24 and identification of L-alpha-amino acids as agonists. The most active agonists were basic L-alpha-amino acids, L-Arg, L-Lys, and L...

  19. Cardioprotective trafficking of caveolin to mitochondria is Gi-protein dependent.

    Science.gov (United States)

    Wang, Jiawan; Schilling, Jan M; Niesman, Ingrid R; Headrick, John P; Finley, J Cameron; Kwan, Evan; Patel, Piyush M; Head, Brian P; Roth, David M; Yue, Yun; Patel, Hemal H

    2014-09-01

    Caveolae are a nexus for protective signaling. Trafficking of caveolin to mitochondria is essential for adaptation to cellular stress though the trafficking mechanisms remain unknown. The authors hypothesized that G protein-coupled receptor/inhibitory G protein (Gi) activation leads to caveolin trafficking to mitochondria. Mice were exposed to isoflurane or oxygen vehicle (30 min, ± 36 h pertussis toxin pretreatment, an irreversible Gi inhibitor). Caveolin trafficking, cardioprotective "survival kinase" signaling, mitochondrial function, and ultrastructure were assessed. Isoflurane increased cardiac caveolae (n = 8 per group; data presented as mean ± SD for Ctrl versus isoflurane; [caveolin-1: 1.78 ± 0.12 vs. 3.53 ± 0.77; P Gi proteins are involved in trafficking caveolin to mitochondria to enhance stress resistance. Agents that target Gi activation and caveolin trafficking may be viable cardioprotective agents.

  20. Postsynaptic VAMP/Synaptobrevin Facilitates Differential Vesicle Trafficking of GluA1 and GluA2 AMPA Receptor Subunits.

    Science.gov (United States)

    Hussain, Suleman; Davanger, Svend

    2015-01-01

    Vertebrate organisms adapt to a continuously changing environment by regulating the strength of synaptic connections between brain cells. Excitatory synapses are believed to increase their strength by vesicular insertion of transmitter glutamate receptors into the postsynaptic plasma membrane. These vesicles, however, have never been demonstrated or characterized. For the first time, we show the presence of small vesicles in postsynaptic spines, often closely adjacent to the plasma membrane and PSD (postsynaptic density). We demonstrate that they harbor vesicle-associated membrane protein 2 (VAMP2/synaptobrevin-2) and glutamate receptor subunit 1 (GluA1). Disrupting VAMP2 by tetanus toxin treatment reduces the concentration of GluA1 in the postsynaptic plasma membrane. GluA1/VAMP2-containing vesicles, but not GluA2/VAMP2-vesicles, are concentrated in postsynaptic spines relative to dendrites. Our results indicate that small postsynaptic vesicles containing GluA1 are inserted directly into the spine plasma membrane through a VAMP2-dependent mechanism.

  1. Postsynaptic VAMP/Synaptobrevin Facilitates Differential Vesicle Trafficking of GluA1 and GluA2 AMPA Receptor Subunits.

    Directory of Open Access Journals (Sweden)

    Suleman Hussain

    Full Text Available Vertebrate organisms adapt to a continuously changing environment by regulating the strength of synaptic connections between brain cells. Excitatory synapses are believed to increase their strength by vesicular insertion of transmitter glutamate receptors into the postsynaptic plasma membrane. These vesicles, however, have never been demonstrated or characterized. For the first time, we show the presence of small vesicles in postsynaptic spines, often closely adjacent to the plasma membrane and PSD (postsynaptic density. We demonstrate that they harbor vesicle-associated membrane protein 2 (VAMP2/synaptobrevin-2 and glutamate receptor subunit 1 (GluA1. Disrupting VAMP2 by tetanus toxin treatment reduces the concentration of GluA1 in the postsynaptic plasma membrane. GluA1/VAMP2-containing vesicles, but not GluA2/VAMP2-vesicles, are concentrated in postsynaptic spines relative to dendrites. Our results indicate that small postsynaptic vesicles containing GluA1 are inserted directly into the spine plasma membrane through a VAMP2-dependent mechanism.

  2. Expression and localization of the omega-3 fatty acid receptor GPR120 in human term placenta.

    Science.gov (United States)

    Lager, S; Ramirez, V I; Gaccioli, F; Jansson, T; Powell, T L

    2014-07-01

    Fatty acids can function as signaling molecules, acting through receptors in the cytosol or on the cell surface. G-Protein Receptor (GPR)120 is a membrane-bound receptor mediating anti-inflammatory and insulin-sensitizing effects of the omega-3 fatty acid docohexaenoic acid (DHA). GPR120 dysfunction is associated with obesity in humans. Cellular localization of GPR120 and the influence of maternal obesity on GPR120 protein expression in the placenta are unknown. Herein we demonstrate that GPR120 is predominantly expressed in the microvillous membrane (MVM) of human placenta and that the expression level of this receptor in MVM is not altered by maternal body mass index (BMI).

  3. Biostructural and pharmacological studies of bicyclic analogues of the 3-isoxazolol glutamate receptor agonist ibotenic acid

    DEFF Research Database (Denmark)

    Frydenvang, Karla Andrea; Pickering, Darryl S; Greenwood, Jeremy R;

    2010-01-01

    We describe an improved synthesis and detailed pharmacological characterization of the conformationally restricted analogue of the naturally occurring nonselective glutamate receptor agonist ibotenic acid (RS)-3-hydroxy-4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridine-7-carboxylic acid (7-HPCA, 5) at A...

  4. The molecular basis of ligand interaction at free fatty acid receptor 4 (FFA4/GPR120)

    DEFF Research Database (Denmark)

    Hudson, Brian D; Shimpukade, Bharat; Milligan, Graeme

    2014-01-01

    The long-chain fatty acid receptor FFA4 (previously GPR120) is receiving substantial interest as a novel target for the treatment of metabolic and inflammatory disease. This study examines for the first time the detailed mode of binding of both long-chain fatty acid and synthetic agonist ligands ...

  5. Arachidonic acid release from PC12 pheochromocytoma cells is regulated by I1-imidazoline receptors.

    Science.gov (United States)

    Ernsberger, P

    1998-10-15

    Rat PC 12 pheochromocytoma cells lack alpha2-adrenergic receptors but express plasma membrane I1-imidazoline receptors. In response to the I1-agonist moxonidine, diglycerides are generated via phosphatidylcholine-selective phospholipase C, and prostaglandin E2 is released. This report characterizes I-receptor-mediated release of arachidonic acid, the precursor to the prostaglandins. PC12 cells were incubated with [3H]arachidonic acid for 24 h and superfused with 0.01% bovine serum albumin in Krebs' physiological buffer at 1 ml/min. Calcium ionophore increased arachidonic acid release only marginally, implying that in PC12 cells arachidonic acid release is not driven by calcium. The I1-agonist moxonidine at concentrations between 10 nM and 1.0 microM rapidly elicited up to two-fold increases in [3H]arachidonic acid release. Guanabenz, a potent alpha2-agonist and I2-ligand, had no effect. The selective I1-antagonist efaroxan blocked the action of moxonidine. The phospholipase A2 inhibitor aristolochic acid had no effect, suggesting that arachidonic acid release may be through an indirect pathway, possibly involving diglycerides. Thus, I1-imidazoline receptors in PC12 cells are coupled to arachidonic acid release through an as yet unknown pathway.

  6. γ-Hydroxybutyric acid (GHB is not an agonist of extrasynaptic GABAA receptors.

    Directory of Open Access Journals (Sweden)

    William M Connelly

    Full Text Available γ-Hydroxybutyric acid (GHB is an endogenous compound and a drug used clinically to treat the symptoms of narcolepsy. GHB is known to be an agonist of GABAB receptors with millimolar affinity, but also binds with much higher affinity to another site, known as the GHB receptor. While a body of evidence has shown that GHB does not bind to GABAA receptors widely, recent evidence has suggested that the GHB receptor is in fact on extrasynaptic α4β1δ GABAA receptors, where GHB acts as an agonist with an EC50 of 140 nM. We investigated three neuronal cell types that express a tonic GABAA receptor current mediated by extrasynaptic receptors: ventrobasal (VB thalamic neurons, dentate gyrus granule cells and striatal medium spiny neurons. Using whole-cell voltage clamp in brain slices, we found no evidence that GHB (10 µM induced any GABAA receptor mediated current in these cell types, nor that it modulated inhibitory synaptic currents. Furthermore, a high concentration of GHB (3 mM was able to produce a GABAB receptor mediated current, but did not induce any other currents. These results suggest either that GHB is not a high affinity agonist at native α4β1δ receptors, or that these receptors do not exist in classical areas associated with extrasynaptic currents.

  7. γ-Hydroxybutyric acid (GHB) is not an agonist of extrasynaptic GABAA receptors.

    Science.gov (United States)

    Connelly, William M; Errington, Adam C; Crunelli, Vincenzo

    2013-01-01

    γ-Hydroxybutyric acid (GHB) is an endogenous compound and a drug used clinically to treat the symptoms of narcolepsy. GHB is known to be an agonist of GABAB receptors with millimolar affinity, but also binds with much higher affinity to another site, known as the GHB receptor. While a body of evidence has shown that GHB does not bind to GABAA receptors widely, recent evidence has suggested that the GHB receptor is in fact on extrasynaptic α4β1δ GABAA receptors, where GHB acts as an agonist with an EC50 of 140 nM. We investigated three neuronal cell types that express a tonic GABAA receptor current mediated by extrasynaptic receptors: ventrobasal (VB) thalamic neurons, dentate gyrus granule cells and striatal medium spiny neurons. Using whole-cell voltage clamp in brain slices, we found no evidence that GHB (10 µM) induced any GABAA receptor mediated current in these cell types, nor that it modulated inhibitory synaptic currents. Furthermore, a high concentration of GHB (3 mM) was able to produce a GABAB receptor mediated current, but did not induce any other currents. These results suggest either that GHB is not a high affinity agonist at native α4β1δ receptors, or that these receptors do not exist in classical areas associated with extrasynaptic currents.

  8. Concomitant Action of Structural Elements and Receptor Phosphorylation Determines Arrestin-3 Interaction with the Free Fatty Acid Receptor FFA4*

    Science.gov (United States)

    Butcher, Adrian J.; Hudson, Brian D.; Shimpukade, Bharat; Alvarez-Curto, Elisa; Prihandoko, Rudi; Ulven, Trond; Milligan, Graeme; Tobin, Andrew B.

    2014-01-01

    In addition to being nutrients, free fatty acids act as signaling molecules by activating a family of G protein-coupled receptors. Among these is FFA4, previously called GPR120, which responds to medium and long chain fatty acids, including health-promoting ω-3 fatty acids, which have been implicated in the regulation of metabolic and inflammatory responses. Here we show, using mass spectrometry, mutagenesis, and phosphospecific antibodies, that agonist-regulated phosphorylation of the human FFA4 receptor occurred primarily at five residues (Thr347, Thr349, Ser350, Ser357, and Ser360) in the C-terminal tail. Mutation of these residues reduced both the efficacy and potency of ligand-mediated arrestin-3 recruitment as well as affecting recruitment kinetics. Combined mutagenesis of all five of these residues was insufficient to fully abrogate interaction with arrestin-3, but further mutagenesis of negatively charged residues revealed additional structural components for the interaction with arrestin-3 within the C-terminal tail of the receptor. These elements consist of the acidic residues Glu341, Asp348, and Asp355 located close to the phosphorylation sites. Receptor phosphorylation thus operates in concert with structural elements within the C-terminal tail of FFA4 to allow for the recruitment of arrestin-3. Importantly, these mechanisms of arrestin-3 recruitment operate independently from Gq/11 coupling, thereby offering the possibility that ligands showing stimulus bias could be developed that exploit these differential coupling mechanisms. Furthermore, this provides a strategy for the design of biased receptors to probe physiologically relevant signaling. PMID:24817122

  9. Combinations of physiologic estrogens with xenoestrogens alter calcium and kinase responses, prolactin release, and membrane estrogen receptor trafficking in rat pituitary cells

    Directory of Open Access Journals (Sweden)

    Watson Cheryl S

    2010-10-01

    Full Text Available Abstract Background Xenoestrogens such as alkylphenols and the structurally related plastic byproduct bisphenol A have recently been shown to act potently via nongenomic signaling pathways and the membrane version of estrogen receptor-α. Though the responses to these compounds are typically measured individually, they usually contaminate organisms that already have endogenous estrogens present. Therefore, we used quantitative medium-throughput screening assays to measure the effects of physiologic estrogens in combination with these xenoestrogens. Methods We studied the effects of low concentrations of endogenous estrogens (estradiol, estriol, and estrone at 10 pM (representing pre-development levels, and 1 nM (representing higher cycle-dependent and pregnancy levels in combinations with the same levels of xenoestrogens in GH3/B6/F10 pituitary cells. These levels of xenoestrogens represent extremely low contamination levels. We monitored calcium entry into cells using Fura-2 fluorescence imaging of single cells. Prolactin release was measured by radio-immunoassay. Extracellular-regulated kinase (1 and 2 phospho-activations and the levels of three estrogen receptors in the cell membrane (ERα, ERβ, and GPER were measured using a quantitative plate immunoassay of fixed cells either permeabilized or nonpermeabilized (respectively. Results All xenoestrogens caused responses at these concentrations, and had disruptive effects on the actions of physiologic estrogens. Xenoestrogens reduced the % of cells that responded to estradiol via calcium channel opening. They also inhibited the activation (phosphorylation of extracellular-regulated kinases at some concentrations. They either inhibited or enhanced rapid prolactin release, depending upon concentration. These latter two dose-responses were nonmonotonic, a characteristic of nongenomic estrogenic responses. Conclusions Responses mediated by endogenous estrogens representing different life stages are

  10. Autophagy and proteins involved in vesicular trafficking.

    Science.gov (United States)

    Amaya, Celina; Fader, Claudio Marcelo; Colombo, María Isabel

    2015-11-14

    Autophagy is an intracellular degradation system that, as a basic mechanism it delivers cytoplasmic components to the lysosomes in order to maintain adequate energy levels and cellular homeostasis. This complex cellular process is activated by low cellular nutrient levels and other stress situations such as low ATP levels, the accumulation of damaged proteins or organelles, or pathogen invasion. Autophagy as a multistep process involves vesicular transport events leading to tethering and fusion of autophagic vesicles with several intracellular compartments. This review summarizes our current understanding of the autophagic pathway with emphasis in the trafficking machinery (i.e. Rabs GTPases and SNAP receptors (SNAREs)) involved in specific steps of the pathway.

  11. Lymphatic Regulation of Cellular Trafficking

    Science.gov (United States)

    Jackson, David G.

    2016-01-01

    Lymphatic vessels play vital roles in immune surveillance and immune regulation by conveying antigen loaded dendritic cells, memory T cells, macrophages and neutrophils from the peripheral tissues to draining lymph nodes where they initiate as well as modify immune responses. Until relatively recently however, there was little understanding of how entry and migration through lymphatic vessels is organized or the specific molecular mechanisms that might be involved. Within the last decade, the situation has been transformed by an explosion of knowledge generated largely through the application of microscopic imaging, transgenic animals, specific markers and function blocking mAbs that is beginning to provide a rational conceptual framework. This article provides a critical review of the recent literature, highlighting seminal discoveries that have revealed the fascinating ultrastructure of leucocyte entry sites in lymphatic vessels, as well as generating controversies over the involvement of integrin adhesion, chemotactic and haptotactic mechanisms in DC entry under normal and inflamed conditions. It also discusses the major changes in lymphatic architecture that occur during inflammation and the different modes of leucocyte entry and trafficking within inflamed lymphatic vessels, as well as presenting a timely update on the likely role of hyaluronan and the major lymphatic endothelial hyaluronan receptor LYVE-1 in leucocyte transit.

  12. RAGE is a nucleic acid receptor that promotes inflammatory responses to DNA

    Science.gov (United States)

    Sirois, Cherilyn M.; Jin, Tengchuan; Miller, Allison L.; Bertheloot, Damien; Nakamura, Hirotaka; Horvath, Gabor L.; Mian, Abubakar; Jiang, Jiansheng; Schrum, Jacob; Bossaller, Lukas; Pelka, Karin; Garbi, Natalio; Brewah, Yambasu; Tian, Jane; Chang, ChewShun; Chowdhury, Partha S.; Sims, Gary P.; Kolbeck, Roland; Coyle, Anthony J.; Humbles, Alison A.

    2013-01-01

    Recognition of DNA and RNA molecules derived from pathogens or self-antigen is one way the mammalian immune system senses infection and tissue damage. Activation of immune signaling receptors by nucleic acids is controlled by limiting the access of DNA and RNA to intracellular receptors, but the mechanisms by which endosome-resident receptors encounter nucleic acids from the extracellular space are largely undefined. In this study, we show that the receptor for advanced glycation end-products (RAGE) promoted DNA uptake into endosomes and lowered the immune recognition threshold for the activation of Toll-like receptor 9, the principal DNA-recognizing transmembrane signaling receptor. Structural analysis of RAGE–DNA complexes indicated that DNA interacted with dimers of the outermost RAGE extracellular domains, and could induce formation of higher-order receptor complexes. Furthermore, mice deficient in RAGE were unable to mount a typical inflammatory response to DNA in the lung, indicating that RAGE is important for the detection of nucleic acids in vivo. PMID:24081950

  13. Phytanic acid and pristanic acid, branched-chain fatty acids associated with Refsum disease and other inherited peroxisomal disorders, mediate intracellular Ca2+ signaling through activation of free fatty acid receptor GPR40.

    Science.gov (United States)

    Kruska, Nicol; Reiser, Georg

    2011-08-01

    The accumulation of the two branched-chain fatty acids phytanic acid and pristanic acid is known to play an important role in several diseases with peroxisomal impairment, like Refsum disease, Zellweger syndrome and α-methylacyl-CoA racemase deficiency. Recent studies elucidated that the toxic activity of phytanic acid and pristanic acid is mediated by multiple mitochondrial dysfunctions, generation of reactive oxygen species and Ca2+ deregulation via the InsP3-Ca2+ signaling pathway in glial cells. However, the exact signaling mechanism through which both fatty acids mediate toxicity is still under debate. Here, we studied the ability of phytanic acid and pristanic acid to activate the free fatty acid receptor GPR40, a G-protein-coupled receptor, which was described to be involved in the Ca2+ signaling of fatty acids. We treated HEK 293 cells expressing the GPR40 receptor with phytanic acid or pristanic acid. This resulted in a significant increase in the intracellular Ca2+ level, similar to the effect seen after treatment with the synthetic GPR40 agonist GW9508. Furthermore, we demonstrate that the GPR40 activation might be due to an interaction of the carboxylate moiety of fatty acids with the receptor. Our findings indicate that the phytanic acid- and pristanic acid-mediated Ca2+ deregulation can involve the activation of GPR40. Therefore, we suppose that activation of GPR40 might be part of the signaling cascade of the toxicity of phytanic and pristanic acids.

  14. Retinoic acid prevents virus-induced airway hyperreactivity and M2 receptor dysfunction via anti-inflammatory and antiviral effects

    OpenAIRE

    Moreno-Vinasco, Liliana; Verbout, Norah G.; Fryer, Allison D.; Jacoby, David B.

    2009-01-01

    Inhibitory M2 muscarinic receptors on airway parasympathetic nerves normally limit acetylcholine release. Viral infections decrease M2 receptor function, increasing vagally mediated bronchoconstriction. Since retinoic acid deficiency causes M2 receptor dysfunction, we tested whether retinoic acid would prevent virus-induced airway hyperreactivity and prevent M2 receptor dysfunction. Guinea pigs infected with parainfluenza virus were hyperreactive to electrical stimulation of the vagus nerves,...

  15. Bile acids, farnesoid X receptor, atherosclerosis and metabolic control

    NARCIS (Netherlands)

    Kuipers, Folkert; Stroeve, Johanna H. M.; Caron, Sandrine; Staels, Bart

    2007-01-01

    Purpose of review Bile acids are amphiphilic molecules synthesized from cholesterol exclusively in the liver that are essential for effective absorption of dietary fat. In addition to this classical role', bile acids act as signalling molecules that control their own metabolism by activating the nuc

  16. Antidepressant-like effects of ascorbic acid and ketamine involve modulation of GABAA and GABAB receptors.

    Science.gov (United States)

    Rosa, Priscila B; Neis, Vivian B; Ribeiro, Camille M; Moretti, Morgana; Rodrigues, Ana Lúcia S

    2016-10-01

    It has been suggested that dysregulation of γ-aminobutyric acid (GABA)-mediated neurotransmission is involved in the etiology of major depressive disorder and in the action of the fast-acting antidepressant ketamine. Considering that recent evidence has suggested that ascorbic acid may exert an antidepressant-like effect through mechanisms similar to ketamine, this study evaluated the involvement of GABAA and GABAB receptors in the antidepressant-like effect of ascorbic acid, comparing the results with those obtained with ketamine. To investigate the involvement of GABAA in the antidepressant-like effect of ascorbic acid and ketamine in the tail suspension test (TST), mice were treated with a sub-effective dose of ascorbic acid (0.1mg/kg, po), ketamine (0.1mg/kg, ip) or vehicle and 30minutes later, a sub-effective dose of muscimol (0.1mg/kg, ip, GABAA receptor agonist) or vehicle was administered. In another set of experiments, mice were treated with ascorbic acid (1mg/kg, po, active dose in the TST) or vehicle and 30minutes later, baclofen (1mg/kg, ip, GABAB receptor agonist) was administered. A similar experimental protocol was performed with ketamine (1mg/kg, ip). The administration of muscimol combined with ascorbic acid or ketamine produced a synergistic antidepressant-like effect in the TST. Moreover, the antidepressant-like effects of ascorbic acid and ketamine were abolished by baclofen. There was no alteration in spontaneous locomotion in any experimental group. Results indicate that the anti-immobility effect of ascorbic acid and ketamine in TST may involve an activation of GABAA receptors and a possible inhibition of GABAB receptors. Copyright © 2016 Institute of Pharmacology, Polish Academy of Sciences. Published by Elsevier Urban & Partner Sp. z o.o. All rights reserved.

  17. Stimulation of acid secretion and phosphoinositol production by rat parietal cell muscarinic M sub 2 receptors

    Energy Technology Data Exchange (ETDEWEB)

    Pfeiffer, A.; Rochlitz, H.; Herz, A.; Paumgartner, G. (Univ. of Munich (West Germany))

    1988-04-01

    The muscarinic receptor system involved in hydrogen production by enriched rat gastric parietal cells was investigated. Muscarinic receptor density determined by (N-methyl-{sup 3}H)scopolamine binding was 8,100/cell. The receptor appeared to be of the M{sub 2} muscarinic receptor subtype, since it had a low affinity (K{sub d} 189 nM) for the M{sub 1} receptor antagonist pirenzepine compared with atropine. Receptor activation by carbachol rapidly augmented levels of polyphosphoinositides, indicating an activation of phospholipase C. The dose-response relations for the increase in inositol phosphates closely paralleled the binding of carbachol to muscarinic receptors. The inositol phosphate response was antagonized by pirenzepine with a K{sub i} of 177 nM. the stimulation of inositol phosphate levels by carbachol correlated well with the stimulation of ({sup 14}C)aminopyrine uptake, determine as an index of acid secretion. The muscarinic agonists oxotremorine, pilocarpine, and bethanechol elicited partial increases in inositol phosphates at maximal drug concentrations, and these partial increases correlated with their ability to stimulate ({sup 14}C)aminopyrine uptake. These data indicate that inositolpolyphosphates may be a second messenger of M{sub 2} receptors stimulating acid secretion.

  18. Role of fatty acid binding proteins and long chain fatty acids in modulating nuclear receptors and gene transcription.

    Science.gov (United States)

    Schroeder, Friedhelm; Petrescu, Anca D; Huang, Huan; Atshaves, Barbara P; McIntosh, Avery L; Martin, Gregory G; Hostetler, Heather A; Vespa, Aude; Landrock, Danilo; Landrock, Kerstin K; Payne, H Ross; Kier, Ann B

    2008-01-01

    Abnormal energy regulation may significantly contribute to the pathogenesis of obesity, diabetes mellitus, cardiovascular disease, and cancer. For rapid control of energy homeostasis, allosteric and posttranslational events activate or alter activity of key metabolic enzymes. For longer impact, transcriptional regulation is more effective, especially in response to nutrients such as long chain fatty acids (LCFA). Recent advances provide insights into how poorly water-soluble lipid nutrients [LCFA; retinoic acid (RA)] and their metabolites (long chain fatty acyl Coenzyme A, LCFA-CoA) reach nuclei, bind their cognate ligand-activated receptors, and regulate transcription for signaling lipid and glucose catabolism or storage: (i) while serum and cytoplasmic LCFA levels are in the 200 mircroM-mM range, real-time imaging recently revealed that LCFA and LCFA-CoA are also located within nuclei (nM range); (ii) sensitive fluorescence binding assays show that LCFA-activated nuclear receptors [peroxisome proliferator-activated receptor-alpha (PPARalpha) and hepatocyte nuclear factor 4alpha (HNF4alpha)] exhibit high affinity (low nM KdS) for LCFA (PPARalpha) and/or LCFA-CoA (PPARalpha, HNF4alpha)-in the same range as nuclear levels of these ligands; (iii) live and fixed cell immunolabeling and imaging revealed that some cytoplasmic lipid binding proteins [liver fatty acid binding protein (L-FABP), acyl CoA binding protein (ACBP), cellular retinoic acid binding protein-2 (CRABP-2)] enter nuclei, bind nuclear receptors (PPARalpha, HNF4alpha, CRABP-2), and activate transcription of genes in fatty acid and glucose metabolism; and (iv) studies with gene ablated mice provided physiological relevance of LCFA and LCFA-CoA binding proteins in nuclear signaling. This led to the hypothesis that cytoplasmic lipid binding proteins transfer and channel lipidic ligands into nuclei for initiating nuclear receptor transcriptional activity to provide new lipid nutrient signaling pathways that

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

    DEFF Research Database (Denmark)

    Milligan, Graeme; Ulven, Trond; Murdoch, Hannah;

    2014-01-01

    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 com...

  20. Mechanisms for the activation of Toll-like receptor 2/4 by saturated fatty acids and inhibition by docosahexaenoic acid.

    Science.gov (United States)

    Hwang, Daniel H; Kim, Jeong-A; Lee, Joo Young

    2016-08-15

    Saturated fatty acids can activate Toll-like receptor 2 (TLR2) and TLR4 but polyunsaturated fatty acids, particularly docosahexaenoic acid (DHA) inhibit the activation. Lipopolysaccharides (LPS) and lipopetides, ligands for TLR4 and TLR2, respectively, are acylated by saturated fatty acids. Removal of these fatty acids results in loss of their ligand activity suggesting that the saturated fatty acyl moieties are required for the receptor activation. X-ray crystallographic studies revealed that these saturated fatty acyl groups of the ligands directly occupy hydrophobic lipid binding domains of the receptors (or co-receptor) and induce the dimerization which is prerequisite for the receptor activation. Saturated fatty acids also induce the dimerization and translocation of TLR4 and TLR2 into lipid rafts in plasma membrane and this process is inhibited by DHA. Whether saturated fatty acids induce the dimerization of the receptors by interacting with these lipid binding domains is not known. Many experimental results suggest that saturated fatty acids promote the formation of lipid rafts and recruitment of TLRs into lipid rafts leading to ligand independent dimerization of the receptors. Such a mode of ligand independent receptor activation defies the conventional concept of ligand induced receptor activation; however, this may enable diverse non-microbial molecules with endogenous and dietary origins to modulate TLR-mediated immune responses. Emerging experimental evidence reveals that TLRs play a key role in bridging diet-induced endocrine and metabolic changes to immune responses.

  1. Role of platelet-derived growth factor/platelet-derived growth factor receptor axis in the trafficking of circulating fibrocytes in pulmonary fibrosis.

    Science.gov (United States)

    Aono, Yoshinori; Kishi, Masami; Yokota, Yuki; Azuma, Momoyo; Kinoshita, Katsuhiro; Takezaki, Akio; Sato, Seidai; Kawano, Hiroshi; Kishi, Jun; Goto, Hisatsugu; Uehara, Hisanori; Izumi, Keisuke; Nishioka, Yasuhiko

    2014-12-01

    Circulating fibrocytes have been reported to migrate into the injured lungs, and contribute to fibrogenesis via CXCL12-CXCR4 axis. In contrast, we report that imatinib mesylate prevented bleomycin (BLM)-induced pulmonary fibrosis in mice by inhibiting platelet-derived growth factor receptor (PDGFR), even when it was administered only in the early phase. The goal of this study was to test the hypothesis that platelet-derived growth factor (PDGF) might directly contribute to the migration of fibrocytes to the injured lungs. PDGFR expression in fibrocytes was examined by flow cytometry and RT-PCR. The migration of fibrocytes was evaluated by using a chemotaxis assay for human fibrocytes isolated from peripheral blood. The numbers of fibrocytes triple-stained for CD45, collagen-1, and CXCR4 were also examined in lung digests of BLM-treated mice. PDGFR mRNA levels in fibrocytes isolated from patients with idiopathic pulmonary fibrosis were investigated by real-time PCR. Fibrocytes expressed both PDGFR-α and -β, and migrated in response to PDGFs. PDGFR inhibitors (imatinib, PDGFR-blocking antibodies) suppressed fibrocyte migration in vitro, and reduced the number of fibrocytes in the lungs of BLM-treated mice. PDGF-BB was a stronger chemoattractant than the other PDGFs in vitro, and anti-PDGFR-β-blocking antibody decreased the numbers of fibrocytes in the lungs compared with anti-PDGFR-α antibody in vivo. Marked expression of PDGFR-β was observed in fibrocytes from patients with idiopathic pulmonary fibrosis compared with healthy subjects. These results suggest that PDGF directly functions as a strong chemoattractant for fibrocytes. In particular, the PDGF-BB-PDGFR-β biological axis might play a critical role in fibrocyte migration into the fibrotic lungs.

  2. HUMAN TRAFFICKING DRUG TRAFFICKING, AND THE DEATH PENALTY

    Directory of Open Access Journals (Sweden)

    Felicity Gerry

    2016-12-01

    Full Text Available Both Australia and Indonesia have made commitments to combatting human trafficking.  Through the experience of Mary Jane Veloso it can be seen that it is most often the vulnerable ‘mule’ that is apprehended by law enforcement and not the powerful leaders of crime syndicates. It is unacceptable that those vulnerable individuals may face execution for acts committed under threat of force, coercion, fraud, deception or abuse of power. For this reason it is vital that a system of victim identification is developed, including better training for law enforcement, legal representatives and members of the judiciary. This paper builds on submissions by authors for Australian Parliamentary Inquiry into Human Trafficking, and focusses on issues arising in the complex cross section of human trafficking, drug trafficking, and the death penalty with particular attention on identifying victims and effective reporting mechanisms in both Australia and Indonesia. It concludes that, in the context of human trafficking both countries could make three main improvements to law and policy, among others, 1 enactment of laws that create clear mandatory protection for human trafficking victims; 2 enactment of criminal laws that provides complete defence for victim of human trafficking; 3 enactment of corporate reporting mechanisms. Systemic protection and support is not sufficiently available without clear legislative protection as this paper suggests together with standardised referral mechanisms and effective financial reporting mechanisms. The implementation can be achieved through collaborative responses and inter-agency coordination with data collection and properly trained specialists.

  3. Unbinding of Retinoic Acid from its Receptor Studied by Steered Molecular Dynamics

    CERN Document Server

    Kosztin, D; Schulten, K; Kosztin, Dorina; Izrailev, Sergei; Schulten, Klaus

    1999-01-01

    Retinoic acid receptor (RAR) is a ligand-dependent transcription factor that regulates the expression of genes involved in cell growth, differentiation, and development. Binding of the retinoic acid hormone to RAR is accompanied by conformational changes in the protein which induce transactivation or transrepression of the target genes. In this paper we present a study of the hormone binding/unbinding process in order to clarify the role of some of the amino acid contacts and identify possible pathways of the all-trans retinoic acid binding/unbinding to/from human retinoic acid receptor (hRAR)-g. Three possible pathways were explored using steered molecular dynamics simulations. Unbinding was induced on a time scale of 1 ns by applying external forces to the hormone. The simulations suggest that the hormone may employ one pathway for binding and an alternative "back door" pathway for unbinding.

  4. Free fatty acid receptor GPR120 and pathogenesis of obesity and type 2 diabetes mellitus.

    Science.gov (United States)

    Mo, Xiu-Lei; Wei, Hong-Kui; Peng, Jian; Tao, Ya-Xiong

    2013-01-01

    G protein-coupled receptor 120 (GPR120) was initially identified as an orphan receptor through mining the human genome databases. In 2005, GPR120 was deorphanized and shown to be a receptor for long-chain free fatty acids. GPR120 regulates various physiological processes, including gut hormone secretion, islet function, food preference, osteoclastogenesis, anti-inflammation, adipogenesis, and appetite control. Recently, a human genetic study conducted in European populations identified a loss-of-function GPR120 mutation associated with obesity and insulin resistance. Therefore, GPR120, the sensing receptor for long-chain free fatty acids, represents a novel drug target for the treatment of obesity and diabetes. Copyright © 2013 Elsevier Inc. All rights reserved.

  5. Nucleic acid sensing pattern recognition receptors in the development of colorectal cancer and colitis.

    Science.gov (United States)

    He, Liangmei; Chen, Yayun; Wu, Yuanbing; Xu, Ying; Zhang, Zixiang; Liu, Zhiping

    2017-07-01

    Colorectal cancer (CRC) is a leading cause of cancer-related deaths that is often associated with inflammation initiated by activation of pattern recognition receptors (PRRs). Nucleic acid sensing PRRs are one of the major subsets of PRRs that sense nucleic acid (DNA and RNA), mainly including some members of Toll-like receptors (TLR3, 7, 8, 9), AIM2-like receptors (AIM2, IFI16), STING, cGAS, RNA polymerase III, and DExD/H box nucleic acid helicases (such as RIG-I like receptors (RIG-I, MDA5, LPG2), DDX1, 3, 5, 7, 17, 21, 41, 60, and DHX9, 36). Activation of these receptors eventually leads to the release of cytokines and activation of immune cells, which are well known to play crucial roles in host defense against intracellular bacterial and virus infection. However, the functions of these nucleic acid sensing PRRs in the other diseases such as CRC and colitis remain largely unknown. Recent studies indicated that nucleic acid sensing PRRs contribute to CRC and/or colitis development, and therapeutic modulation of nucleic acid sensing PRRs may reduce the risk of CRC development. However, until now, a comprehensive review on the role of nucleic acid sensing PRRs in CRC and colitis is still lacking. This review provided an overview of the roles as well as the mechanisms of these nucleic acid sensing PRRs (AIM2, STING, cGAS, RIG-I and its downstream molecules, DDX3, 5, 6,17, and DHX9, 36) in CRC and colitis, which may aid the diagnosis, therapy, and prognostic prediction of CRC and colitis.

  6. Bile acid-induced arrhythmia is mediated by muscarinic M2 receptors in neonatal rat cardiomyocytes.

    Directory of Open Access Journals (Sweden)

    Siti H Sheikh Abdul Kadir

    Full Text Available BACKGROUND: Intrahepatic cholestasis of pregnancy (ICP is a common disease affecting up to 5% of pregnancies and which can cause fetal arrhythmia and sudden intrauterine death. We previously demonstrated that bile acid taurocholate (TC, which is raised in the bloodstream of ICP, can acutely alter the rate and rhythm of contraction and induce abnormal calcium destabilization in cultured neonatal rat cardiomyocytes (NRCM. Apart from their hepatic functions bile acids are ubiquitous signalling molecules with diverse systemic effects mediated by either the nuclear receptor FXR or by a recently discovered G-protein coupled receptor TGR5. We aim to investigate the mechanism of bile-acid induced arrhythmogenic effects in an in-vitro model of the fetal heart. METHODS AND RESULTS: Levels of bile acid transporters and nuclear receptor FXR were studied by quantitative real time PCR, western blot and immunostaining, which showed low levels of expression. We did not observe functional involvement of the canonical receptors FXR and TGR5. Instead, we found that TC binds to the muscarinic M(2 receptor in NRCM and serves as a partial agonist of this receptor in terms of inhibitory effect on intracellular cAMP and negative chronotropic response. Pharmacological inhibition and siRNA-knockdown of the M(2 receptor completely abolished the negative effect of TC on contraction, calcium transient amplitude and synchronisation in NRCM clusters. CONCLUSION: We conclude that in NRCM the TC-induced arrhythmia is mediated by the partial agonism at the M(2 receptor. This mechanism might serve as a promising new therapeutic target for fetal arrhythmia.

  7. Treatment of type 2 diabetes by free Fatty Acid receptor agonists

    DEFF Research Database (Denmark)

    Watterson, Kenneth R; Hudson, Brian D; Ulven, Trond

    2014-01-01

    Dietary free fatty acids (FFAs), such as ω-3 fatty acids, regulate metabolic and anti-inflammatory processes, with many of these effects attributed to FFAs interacting with a family of G protein-coupled receptors. Selective synthetic ligands for free fatty acid receptors (FFA1-4) have consequently...... been developed as potential treatments for type 2 diabetes (T2D). In particular, clinical studies show that Fasiglifam, an agonist of the long-chain FFA receptor, FFA1, improved glycemic control and reduced HbA1c levels in T2D patients, with a reduced risk of hypoglycemia. However, this ligand...... was removed from clinical trials due to potential liver toxicity and determining if this is a target or a ligand-specific feature is now of major importance. Pre-clinical studies also show that FFA4 agonism increases insulin sensitivity, induces weight loss, and reduces inflammation and the metabolic and anti...

  8. Imaging Cancer Cells Expressing the Folate Receptor with Carbon Dots Produced from Folic Acid.

    Science.gov (United States)

    Bhunia, Susanta Kumar; Maity, Amit Ranjan; Nandi, Sukhendu; Stepensky, David; Jelinek, Raz

    2016-04-01

    Development of new imaging tools for cancer cells in vitro and in vitro is important for advancing cancer research, elucidating drug effects upon cancer cells, and studying cellular processes. We showed that fluorescent carbon dots (C-dots) synthesized from folic acid can serve as an effective vehicle for imaging cancer cells expressing the folate receptor on their surface. The C-dots, synthesized through a simple one-step process from folic acid as the carbon source, exhibited selectivity towards cancer cells displaying the folate receptor, making such cells easily distinguishable in fluorescence microscopy imaging. Biophysical measurements and competition experiments both confirmed the specific targeting and enhanced uptake of C-dots by the folate receptor-expressing cells. The folic acid-derived C-dots were not cytotoxic, and their use in bioimaging applications could aid biological studies of cancer cells, identification of agonists/antagonists, and cancer diagnostics.

  9. Expression of functional receptors by the human γ-aminobutyric acid A γ2 subunit

    Science.gov (United States)

    Martínez-Torres, Ataúlfo; Miledi, Ricardo

    2004-01-01

    γ-Aminobutyric acid A (GABAA) receptors are heteromeric membrane proteins formed mainly by various combinations of α, β, and γ subunits; and it is commonly thought that the γ2 subunit alone does not form functional receptors. In contrast, we found that cDNA encoding the γ2L subunit of the human GABAA receptor, injected alone into Xenopus oocytes, expressed functional GABA receptors whose properties were investigated by using the two-microelectrode voltage-clamp technique. GABA elicited desensitizing membrane currents that recovered after a few minutes' wash. Repetitive applications of GABA induced a “run-up” of GABA currents that nearly doubled the amplitude of the first response. The GABA currents inverted direction at about -30 mV, indicating that they are carried mainly by Cl- ions. The homomeric γ2L receptors were also activated by β-alanine > taurine > glycine, and, like some types of heteromeric GABAA receptors, the γ2L receptors were blocked by bicuculline and were potentiated by pentobarbital and flunitrazepam. These results indicate that the human γ2L subunit is capable of forming fully functional GABA receptors by itself in Xenopus oocytes and suggest that the roles proposed for the various subunits that make up the heteromeric GABAA receptors in situ require further clarification. PMID:14981251

  10. Optical control of trimeric P2X receptors and acid-sensing ion channels.

    Science.gov (United States)

    Browne, Liam E; Nunes, João P M; Sim, Joan A; Chudasama, Vijay; Bragg, Laricia; Caddick, Stephen; North, R Alan

    2014-01-07

    P2X receptors are trimeric membrane proteins that function as ion channels gated by extracellular ATP. We have engineered a P2X2 receptor that opens within milliseconds by irradiation at 440 nm, and rapidly closes at 360 nm. This requires bridging receptor subunits via covalent attachment of 4,4'-bis(maleimido)azobenzene to a cysteine residue (P329C) introduced into each second transmembrane domain. The cis-trans isomerization of the azobenzene pushes apart the outer ends of the transmembrane helices and opens the channel in a light-dependent manner. Light-activated channels exhibited similar unitary currents, rectification, calcium permeability, and dye uptake as P2X2 receptors activated by ATP. P2X3 receptors with an equivalent mutation (P320C) were also light sensitive after chemical modification. They showed typical rapid desensitization, and they could coassemble with native P2X2 subunits in pheochromocytoma cells to form light-activated heteromeric P2X2/3 receptors. A similar approach was used to open and close human acid-sensing ion channels (ASICs), which are also trimers but are unrelated in sequence to P2X receptors. The experiments indicate that the opening of the permeation pathway requires similar and substantial movements of the transmembrane helices in both P2X receptors and ASICs, and the method will allow precise optical control of P2X receptors or ASICs in intact tissues.

  11. Trafficking and cell migration.

    Science.gov (United States)

    Ulrich, Florian; Heisenberg, Carl-Philipp

    2009-07-01

    The migration of single cells and epithelial sheets is of great importance for gastrulation and organ formation in developing embryos and, if misregulated, can have dire consequences e.g. during cancer metastasis. A keystone of cell migration is the regulation of adhesive contacts, which are dynamically assembled and disassembled via endocytosis. Here, we discuss some of the basic concepts about the function of endocytic trafficking during cell migration: transport of integrins from the cell rear to the leading edge in fibroblasts; confinement of signalling to the front of single cells by endocytic transport of growth factors; regulation of movement coherence in multicellular sheets by cadherin turnover; and shaping of extracellular chemokine gradients. Taken together, endocytosis enables migrating cells and tissues to dynamically modulate their adhesion and signalling, allowing them to efficiently migrate through their extracellular environment.

  12. N-Hydroxypyrazolyl glycine derivatives as selective N-methyl-D-aspartic acid receptor ligands

    DEFF Research Database (Denmark)

    Clausen, Rasmus Prætorius; Christensen, Caspar; Hansen, Kasper Bø;

    2008-01-01

    A series of analogues based on N-hydroxypyrazole as a bioisostere for the distal carboxylate group of aspartate have been designed, synthesized, and pharmacologically characterized. Affinity studies on the major glutamate receptor subgroups show that these 4-substituted N-hydroxypyrazol-5-yl...... glycine (NHP5G) derivatives are selectively recognized by N-methyl- d-aspartic acid (NMDA) receptors and that the ( R)-enantiomers are preferred. Moreover, several of the compounds are able to discriminate between individual subtypes among the NMDA receptors, providing new pharmacological tools...

  13. 溶血磷脂酸受体2%Research progress on lysophosphatidic acid receptor 2

    Institute of Scientific and Technical Information of China (English)

    龚晓华; 饶勇; 马卫列; 张志珍

    2013-01-01

    Lysophosphatidic acid receptor 2 (LPA2), also known as endothelial differentiation gene receptor 4 (EDG4), is a G-protein coupled receptor (GPCR) activated by lysophosphatidic acid. It has high affinity to lysophosphatidic acid, and can mediate many kinds of cellular activities. Recent researches have shown that LPA2/EDG4 is overexpressed in ovarian cancer cells. In addition, it is closely connected with breast carcinoma, colon cancer, atherosclerosis, respiratory disease and hypertensivedisorder complicating pregnancy.%溶血磷脂酸受体2 (lysophosphatidic acid receptor 2,LPA2),也称内皮分化基因受体4(endothelial differentiation gene receptor4,EDG4),是溶血磷脂酸G蛋白偶联受体类的一种,对溶血磷脂酸有较高亲和力,可介导多种细胞活动.近年研究发现,LPA2/EDG4在卵巢癌细胞中过表达,同时与乳腺癌、结直肠癌、动脉粥样硬化、呼吸道疾病、妊娠性高血压等有着密切关系.

  14. Clinical relevance of the bile acid receptor TGR5 in metabolism

    DEFF Research Database (Denmark)

    van Nierop, F Samuel; Scheltema, Matthijs J; Eggink, Hannah M

    2016-01-01

    The bile acid receptor TGR5 (also known as GPBAR1) is a promising target for the development of pharmacological interventions in metabolic diseases, including type 2 diabetes, obesity, and non-alcoholic steatohepatitis. TGR5 is expressed in many metabolically active tissues, but complex enterohep......The bile acid receptor TGR5 (also known as GPBAR1) is a promising target for the development of pharmacological interventions in metabolic diseases, including type 2 diabetes, obesity, and non-alcoholic steatohepatitis. TGR5 is expressed in many metabolically active tissues, but complex...

  15. Natural and synthetic sialic acid-containing inhibitors of influenza virus receptor binding.

    Science.gov (United States)

    Matrosovich, Mikhail; Klenk, Hans-Dieter

    2003-01-01

    Influenza viruses attach to susceptible cells via multivalent interactions of their haemagglutinins with sialyloligosaccharide moieties of cellular glycoconjugates. Soluble macromolecules containing sialic acid from animal sera and mucosal fluids can act as decoy receptors and competitively inhibit virus-mediated haemagglutination and infection. Although a role for these natural inhibitors in the innate anti-influenza immunity is still not clear, studies are in progress on the design of synthetic sialic acid-containing inhibitors of receptor binding which could be used as anti-influenza drugs.

  16. Boronic acids as probes for investigation of allosteric modulation of the chemokine receptor CXCR3.

    Science.gov (United States)

    Bernat, Viachaslau; Admas, Tizita Haimanot; Brox, Regine; Heinemann, Frank W; Tschammer, Nuska

    2014-11-21

    The chemokine receptor CXCR3 is a G protein-coupled receptor, which conveys extracellular signals into cells by changing its conformation upon agonist binding. To facilitate the mechanistic understanding of allosteric modulation of CXCR3, we combined computational modeling with the synthesis of novel chemical tools containing boronic acid moiety, site-directed mutagenesis, and detailed functional characterization. The design of boronic acid derivatives was based on the predictions from homology modeling and docking. The choice of the boronic acid moiety was dictated by its unique ability to interact with proteins in a reversible covalent way, thereby influencing conformational dynamics of target biomolecules. During the synthesis of the library we have developed a novel approach for the purification of drug-like boronic acids. To validate the predicted binding mode and to identify amino acid residues responsible for the transduction of signal through CXCR3, we conducted a site-directed mutagenesis study. With the use of allosteric radioligand RAMX3 we were able to establish the existence of a second allosteric binding pocket in CXCR3, which enables different binding modes of structurally closely related allosteric modulators of CXCR3. We have also identified residues Trp109(2.60) and Lys300(7.35) inside the transmembrane bundle of the receptor as crucial for the regulation of the G protein activation. Furthermore, we report the boronic acid 14 as the first biased negative allosteric modulator of the receptor. Overall, our data demonstrate that boronic acid derivatives represent an outstanding tool for determination of key receptor-ligand interactions and induction of ligand-biased signaling.

  17. Regulation of expression of citrate synthase by the retinoic acid receptor-related orphan receptor α (RORα.

    Directory of Open Access Journals (Sweden)

    Christine Crumbley

    Full Text Available The retinoic acid receptor-related orphan receptor α (RORα is a member of the nuclear receptor superfamily of transcription factors that plays an important role in regulation of the circadian rhythm and metabolism. Mice lacking a functional RORα display a range of metabolic abnormalities including decreased serum cholesterol and plasma triglycerides. Citrate synthase (CS is a key enzyme of the citric acid cycle that provides energy for cellular function. Additionally, CS plays a critical role in providing citrate derived acetyl-CoA for lipogenesis and cholesterologenesis. Here, we identified a functional RORα response element (RORE in the promoter of the CS gene. ChIP analysis demonstrates RORα occupancy of the CS promoter and a putative RORE binds to RORα effectively in an electrophoretic mobility shift assay and confers RORα responsiveness to a reporter gene in a cotransfection assay. We also observed a decrease in CS gene expression and CS enzymatic activity in the staggerer mouse, which has a mutation of in the Rora gene resulting in nonfunctional RORα protein. Furthermore, we found that SR1001 a RORα inverse agonist eliminated the circadian pattern of expression of CS mRNA in mice. These data suggest that CS is a direct RORα target gene and one mechanism by which RORα regulates lipid metabolism is via regulation of CS expression.

  18. Illicit Trafficking of Natural Radionuclides

    Science.gov (United States)

    Friedrich, Steinhäusler; Lyudmila, Zaitseva

    2008-08-01

    Natural radionuclides have been subject to trafficking worldwide, involving natural uranium ore (U 238), processed uranium (yellow cake), low enriched uranium (20% U 235), radium (Ra 226), polonium (Po 210), and natural thorium ore (Th 232). An important prerequisite to successful illicit trafficking activities is access to a suitable logistical infrastructure enabling an undercover shipment of radioactive materials and, in case of trafficking natural uranium or thorium ore, capable of transporting large volumes of material. Covert en route diversion of an authorised uranium transport, together with covert diversion of uranium concentrate from an operating or closed uranium mines or mills, are subject of case studies. Such cases, involving Israel, Iran, Pakistan and Libya, have been analyzed in terms of international actors involved and methods deployed. Using international incident data contained in the Database on Nuclear Smuggling, Theft and Orphan Radiation Sources (DSTO) and international experience gained from the fight against drug trafficking, a generic Trafficking Pathway Model (TPM) is developed for trafficking of natural radionuclides. The TPM covers the complete trafficking cycle, ranging from material diversion, covert material transport, material concealment, and all associated operational procedures. The model subdivides the trafficking cycle into five phases: (1) Material diversion by insider(s) or initiation by outsider(s); (2) Covert transport; (3) Material brokerage; (4) Material sale; (5) Material delivery. An Action Plan is recommended, addressing the strengthening of the national infrastructure for material protection and accounting, development of higher standards of good governance, and needs for improving the control system deployed by customs, border guards and security forces.

  19. Nuclear hormone receptor NHR-49 controls fat consumption and fatty acid composition in C. elegans.

    Directory of Open Access Journals (Sweden)

    Marc R Van Gilst

    2005-02-01

    Full Text Available Mammalian nuclear hormone receptors (NHRs, such as liver X receptor, farnesoid X receptor, and peroxisome proliferator-activated receptors (PPARs, precisely control energy metabolism. Consequently, these receptors are important targets for the treatment of metabolic diseases, including diabetes and obesity. A thorough understanding of NHR fat regulatory networks has been limited, however, by a lack of genetically tractable experimental systems. Here we show that deletion of the Caenorhabditis elegans NHR gene nhr-49 yielded worms with elevated fat content and shortened life span. Employing a quantitative RT-PCR screen, we found that nhr-49 influenced the expression of 13 genes involved in energy metabolism. Indeed, nhr-49 served as a key regulator of fat usage, modulating pathways that control the consumption of fat and maintain a normal balance of fatty acid saturation. We found that the two phenotypes of the nhr-49 knockout were linked to distinct pathways and were separable: The high-fat phenotype was due to reduced expression of enzymes in fatty acid beta-oxidation, and the shortened adult life span resulted from impaired expression of a stearoyl-CoA desaturase. Despite its sequence relationship with the mammalian hepatocyte nuclear factor 4 receptor, the biological activities of nhr-49 were most similar to those of the mammalian PPARs, implying an evolutionarily conserved role for NHRs in modulating fat consumption and composition. Our findings in C. elegans provide novel insights into how NHR regulatory networks are coordinated to govern fat metabolism.

  20. Comparative distribution of human and avian type sialic acid influenza receptors in the pig

    Directory of Open Access Journals (Sweden)

    Perez Belinda

    2010-01-01

    Full Text Available Abstract Background A major determinant of influenza infection is the presence of virus receptors on susceptible host cells to which the viral haemagglutinin is able to bind. Avian viruses preferentially bind to sialic acid α2,3-galactose (SAα2,3-Gal linked receptors, whereas human strains bind to sialic acid α2,6-galactose (SAα2,6-Gal linked receptors. To date, there has been no detailed account published on the distribution of SA receptors in the pig, a model host that is susceptible to avian and human influenza subtypes, thus with potential for virus reassortment. We examined the relative expression and spatial distribution of SAα2,3-GalG(1-3GalNAc and SAα2,6-Gal receptors in the major organs from normal post-weaned pigs by binding with lectins Maackia amurensis agglutinins (MAA II and Sambucus nigra agglutinin (SNA respectively. Results Both SAα2,3-Gal and SAα2,6-Gal receptors were extensively detected in the major porcine organs examined (trachea, lung, liver, kidney, spleen, heart, skeletal muscle, cerebrum, small intestine and colon. Furthermore, distribution of both SA receptors in the pig respiratory tract closely resembled the published data of the human tract. Similar expression patterns of SA receptors between pig and human in other major organs were found, with exception of the intestinal tract. Unlike the limited reports on the scarcity of influenza receptors in human intestines, we found increasing presence of SAα2,3-Gal and SAα2,6-Gal receptors from duodenum to colon in the pig. Conclusions The extensive presence of SAα2,3-Gal and SAα2,6-Gal receptors in the major organs examined suggests that each major organ may be permissive to influenza virus entry or infection. The high similarity of SA expression patterns between pig and human, in particular in the respiratory tract, suggests that pigs are not more likely to be potential hosts for virus reassortment than humans. Our finding of relative abundance of SA receptors

  1. Role of LIMP-2 in the intracellular trafficking of β-glucosidase in different human cellular models.

    Science.gov (United States)

    Malini, Erika; Zampieri, Stefania; Deganuto, Marta; Romanello, Milena; Sechi, Annalisa; Bembi, Bruno; Dardis, Andrea

    2015-09-01

    Acid β-glucosidase (GCase), the enzyme deficient in Gaucher disease (GD), is transported to lysosomes by the lysosomal integral membrane protein (LIMP)-2. In humans, LIMP-2 deficiency leads to action myoclonus-renal failure (AMRF) syndrome. GD and AMRF syndrome share some clinical features. However, they are different from clinical and biochemical points of view, suggesting that the role of LIMP-2 in the targeting of GCase would be different in different tissues. Besides, the role of LIMP-2 in the uptake and trafficking of the human recombinant (hr)GCase used in the treatment of GD is unknown. Thus, we compared GCase activity and intracellular localization in immortalized lymphocytes, fibroblasts, and a neuronal model derived from multipotent adult stem cells, from a patient with AMRF syndrome, patients with GD, and control subjects. In fibroblasts and neuronlike cells, GCase targeting to the lysosomes is completely dependent on LIMP-2, whereas in blood cells, GCase is partially targeted to lysosomes by a LIMP-2-independent mechanism. Although hrGCase cellular uptake is independent of LIMP-2, its trafficking to the lysosomes is mediated by this receptor. These data provide new insights into the mechanisms involved in the intracellular trafficking of GCase and in the pathogeneses of GD and AMRF syndrome.

  2. Sex trafficking in South Asia.

    Science.gov (United States)

    Huda, S

    2006-09-01

    Economic and social inequalities and political conflicts have led to the movement of persons within each country and across the borders in South Asia. Globalization has encouraged free mobility of capital, technology, experts and sex tourism. Illiteracy, dependency, violence, social stigma, cultural stereotypes, gender disparity and endemic poverty, among other factors, place women and children in powerless, non-negotiable situations that have contributed to the emergence and breeding of the cavernous problem of sex trafficking in the entire region. This alarming spread of sex trafficking has fuelled the spread of HIV infection in South Asia, posing a unique and serious threat to community health, poverty alleviation and other crucial aspects of human development. Although the SAARC (South Asian Association for Regional Cooperation) Convention on Trafficking in Women and Children has been an important breakthrough, most of the countries in the region do not have anti-trafficking legislation or means to protect the victims. Countries of the region should make a concerted effort to treat trafficking victims as "victims" of human rights violations in all anti-trafficking strategies and actions.

  3. Appearance and cellular distribution of lectin-like receptors for alpha 1-acid glycoprotein in the developing rat testis

    DEFF Research Database (Denmark)

    Andersen, U O; Bøg-Hansen, T C; Kirkeby, S

    1996-01-01

    glycoprotein glycoforms to their receptors is inhibited by steroids. Testosterone, oestradiol and progesterone inhibited the binding of alpha 1-acid glycoprotein glycoform A to its receptor. Cortisone, aldosterone, oestradiol and progesterone inhibited the binding of alpha 1-acid glycoprotein glycoforms B...

  4. Zonal variation in the distribution of an alpha 1-acid glycoprotein glycoform receptor in human adrenal cortex

    DEFF Research Database (Denmark)

    Andersen, U O; Bøg-Hansen, T C; Kirkeby, S

    1999-01-01

    specific receptor. The binding of alpha 1-acid glycoprotein glycoform B and alpha 1-acid glycoprotein glycoform C to the glycoform specific receptor is inhibited by the steroid hormones cortisone, aldosterone, estradiol and progesterone but not by testosterone. The pronounced changes in the distribution...

  5. Extracellular loop 2 of the free Fatty Acid receptor 2 mediates allosterism of a phenylacetamide ago-allosteric modulator

    DEFF Research Database (Denmark)

    Smith, Nicola J; Ward, Richard J; Stoddart, Leigh A;

    2011-01-01

    Allosteric agonists are powerful tools for exploring the pharmacology of closely related G protein-coupled receptors that have nonselective endogenous ligands, such as the short chain fatty acids at free fatty acid receptors 2 and 3 (FFA2/GPR43 and FFA3/GPR41, respectively). We explored the molec...

  6. Excitatory amino acid neurotoxicity and modulation of glutamate receptor expression in organotypic brain slice cultures

    DEFF Research Database (Denmark)

    Zimmer, J; Kristensen, Bjarne Winther; Jakobsen, B

    2000-01-01

    Using organotypic slice cultures of hippocampus and cortex-striatum from newborn to 7 day old rats, we are currently studying the excitotoxic effects of kainic acid (KA), AMPA and NMDA and the neuroprotective effects of glutamate receptor blockers, like NBQX. For detection and quantitation of the...

  7. Rac activation by lysophosphatidic acid LPA1 receptors through the guanine nucleotide exchange factor Tiam1

    NARCIS (Netherlands)

    Van Leeuwen, Frank N; Olivo, Cristina; Grivell, Shula; Giepmans, Ben N G; Collard, John G; Moolenaar, Wouter H

    2003-01-01

    Lysophosphatidic acid (LPA) is a serum-borne phospholipid that activates its own G protein-coupled receptors present in numerous cell types. In addition to stimulating cell proliferation, LPA also induces cytoskeletal changes and promotes cell migration in a RhoA- and Rac-dependent manner. Whereas R

  8. Treatment of Type 2 Diabetes by Free Fatty Acid Receptor Agonists

    Science.gov (United States)

    Watterson, Kenneth R.; Hudson, Brian D.; Ulven, Trond; Milligan, Graeme

    2014-01-01

    Dietary free fatty acids (FFAs), such as ω-3 fatty acids, regulate metabolic and anti-inflammatory processes, with many of these effects attributed to FFAs interacting with a family of G protein-coupled receptors. Selective synthetic ligands for free fatty acid receptors (FFA1-4) have consequently been developed as potential treatments for type 2 diabetes (T2D). In particular, clinical studies show that Fasiglifam, an agonist of the long-chain FFA receptor, FFA1, improved glycemic control and reduced HbA1c levels in T2D patients, with a reduced risk of hypoglycemia. However, this ligand was removed from clinical trials due to potential liver toxicity and determining if this is a target or a ligand-specific feature is now of major importance. Pre-clinical studies also show that FFA4 agonism increases insulin sensitivity, induces weight loss, and reduces inflammation and the metabolic and anti-inflammatory effects of short chain fatty acids (SCFAs) are linked with FFA2 and FFA3 activation. In this review, we therefore show that FFA receptor agonism is a potential clinical target for T2D treatment and discuss ongoing drug development programs within industry and academia aimed at improving the safety and effectiveness of these potential treatments. PMID:25221541

  9. Excitatory amino acid receptors in the ventral tegmental area regulate dopamine release in the ventral striatum

    NARCIS (Netherlands)

    Karreman, M; Westerink, BHC; Moghaddam, B

    1996-01-01

    The role of excitatory amino acid (EAA) receptors located in the ventral tegmental area (VTA) in tonic and phasic regulation of dopamine release in the ventral striatum was investigated. Microdialysis in conscious rats was used to assess dopamine release primarily from the nucleus accumbens shell re

  10. Design and synthesis of novel tweezer anion receptors based on deoxycholic acid

    Institute of Scientific and Technical Information of China (English)

    Xing Li Liu; Zhi Gang Zhao; Shu Hua Chen

    2007-01-01

    A novel type of molecular tweezer receptors based on deoxycholic acid has been designed and synthesized and their binding properties were examined by UV-vis spectral titration. These molecular tweezers showed a high selectivity toward F- over Cl-,Br-, I-, AcO-, H2PO4-.

  11. The iron-chelating agent picolinic acid enhances transferrin receptors expression in human erythroleukaemic cell lines.

    Science.gov (United States)

    Testa, U; Louache, F; Titeux, M; Thomopoulos, P; Rochant, H

    1985-07-01

    Picolinic acid, a metal chelating molecule, was administered to human erythroleukaemic cell lines (K 562 and HEL) that were grown in serum-containing media. Picolinic acid inhibited both iron uptake and cell growth. Furthermore, picolinic acid was shown to markedly decrease the level of ferritin in the cells. In spite of the inhibition of cell growth, picolinic acid induced a marked increase in the transferrin-binding capacity of the cells. This phenomenon was due to a two-five-fold enhancement of the rate of transferrin receptor biosynthesis. Other iron-chelating compounds, capable of reducing the level of intracellular iron, also elicited a marked enhancement of the transferrin-binding capacity of the cells. However, the addition of iron, as ferric ammonium citrate, in the culture medium elicited a marked increase in the level of ferritin and a strong decrease in the transferrin-binding capacity of the cells. On the basis of these data we propose that a feed-back mechanism is involved in the regulation of transferrin receptors: when the cells accumulate iron they decrease the number of transferrin receptors in order to prevent further accumulation of iron; when no or low iron is available to the cells, the number of transferrin receptors markedly increases as a compensatory mechanism.

  12. Regulation of bile acid synthesis by the nuclear receptor Rev-erb alpha

    NARCIS (Netherlands)

    Duez, Helene; Van Der Veen, Jelske N.; Duhem, Christian; Pourcet, Benoit; Touvier, Thierry; Fontaine, Coralie; Derudas, Bruno; Bauge, Eric; Havinga, Rick; Bloks, Vincent W.; Wolters, Henk; Van Der Sluijs, Fjodor H.; Vennstrom, Bjorn; Kuipers, Folkert; Staels, Bart

    2008-01-01

    Background & Aims: Conversion into bile acids represents an important route to remove excess cholesterol from the body. Rev-erb alpha is a nuclear receptor that participates as one of the clock genes in the control of circadian rhythmicity and plays a regulatory role in lipid metabolism and adipogen

  13. Identification of diarylsulfonamides as agonists of the free fatty acid receptor 4 (FFA4/GPR120).

    Science.gov (United States)

    Sparks, Steven M; Chen, Grace; Collins, Jon L; Danger, Dana; Dock, Steven T; Jayawickreme, Channa; Jenkinson, Stephen; Laudeman, Christopher; Leesnitzer, M Anthony; Liang, Xi; Maloney, Patrick; McCoy, David C; Moncol, David; Rash, Vincent; Rimele, Thomas; Vulimiri, Padmaja; Way, James M; Ross, Sean

    2014-07-15

    The exploration of a diarylsulfonamide series of free fatty acid receptor 4 (FFA4/GPR120) agonists is described. This work led to the identification of selective FFA4 agonist 8 (GSK137647A) and selective FFA4 antagonist 39. The in vitro profile of compounds 8 and 39 is presented herein.

  14. Myeloid differentiation and retinoblastoma phosphorylation changes in HL-60 cells induced by retinoic acid receptor- and retinoid X receptor-selective retinoic acid analogs.

    Science.gov (United States)

    Brooks, S C; Kazmer, S; Levin, A A; Yen, A

    1996-01-01

    The ability of subtypes of retinoic acid receptors (RARs) and retinoid X receptors (RXRs) singly and in combination to elicit myeloid differentiation, G1/0-specific growth arrest, and retinoblastoma (RB) tumor suppressor protein dephosphorylation was determined in the human myeloblastic leukemia cell line HL-60 using subtype-selective retinoic acid (RA) analogs. RA analogs that selectively bind only to RARs (Am580 and/or TTNPB) or to RXRs (Ro 25-6603, SR11237, and/or SR11234) did not elicit the above-mentioned three cellular responses. In contrast, simultaneous treatment with both an RAR-selective ligand (Am580 or TTNPB) and an RXR-selective ligand (Ro 25-6603, SR11237, or SR11234) induced all three cellular processes. An RAR alpha-selective ligand used with an RXR-selective ligand generated the same responses as did all-trans RA or 9-cis RA, which affect both families of receptors, suggesting an important role for RAR alpha among RAR subtypes in eliciting cellular response. Consistent with this finding, the RAR alpha antagonist, Ro 41-5253, reduced the level of the cellular responses elicited by treatment with an RAR alpha-selective ligand plus RXR-selective ligand. The coupling of the shift of RB to its hypophosphorylated form with G1/0 arrest and differentiation in response to ligands is consistent with a possible role of RB as a downstream target or effector of RAR alpha and RXR in combination.

  15. To discuss illicit nuclear trafficking

    Energy Technology Data Exchange (ETDEWEB)

    Balatsky, Galya I [Los Alamos National Laboratory; Severe, William R [Los Alamos National Laboratory; Wallace, Richard K [Los Alamos National Laboratory

    2010-01-01

    The Illicit nuclear trafficking panel was conducted at the 4th Annual INMM workshop on Reducing the Risk from Radioactive and Nuclear Materials on February 2-3, 2010 in Washington DC. While the workshop occurred prior to the Nuclear Security Summit, April 12-13 2010 in Washington DC, some of the summit issues were raised during the workshop. The Communique of the Washington Nuclear Security Summit stated that 'Nuclear terrorism is one of the most challenging threats to international security, and strong nuclear security measures are the most effective means to prevent terrorists, criminals, or other unauthorized actors from acquiring nuclear materials.' The Illicit Trafficking panel is one means to strengthen nuclear security and cooperation at bilateral, regional and multilateral levels. Such a panel promotes nuclear security culture through technology development, human resources development, education and training. It is a tool which stresses the importance of international cooperation and coordination of assistance to improve efforts to prevent and respond to incidents of illicit nuclear trafficking. Illicit trafficking panel included representatives from US government, an international organization (IAEA), private industry and a non-governmental organization to discuss illicit nuclear trafficking issues. The focus of discussions was on best practices and challenges for addressing illicit nuclear trafficking. Terrorism connection. Workshop discussions pointed out the identification of terrorist connections with several trafficking incidents. Several trafficking cases involved real buyers (as opposed to undercover law enforcement agents) and there have been reports identifying individuals associated with terrorist organizations as prospective plutonium buyers. Some specific groups have been identified that consistently search for materials to buy on the black market, but no criminal groups were identified that specialize in nuclear materials or isotope

  16. Ubiquitin/proteasome pathway regulates levels of retinoic acid receptor gamma and retinoid X receptor alpha in human keratinocytes.

    Science.gov (United States)

    Boudjelal, M; Wang, Z; Voorhees, J J; Fisher, G J

    2000-04-15

    Repeated exposure of human skin to solar UV radiation leads to premature aging (photoaging) and skin cancer. UV-induced skin damage can be ameliorated by all-trans retinoic acid treatment. The actions of retinoic acid in skin keratinocytes are mediated primarily by nuclear retinoic acid receptor gamma (RARgamma) and retinoid X receptor alpha (RXRalpha). We found that exposure of cultured primary human keratinocytes to UV irradiation (30 mJ/cm2) substantially reduced (50-90%) RARgamma and RXRalpha mRNA and protein within 8 h. The rates of disappearance of RARgamma and RXRalpha proteins after UV exposure or treatment with the protein synthesis inhibitor cycloheximide were similar. UV irradiation did not increase the rate of breakdown of RARgamma or RXRalpha but rather reduced their rate of synthesis. The addition of proteasome inhibitors MG132 and LLvL, but not the lysosomal inhibitor E64, prevented loss of RARgamma and RXRalpha proteins after exposure of keratinocytes to either UV radiation or cycloheximide. Soluble extracts from nonirradiated or UV-irradiated keratinocytes possessed similar levels of proteasome activity that degraded RARgamma and RXRalpha proteins in vitro. Furthermore, RARgamma and RXRalpha were polyubiquitinated in intact cells. RXRalpha was found to contain two proline, glutamate/aspartate, serine, and threonine (PEST) motifs, which confer rapid turnover of many short-lived regulatory proteins that are degraded by the ubiquitin/proteasome pathway. However, the PEST motifs in RXRalpha did not function to regulate its stability, because deletion of the PEST motifs individually or together did not alter ubiquitination or proteasome-mediated degradation of RXRalpha. These results demonstrate that loss of RARgamma and RXRalpha proteins after UV irradiation results from degradation via the ubiquitin/proteasome pathway. Taken together, the data here indicate that ubiquitin/proteasome-mediated breakdown is an important mechanism regulating the levels of

  17. Novel class of amino acid antagonists at non-N-methyl-D-aspartic acid excitatory amino acid receptors. Synthesis, in vitro and in vivo pharmacology, and neuroprotection

    Energy Technology Data Exchange (ETDEWEB)

    Krogsgaard-Larsen, P.; Ferkany, J.W.; Nielsen, E.O.; Madsen, U.; Ebert, B.; Johansen, J.S.; Diemer, N.H.; Bruhn, T.; Beattie, D.T.; Curtis, D.R. (Royal Danish School of Pharmacy, Copenhagen (Denmark))

    1991-01-01

    The isoxazole amino acid 2-amino-3-(3-hydroxy-5-methylisoxazol-4-yl) propionic acid (AMPA) (1), which is a highly selective agonist at the AMPA subtype of excitatory amino acid (EAA) receptors, has been used as a lead for the development of two novel EAA receptor antagonists. One of the compounds, 2-amino-3-(3-(carboxymethoxy)-5-methylisoxazol-4-yl)propionic acid (AMOA, 7), was synthesized via O-alkylation by ethyl chloroacetate of the amino acid protected AMPA derivative 4. The other compound, 2-amino-3-(2-(3-hydroxy-5-methylisoxazol-4-yl)-methyl-5-methyl-3-+ ++oxoisoxazolin -4-yl)propionic acid (AMNH, 14) was synthesized with use of 4-(chloromethyl)-3-methoxy-5-methylisoxazole (8) as the starting material. The intermediate 4-(chloromethyl)-2-(3-methoxy-5-methylisoxazol-4-yl)methyl-5-me thylisoxazolin- 3-one (11) was converted into the acetamidomalonate (12), which was stepwise deprotected to give 14. Compounds 7 and 14 were stable in aqueous solution at pH values close to physiological pH. Neither 7 nor 14 showed detectable affinities for the receptor, ion channel, or modulatory sites of the N-methyl-D-aspartic acid (NMDA) receptor complex. Quantitative receptor autoradiographic and conventional binding techniques were used to study the affinities of 7 and 14 for non-NMDA receptor sites. Both compounds were inhibitors of the binding of (3H)AMPA (IC50 = 90 and 29 microM, respectively). Compounds 14 and 7 were both very weak inhibitors of the high-affinity binding of radioactive kainic acid ((3H)KAIN). Compound 14, but not 7, was, however, shown to be an inhibitor of low-affinity (3H)KAIN binding as determined in the presence of 100 mM calcium chloride. In the rat cortical slice preparation, 7 was shown to antagonize excitation induced by 1 with some selectivity, whereas 14 proved to be a rather selective antagonist of KAIN-induced excitation.

  18. Ganglioside Regulation of AMPA Receptor Trafficking

    OpenAIRE

    Prendergast, Jillian; Umanah, George K. E.; Yoo, Seung-Wan; Lagerlöf, Olof; Motari, Mary G.; Cole, Robert N.; Huganir, Richard L.; Dawson, Ted M.; Dawson, Valina L.; Schnaar, Ronald L.

    2014-01-01

    Gangliosides are major cell-surface determinants on all vertebrate neurons. Human congenital disorders of ganglioside biosynthesis invariably result in intellectual disability and are often associated with intractable seizures. To probe the mechanisms of ganglioside functions, affinity-captured ganglioside-binding proteins from rat cerebellar granule neurons were identified by quantitative proteomic mass spectrometry. Of the six proteins that bound selectively to the major brain ganglioside G...

  19. l-Amino acid sensing by the extracellular Ca2+-sensing receptor

    OpenAIRE

    Conigrave, Arthur D; Quinn, Stephen J.; Brown, Edward M.

    2000-01-01

    The extracellular calcium (Ca2+o)-sensing receptor (CaR) recognizes and responds to (i.e., “senses”) Ca2+o as its principal physiological ligand. In the present studies, we document that the CaR is activated not only by extracellular calcium ions but also by amino acids, establishing its capacity to sense nutrients of two totally different classes. l-Amino acids, especially aromatic amino acids, including l-phenylalanine and l-tryptophan, stereoselectively mobilized Ca2+ ions in the presence ...

  20. Phosphorylation and Internalization of Lysophosphatidic Acid Receptors LPA1, LPA2, and LPA3.

    Directory of Open Access Journals (Sweden)

    Rocío Alcántara-Hernández

    Full Text Available The lysophosphatidic acid receptors LPA1, LPA2, and LPA3 were individually expressed in C9 cells and their signaling and regulation were studied. Agonist-activation increases intracellular calcium concentration in a concentration-dependent fashion. Phorbol myristate acetate markedly inhibited LPA1- and LPA3-mediated effect, whereas that mediated by LPA2 was only partially diminished; the actions of the phorbol ester were inhibited by bisindolylmaleimide I and by overnight incubation with the protein kinase C activator, which leads to down regulation of this protein kinase. Homologous desensitization was also observed for the three LPA receptors studied, with that of LPA2 receptors being consistently of lesser magnitude; neither inhibition nor down-regulation of protein kinase C exerted any effect on homologous desensitization. Activation of LPA1-3 receptors induced ERK 1/2 phosphorylation; this effect was markedly attenuated by inhibition of epidermal growth factor receptor tyrosine kinase activity, suggesting growth factor receptor transactivation in this effect. Lysophosphatidic acid and phorbol myristate acetate were able to induce LPA1-3 phosphorylation, in time- and concentration-dependent fashions. It was also clearly observed that agonists and protein kinase C activation induced internalization of these receptors. Phosphorylation of the LPA2 subtype required larger concentrations of these agents and its internalization was less intense than that of the other subtypes.Our data show that these three LPA receptors are phosphoproteins whose phosphorylation state is modulated by agonist-stimulation and protein kinase C-activation and that differences in regulation and cellular localization exist, among the subtypes.

  1. Transport of cationic amino acids by the mouse ecotropic retrovirus receptor.

    Science.gov (United States)

    Kim, J W; Closs, E I; Albritton, L M; Cunningham, J M

    1991-08-22

    Susceptibility of rodent cells to infection by ecotropic murine leukaemia viruses (MuLV) is determined by binding of the virus envelope to a membrane receptor that has multiple membrane-spanning domains. Cells infected by ecotropic MuLV synthesize envelope protein, gp70, which binds to this receptor, thereby preventing additional infections. The consequences of envelope-MuLV receptor binding for the infected host cell have not been directly determined, partly because the cellular function of the MuLV receptor protein is unknown. Here we report a coincidence in the positions of the first eight putative membrane-spanning domains found in the virus receptor and in two related proteins, the arginine and histidine permeases of Saccharomyces cerevisiae (Fig. 1), but not in any other proteins identified by computer-based sequence comparison of the GenBank data base. Xenopus oocytes injected with receptor-encoding messenger RNA show increased uptake of L-arginine, L-lysine and L-ornithine. The transport properties and the expression pattern of the virus receptor behave in ways previously attributed to y+, the principal transporter of cationic L-amino acids in mammalian cells.

  2. Uptake and intracellular trafficking of superantigens in dendritic cells.

    Directory of Open Access Journals (Sweden)

    María B Ganem

    Full Text Available Bacterial superantigens (SAgs are exotoxins produced mainly by Staphylococcus aureus and Streptococcus pyogenes that can cause toxic shock syndrome (TSS. According to current paradigm, SAgs interact directly and simultaneously with T cell receptor (TCR on the T cell and MHC class II (MHC-II on the antigen-presenting cell (APC, thereby circumventing intracellular processing to trigger T cell activation. Dendritic cells (DCs are professional APCs that coat nearly all body surfaces and are the most probable candidate to interact with SAgs. We demonstrate that SAgs are taken up by mouse DCs without triggering DC maturation. SAgs were found in intracellular acidic compartment of DCs as biologically active molecules. Moreover, SAgs co-localized with EEA1, RAB-7 and LAMP-2, at different times, and were then recycled to the cell membrane. DCs loaded with SAgs are capable of triggering in vitro lymphocyte proliferation and, injected into mice, stimulate T cells bearing the proper TCR in draining lymph nodes. Transportation and trafficking of SAgs in DCs might increase the local concentration of these exotoxins where they will produce the highest effect by promoting their encounter with both MHC-II and TCR in lymph nodes, and may explain how just a few SAg molecules can induce the severe pathology associated with TSS.

  3. Tetrahydro-iso-alpha Acids Antagonize Estrogen Receptor Alpha Activity in MCF-7 Breast Cancer Cells

    Directory of Open Access Journals (Sweden)

    Maëlle Lempereur

    2016-01-01

    Full Text Available Tetrahydro-iso-alpha acids commonly called THIAA or Tetra are modified hop acids extracted from hop (Humulus lupulus L. which are frequently used in brewing industry mainly in order to provide beer bitterness and foam stability. Interestingly, molecular structure of tetrahydro-iso-alpha acids is close to a new type of estrogen receptor alpha (ERα antagonists aimed at disrupting the binding of coactivators containing an LxxLL motif (NR-box. In this work we show that THIAA decreases estradiol-stimulated proliferation of MCF-7 (ERα-positive breast cancer cells. Besides, we show that it inhibits ERα transcriptional activity. Interestingly, this extract fails to compete with estradiol for ERα binding and does not significantly impact the receptor turnover rate in MCF-7 cells, suggesting that it does not act like classical antiestrogens. Hence, we demonstrate that THIAA is able to antagonize ERα estradiol-induced recruitment of the LxxLL binding motif.

  4. The human receptor for urokinase plasminogen activator. NH2-terminal amino acid sequence and glycosylation variants

    DEFF Research Database (Denmark)

    Behrendt, N; Rønne, E; Ploug, M;

    1990-01-01

    -PA. The purified protein shows a single 55-60 kDa band after sodium dodecyl sulfate-polyacrylamide gel electrophoresis and silver staining. It is a heavily glycosylated protein, the deglycosylated polypeptide chain comprising only 35 kDa. The glycosylated protein contains N-acetyl-D-glucosamine and sialic acid......, but no N-acetyl-D-galactosamine. Glycosylation is responsible for substantial heterogeneity in the receptor on phorbol ester-stimulated U937 cells, and also for molecular weight variations among various cell lines. The amino acid composition and the NH2-terminal amino acid sequence are reported....... The protein has a high content of cysteine residues. The NH2-terminal sequence is not closely related to any known sequence. The identification of the purified and sequenced protein with the human u-PA receptor is based on the following findings: 1) the ability of the purified protein to bind u-PA and its...

  5. Relationship between structure, conformational flexibility, and biological activity of agonists and antagonists at the N-methyl-D-aspartic acid subtype of excitatory amino acid receptors

    DEFF Research Database (Denmark)

    Madsen, U; Brehm, L; Schaumburg, Kjeld;

    1990-01-01

    The relationship between conformational flexibility and agonist or antagonist actions at the N-Methyl-D-aspartic acid (NMDA) subtype of central L-glutamic acid (GLU) receptors of a series of racemic piperidinedicarboxylic acids (PDAs) was studied. The conformational analyses were based on 1H NMR...... receptors. Each of the three cyclic acidic amino acids showing NMDA agonist activities was found to exist as an equilibrium mixture of two conformers in aqueous solution. In contrast, the NMDA antagonists cis-2,3-PDA and cis-2,4-PDA as well as the inactive compounds trans-2,5-PDA and cis-2,6-PDA were shown...

  6. Structure-dependent effects of pyridine derivatives on mechanisms of intestinal fatty acid uptake: regulation of nicotinic acid receptor and fatty acid transporter expression.

    Science.gov (United States)

    Riedel, Annett; Lang, Roman; Rohm, Barbara; Rubach, Malte; Hofmann, Thomas; Somoza, Veronika

    2014-07-01

    Pyridines are widely distributed in foods. Nicotinic acid (NA), a carboxylated pyridine derivative, inhibits lipolysis in adipocytes by activation of the orphan NA receptor (HM74A) and is applied to treat hyperlipidemia. However, knowledge on the impact of pyridine derivatives on intestinal lipid metabolism is scarce. This study was performed to identify the structural determinants of pyridines for their effects on fatty acid uptake in enterocyte-like Caco-2 cells and to elucidate the mechanisms of action. The impact of 17 pyridine derivatives on fatty acid uptake was tested. Multiple regression analysis revealed the presence of a methyl group to be the structural determinant at 0.1 mM, whereas at 1 mM, the presence of a carboxylic group and the N-methylation presented further structural characteristics to affect the fatty acid uptake. NA, showing a stimulating effect on FA uptake, and N-methyl-4-phenylpyridinium (MPP), inhibiting FA uptake, were selected for mechanistic studies. Gene expression of the fatty acid transporters CD36, FATP2 and FATP4, and the lipid metabolism regulating transcription factors peroxisome proliferator-activated receptor (PPAR) α and PPARγ was up-regulated upon NA treatment. Caco-2 cells were demonstrated to express the low-affinity NA receptor HM74 of which the gene expression was up-regulated upon NA treatment. We hypothesize that the NA-induced fatty acid uptake might result from NA receptor activation and related intracellular signaling cascades. In contrast, MPP increased transepithelial electrical resistance. We therefore conclude that NA and MPP, both sharing the pyridine motif core, exhibit their contrary effects on intestinal FA uptake by activation of different mechanisms.

  7. Obeticholic acid, a selective farnesoid X receptor agonist, regulates bile acid homeostasis in sandwich-cultured human hepatocytes.

    Science.gov (United States)

    Zhang, Yuanyuan; Jackson, Jonathan P; St Claire, Robert L; Freeman, Kimberly; Brouwer, Kenneth R; Edwards, Jeffrey E

    2017-08-01

    Farnesoid X receptor (FXR) is a master regulator of bile acid homeostasis through transcriptional regulation of genes involved in bile acid synthesis and cellular membrane transport. Impairment of bile acid efflux due to cholangiopathies results in chronic cholestasis leading to abnormal elevation of intrahepatic and systemic bile acid levels. Obeticholic acid (OCA) is a potent and selective FXR agonist that is 100-fold more potent than the endogenous ligand chenodeoxycholic acid (CDCA). The effects of OCA on genes involved in bile acid homeostasis were investigated using sandwich-cultured human hepatocytes. Gene expression was determined by measuring mRNA levels. OCA dose-dependently increased fibroblast growth factor-19 (FGF-19) and small heterodimer partner (SHP) which, in turn, suppress mRNA levels of cholesterol 7-alpha-hydroxylase (CYP7A1), the rate-limiting enzyme for de novo synthesis of bile acids. Consistent with CYP7A1 suppression, total bile acid content was decreased by OCA (1 μmol/L) to 42.7 ± 20.5% relative to control. In addition to suppressing de novo bile acids synthesis, OCA significantly increased the mRNA levels of transporters involved in bile acid homeostasis. The bile salt excretory pump (BSEP), a canalicular efflux transporter, increased by 6.4 ± 0.8-fold, and the basolateral efflux heterodimer transporters, organic solute transporter α (OSTα ) and OSTβ increased by 6.4 ± 0.2-fold and 42.9 ± 7.9-fold, respectively. The upregulation of BSEP and OSTα and OSTβ, by OCA reduced the intracellular concentrations of d8 -TCA, a model bile acid, to 39.6 ± 8.9% relative to control. These data demonstrate that OCA does suppress bile acid synthesis and reduce hepatocellular bile acid levels, supporting the use of OCA to treat bile acid-induced toxicity observed in cholestatic diseases. © 2017 Intercept Pharmaceuticals. Pharmacology Research & Perspectives published by John Wiley & Sons Ltd, British Pharmacological Society and

  8. Allspice and Clove As Source of Triterpene Acids Activating the G Protein-Coupled Bile Acid Receptor TGR5

    Directory of Open Access Journals (Sweden)

    Angela Ladurner

    2017-07-01

    Full Text Available Worldwide, metabolic diseases such as obesity and type 2 diabetes have reached epidemic proportions. A major regulator of metabolic processes that gained interest in recent years is the bile acid receptor TGR5 (Takeda G protein-coupled receptor 5. This G protein-coupled membrane receptor can be found predominantly in the intestine, where it is mainly responsible for the secretion of the incretins glucagon-like peptide 1 (GLP-1 and peptide YY (PYY. The aim of this study was (i to identify plant extracts with TGR5-activating potential, (ii to narrow down their activity to the responsible constituents, and (iii to assess whether the intestinal microbiota produces transformed metabolites with a different activity profile. Chenodeoxycholic acid (CDCA served as positive control for both, the applied cell-based luciferase reporter gene assay for TGR5 activity and the biotransformation assay using mouse fecal slurry. The suitability of the workflow was demonstrated by the biotransformation of CDCA to lithocholic acid resulting in a distinct increase in TGR5 activity. Based on a traditional Tibetan formula, 19 plant extracts were selected and investigated for TGR5 activation. Extracts from the commonly used spices Syzygium aromaticum (SaroE, clove, Pimenta dioica (PdioE, allspice, and Kaempferia galanga (KgalE, aromatic ginger significantly increased TGR5 activity. After biotransformation, only KgalE showed significant differences in its metabolite profile, which, however, did not alter its TGR5 activity compared to non-transformed KgalE. UHPLC-HRMS (high-resolution mass spectrometry analysis revealed triterpene acids (TTAs as the main constituents of the extracts SaroE and PdioE. Identification and quantification of TTAs in these two extracts as well as comparison of their TGR5 activity with reconstituted TTA mixtures allowed the attribution of the TGR5 activity to TTAs. EC50s were determined for the main TTAs, i.e., oleanolic acid (2.2 ± 1.6

  9. Allspice and Clove As Source of Triterpene Acids Activating the G Protein-Coupled Bile Acid Receptor TGR5.

    Science.gov (United States)

    Ladurner, Angela; Zehl, Martin; Grienke, Ulrike; Hofstadler, Christoph; Faur, Nadina; Pereira, Fátima C; Berry, David; Dirsch, Verena M; Rollinger, Judith M

    2017-01-01

    Worldwide, metabolic diseases such as obesity and type 2 diabetes have reached epidemic proportions. A major regulator of metabolic processes that gained interest in recent years is the bile acid receptor TGR5 (Takeda G protein-coupled receptor 5). This G protein-coupled membrane receptor can be found predominantly in the intestine, where it is mainly responsible for the secretion of the incretins glucagon-like peptide 1 (GLP-1) and peptide YY (PYY). The aim of this study was (i) to identify plant extracts with TGR5-activating potential, (ii) to narrow down their activity to the responsible constituents, and (iii) to assess whether the intestinal microbiota produces transformed metabolites with a different activity profile. Chenodeoxycholic acid (CDCA) served as positive control for both, the applied cell-based luciferase reporter gene assay for TGR5 activity and the biotransformation assay using mouse fecal slurry. The suitability of the workflow was demonstrated by the biotransformation of CDCA to lithocholic acid resulting in a distinct increase in TGR5 activity. Based on a traditional Tibetan formula, 19 plant extracts were selected and investigated for TGR5 activation. Extracts from the commonly used spices Syzygium aromaticum (SaroE, clove), Pimenta dioica (PdioE, allspice), and Kaempferia galanga (KgalE, aromatic ginger) significantly increased TGR5 activity. After biotransformation, only KgalE showed significant differences in its metabolite profile, which, however, did not alter its TGR5 activity compared to non-transformed KgalE. UHPLC-HRMS (high-resolution mass spectrometry) analysis revealed triterpene acids (TTAs) as the main constituents of the extracts SaroE and PdioE. Identification and quantification of TTAs in these two extracts as well as comparison of their TGR5 activity with reconstituted TTA mixtures allowed the attribution of the TGR5 activity to TTAs. EC50s were determined for the main TTAs, i.e., oleanolic acid (2.2 ± 1.6 μM), ursolic

  10. Molecular recognition of amino acids with some fluorescent ditopic pyrylium- and pyridinium-based crown ether receptors

    Science.gov (United States)

    Moghimi, A.; Maddah, B.; Yari, A.; Shamsipur, M.; Boostani, M.; Fall Rastegar, M.; Ghaderi, A. R.

    2005-10-01

    The molecular recognition of L-amino acids such as asparagine, glutamine, lysine and arginine with some crownpyryliums, CP's, and a crownpyridinium compound, as receptors, were examined in methanol. 1H NMR spectroscopy was used to examine the structural stability of the receptors in the presence of the amino acids. The fluorimetric titration of the receptors by specified amino acids, other than arginine, was followed within a few minutes and the stoichiometry and stability of the resulting amino acid complexes were evaluated. The data analysis clearly demonstrated the critical role of the terminal amino group to carboxylic acid distance of amino acids for their proper fixation on the receptor molecules. Ion pairing for the two oppositely charged carboxylate anion and pyrylium (or pyridinium) cation, as well as the hydrogen bonding between crown ethers' oxygens and ammonium hydrogens are expected as the main interaction sources in the host-guest complexations.

  11. Evolution of neurotransmitter gamma-aminobutyric acid,glutamate and their receptors

    Institute of Scientific and Technical Information of China (English)

    Zhiheng GOU; Xiao WANG; Wen WANG

    2012-01-01

    Gamma-aminobutyric acid (GABA) and glutamate are two important amino acid neurotransmitters widely present in the nervous systems of ammals,insects,round worm,and platyhelminths,while their receptors are quite diversified across different animal phyla.However,the evolutionary mechanisms between the two conserved neurotransmitters and their diversified receptors remain elusive,and antagonistic interactions between GABA and glutamate signal transduction systems,in particular,have begun to attract significant attention.In this review,we summarize the extant results on the origin and evolution of GABA and glutamate,as well as their receptors,and analyze possible evolutionary processes and phylogenetic relationships of various GABAs and glutamate receptors.We further discuss the evolutionary history of Excitatory/Neutral Amino Acid Transporter (EAAT),a transport protein,which plays an important role in the GABA-glutamate "yin and yang" balanced regulation.Finally,based on current advances,we propose several potential directions of future research.

  12. Retinal pigment epithelial acid lipase activity and lipoprotein receptors: effects of dietary omega-3 fatty acids.

    OpenAIRE

    2002-01-01

    PURPOSE: To show that fish oil-derived omega-3 polyunsaturated fatty acids, delivered to the retinal pigment epithelium (RPE) by circulating low-density lipoproteins (LDL), enhance already considerable RPE lysosomal acid lipase activity, providing for more efficient hydrolysis of intralysosomal RPE lipids, an effect that may help prevent development of age-related macular degeneration (ARMD). METHODS: Colorimetric biochemical and histochemical techniques were used to demonstrate RPE acid lipa...

  13. Gamma-aminobutyric acid promotes human hepatocellular carcinoma growth through overexpressed gamma-aminobutyric acid A receptor α3 subunit

    Institute of Scientific and Technical Information of China (English)

    2008-01-01

    AIM: To investigate the expression pattern of gamma-aminobutyric acid A (GABAA) receptors in hepatocellular carcinoma (HCC) and indicate the relationship among gamma-aminobutyric acid (GABA), gamrna-aminobutyric acid A receptor α3 subunit (GABRA3) and HCC.METHODS: HCC cell line Chang, HepG2, normal liver cell line L-02 and 8 samples of HCC tissues and paired non-cancerous tissues were analyzed with semiquantitative polymerase chain reaction (PCR) for the expression of GABAA receptors. HepG2 cells were treated with gamma-aminobutyric acid (GABA) at serial concentrations (0, 1, 10, 20, 40 and 60 μmol/L), and their proliferating abilities were analyzed with the 3-(4, 5-methylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay, cell doubling time test, colon formation assay, cell cycle analysis and tumor planted in nude mice. Small interfering RNA was used for knocking down the endogenous GABRA3 in HepG2. oliferating abilities of these cells treated with or without GABA were analyzed.RESULTS: We identified the overexpression of GABRA3 in HCC cells. Knockdown of endogenous GABRA3 expression in HepG2 attenuated HCC cell growth, suggesting its role in HCC cell viability. We determined the in vitro and in vivo effect of GABA in the proliferation of GABRA3-positive cell lines, and found that GABA increased HCC growth in a dose-dependent manner. Notably, the addition of GABA into the cell culture medium promoted the proliferation of GABRA3-expressing HepG2 cells, but not GABRA3-knockdown HepG2 cells. This means that GABA stimulates HepG2 cell growth through GABRA3. CONCLUSION: GABA and GABRA3 play important roles in HCC development and progression and can be a promising molecular target for the development of new diagnostic and therapeutic strategies for HCC.

  14. Bile Acid Nuclear Receptor Farnesoid X Receptor: Therapeutic Target for Nonalcoholic Fatty Liver Disease

    Science.gov (United States)

    Kim, Sun-Gi; Kim, Byung-Kwon; Kim, Kyumin

    2016-01-01

    Nonalcoholic fatty liver disease (NAFLD) is one of the causes of fatty liver, occurring when fat is accumulated in the liver without alcohol consumption. NAFLD is the most common liver disorder in advanced countries. NAFLD is a spectrum of pathology involving hepatic steatosis with/without inflammation and nonalcoholic steatohepatitis with accumulation of hepatocyte damage and hepatic fibrosis. Recent studies have revealed that NAFLD results in the progression of cryptogenic cirrhosis that leads to hepatocarcinoma and cardiovascular diseases such as heart failure. The main causes of NAFLD have not been revealed yet, metabolic syndromes including obesity and insulin resistance are widely accepted for the critical risk factors for the pathogenesis of NAFLD. Nuclear receptors (NRs) are transcriptional factors that sense environmental or hormonal signals and regulate expression of genes, involved in cellular growth, development, and metabolism. Several NRs have been reported to regulate genes involved in energy and xenobiotic metabolism and inflammation. Among various NRs, farnesoid X receptor (FXR) is abundantly expressed in the liver and a key regulator to control various metabolic processes in the liver. Recent studies have shown that NAFLD is associated with inappropriate function of FXR. The impact of FXR transcriptional activity in NAFLD is likely to be potential therapeutic strategy, but still requires to elucidate underlying potent therapeutic mechanisms of FXR for the treatment of NAFLD. This article will focus the physiological roles of FXR and establish the correlation between FXR transcriptional activity and the pathogenesis of NAFLD. PMID:28029021

  15. Bile Acid Nuclear Receptor Farnesoid X Receptor: Therapeutic Target for Nonalcoholic Fatty Liver Disease

    Directory of Open Access Journals (Sweden)

    Sun-Gi Kim

    2016-12-01

    Full Text Available Nonalcoholic fatty liver disease (NAFLD is one of the causes of fatty liver, occurring when fat is accumulated in the liver without alcohol consumption. NAFLD is the most common liver disorder in advanced countries. NAFLD is a spectrum of pathology involving hepatic steatosis with/without inflammation and nonalcoholic steatohepatitis with accumulation of hepatocyte damage and hepatic fibrosis. Recent studies have revealed that NAFLD results in the progression of cryptogenic cirrhosis that leads to hepatocarcinoma and cardiovascular diseases such as heart failure. The main causes of NAFLD have not been revealed yet, metabolic syndromes including obesity and insulin resistance are widely accepted for the critical risk factors for the pathogenesis of NAFLD. Nuclear receptors (NRs are transcriptional factors that sense environmental or hormonal signals and regulate expression of genes, involved in cellular growth, development, and metabolism. Several NRs have been reported to regulate genes involved in energy and xenobiotic metabolism and inflammation. Among various NRs, farnesoid X receptor (FXR is abundantly expressed in the liver and a key regulator to control various metabolic processes in the liver. Recent studies have shown that NAFLD is associated with inappropriate function of FXR. The impact of FXR transcriptional activity in NAFLD is likely to be potential therapeutic strategy, but still requires to elucidate underlying potent therapeutic mechanisms of FXR for the treatment of NAFLD. This article will focus the physiological roles of FXR and establish the correlation between FXR transcriptional activity and the pathogenesis of NAFLD.

  16. Saturated fatty acids inhibit hepatic insulin action by modulating insulin receptor expression and post-receptor signalling.

    Science.gov (United States)

    Ruddock, Mark W; Stein, Andrew; Landaker, Edwin; Park, Jun; Cooksey, Robert C; McClain, Donald; Patti, Mary-Elizabeth

    2008-11-01

    Free fatty acids (FFAs) are proposed to play a pathogenic role in both peripheral and hepatic insulin resistance. We have examined the effect of saturated FFA on insulin signalling (100 nM) in two hepatocyte cell lines. Fao hepatoma cells were treated with physiological concentrations of sodium palmitate (0.25 mM) (16:0) for 0.25-48 h. Palmitate decreased insulin receptor (IR) protein and mRNA expression in a dose- and time-dependent manner (35% decrease at 12 h). Palmitate also reduced insulin-stimulated IR and IRS-2 tyrosine phosphorylation, IRS-2-associated PI 3-kinase activity, and phosphorylation of Akt, p70 S6 kinase, GSK-3 and FOXO1A. Palmitate also inhibited insulin action in hepatocytes derived from wild-type IR (+/+) mice, but was ineffective in IR-deficient (-/-) cells. The effects of palmitate were reversed by triacsin C, an inhibitor of fatty acyl CoA synthases, indicating that palmitoyl CoA ester formation is critical. Neither the non-metabolized bromopalmitate alone nor the medium chain fatty acid octanoate (8:0) produced similar effects. However, the CPT-1 inhibitor (+/-)-etomoxir and bromopalmitate (in molar excess) reversed the effects of palmitate. Thus, the inhibition of insulin signalling by palmitate in hepatoma cells is dependent upon oxidation of fatty acyl-CoA species and requires intact insulin receptor expression.

  17. Trafficking: a perspective from Asia.

    Science.gov (United States)

    Skeldon, R

    2000-01-01

    The main theme of this article is market development and trafficking as a business. It touches upon most of the aspects of the phenomenon, which have been encountered elsewhere, and translates them into the relatively unfamiliar context of many of the Asian and South-East Asian economies. Equally, the literature cited is also probably unfamiliar. Themes touched upon include democratization, inter-state relations, human rights, and scale and perspectives, together with the problems of definitions, theory, and the reliability of data. The directions and characteristics of trafficking flows together with routes and border control are also considered. Coordinated official responses to criminality and criminal organizations, as well as to trafficked individuals, are beginning to emerge. There is a note of caution sounded that contextual and cultural perspectives, particularly on sex workers, must be viewed somewhat differently to those in Western societies. The article concludes that as long as countries in Asia maintain their policies of restrictive immigration, trafficking can be expected to continue and almost certainly increase. This is because accelerating development creates demand for labor at various skill levels and because even in times of recession migrants and brokers will seek to side-step attempts to expel immigrants and restrict access to labor markets. The elimination of trafficking is unlikely to be realistically achieved through legislation and declarations of intent but by improvements in the socioeconomic status of the population.

  18. Free fatty acid receptors as therapeutic targets for the treatment of diabetes

    Directory of Open Access Journals (Sweden)

    Atsuhiko eIchimura

    2014-11-01

    Full Text Available Nutrition regulates energy balance; however, dysfunction of energy balance can cause metabolic disorders, such as obesity and diabetes. Fatty acids are an essential energy source and signaling molecules that regulate various cellular processes and physiological functions. Recently, several orphan G protein-coupled receptors were indentified as free fatty acid receptors (FFARs. GPR40/FFAR1 and GPR120/FFAR4 are activated by medium- and/or long-chain fatty acids, whereas GPR41/FFAR3 and GPR43/FFAR2 are activated by short-chain fatty acids. FFARs are regarded as targets for novel drugs to treat metabolic disorders, such as obesity and type 2 diabetes, because recent studies have showed that these receptors are involved in the energy metabolism in various tissues, including adipose, intestinal, and immune tissue. In this review, we summarize physiological roles of the FFARs, provide a comprehensive overview of energy regulation by FFARs, and discuss new prospects for treatment of metabolic disorders.

  19. Trafficking in persons: a health concern?

    Directory of Open Access Journals (Sweden)

    Cathy Zimmerman

    Full Text Available Human trafficking is a phenomenon that has now been documented in most regions in the world. Although trafficking of women and girls for sexual exploitation is the most commonly recognised form of trafficking, it is widely acknowledged that human trafficking also involves men, women and children who are trafficked for various forms of labour exploitation and into other abusive circumstances. Despite the violence and harm inherent in most trafficking situations, there remains extremely little evidence on the individual and public health implications of any form of human trafficking. The Brazilian government has recently launched a national plan to combat human trafficking. However, because the health risks associated with human trafficking have not been well-recognised or documented, there is extremely limited reliable data on the health needs of trafficked persons to inform policy and practices.. Brazilian policy-makers and service providers should be encouraged to learn about the likely range of health impacts of trafficking, and incorporate this into anti-trafficking protection and response strategies. As well as prevention activities, the government, international and local organisations should work together with the public health research community to study the health needs of trafficked persons and explore opportunities to provide safe and appropriate services to victims in need of care.

  20. Trafficking in persons: a health concern?

    Science.gov (United States)

    Zimmerman, Cathy; Kiss, Ligia; Houssain, Mazeda; Watts, Charlotte

    2009-01-01

    Human trafficking is a phenomenon that has now been documented in most regions in the world. Although trafficking of women and girls for sexual exploitation is the most commonly recognised form of trafficking, it is widely acknowledged that human trafficking also involves men, women and children who are trafficked for various forms of labour exploitation and into other abusive circumstances. Despite the violence and harm inherent in most trafficking situations, there remains extremely little evidence on the individual and public health implications of any form of human trafficking. The Brazilian government has recently launched a national plan to combat human trafficking. However, because the health risks associated with human trafficking have not been well-recognised or documented, there is extremely limited reliable data on the health needs of trafficked persons to inform policy and practices.. Brazilian policy-makers and service providers should be encouraged to learn about the likely range of health impacts of trafficking, and incorporate this into anti-trafficking protection and response strategies. As well as prevention activities, the government, international and local organisations should work together with the public health research community to study the health needs of trafficked persons and explore opportunities to provide safe and appropriate services to victims in need of care.

  1. N-Glycosylation of the Na+-Taurocholate Cotransporting Polypeptide (NTCP) Determines Its Trafficking and Stability and Is Required for Hepatitis B Virus Infection

    Science.gov (United States)

    Appelman, Monique D.; Chakraborty, Anindita; Protzer, Ulrike; McKeating, Jane A.

    2017-01-01

    The sodium/bile acid cotransporter NTCP was recently identified as a receptor for hepatitis B virus (HBV). NTCP is glycosylated and the role of glycans in protein trafficking or viral receptor activity is not known. NTCP contains two N-linked glycosylation sites and asparagine amino acid residues N5 and N11 were mutated to a glutamine to generate NTCP with a single glycan (NTCP-N5Q or NTCP- N11Q) or no glycans (NTCP- N5,11Q). HepG2 cells expressing NTCP with a single glycan supported HBV infection at a comparable level to NTCP-WT. The physiological function of NTCP, the uptake of bile acids, was also not affected in cells expressing these single glycosylation variants, consistent with their trafficking to the plasma membrane. However, glycosylation-deficient NTCP (NTCP-N5,11Q) failed to support HBV infection, showed minimal cellular expression and was degraded in the lysosome. This affected the physiological bile acid transporter function of NTCP-N5,11Q in a similar fashion. In conclusion, N-glycosylation is required for efficient NTCP localization at the plasma membrane and subsequent HBV infection and these characteristics are preserved in NTCP carrying a single carbohydrate moiety. PMID:28125599

  2. Triphenyl phosphate-induced developmental toxicity in zebrafish: Potential role of the retinoic acid receptor

    Energy Technology Data Exchange (ETDEWEB)

    Isales, Gregory M.; Hipszer, Rachel A.; Raftery, Tara D. [Department of Environmental Health Sciences, Arnold School of Public Health, University of South Carolina, Columbia, SC (United States); Chen, Albert; Stapleton, Heather M. [Division of Environmental Sciences and Policy, Nicholas School of the Environment, Duke University, Durham, NC (United States); Volz, David C., E-mail: volz@mailbox.sc.edu [Department of Environmental Health Sciences, Arnold School of Public Health, University of South Carolina, Columbia, SC (United States)

    2015-04-15

    Highlights: • Triphenyl phosphate-induced toxicity in zebrafish embryos is enhanced in the presence of a retinoic acid receptor antagonist. • Triphenyl phosphate uptake or metabolism within zebrafish embryos is not altered in the presence of a retinoic acid receptor antagonist. • Triphenyl phosphate decreases expression of cytochrome P450 26a1 in zebrafish embryos. • Triphenyl phosphate inhibits retinoic acid-induced activation of human retinoic acid receptors. - Abstract: Using zebrafish as a model, we previously reported that developmental exposure to triphenyl phosphate (TPP) – a high-production volume organophosphate-based flame retardant – results in dioxin-like cardiac looping impairments that are independent of the aryl hydrocarbon receptor. Using a pharmacologic approach, the objective of this study was to investigate the potential role of retinoic acid receptor (RAR) – a nuclear receptor that regulates vertebrate heart morphogenesis – in mediating TPP-induced developmental toxicity in zebrafish. We first revealed that static exposure of zebrafish from 5–72 h post-fertilization (hpf) to TPP in the presence of non-toxic concentrations of an RAR antagonist (BMS493) significantly enhanced TPP-induced toxicity (relative to TPP alone), even though identical non-toxic BMS493 concentrations mitigated retinoic acid (RA)-induced toxicity. BMS493-mediated enhancement of TPP toxicity was not a result of differential TPP uptake or metabolism, as internal embryonic doses of TPP and diphenyl phosphate (DPP) – a primary TPP metabolite – were not different in the presence or absence of BMS493. Using real-time PCR, we then quantified the relative change in expression of cytochrome P450 26a1 (cyp26a1) – a major target gene for RA-induced RAR activation in zebrafish – and found that RA and TPP exposure resulted in a ∼5-fold increase and decrease in cyp26a1 expression, respectively, relative to vehicle-exposed embryos. To address whether TPP may

  3. Roles of the Mevalonate Pathway and Cholesterol Trafficking in Pulmonary Host Defense.

    Science.gov (United States)

    Gabor, Kristin A; Fessler, Michael B

    2017-01-01

    The mevalonic acid synthesis pathway, cholesterol, and lipoproteins play fundamental roles in lung physiology and the innate immune response. Recent literature investigating roles for cholesterol synthesis and trafficking in host defense against respiratory infection was critically reviewed. The innate immune response and the cholesterol biosynthesis/trafficking network regulate one another, with important implications for pathogen invasion and host defense in the lung. The activation of pathogen recognition receptors and downstream cellular host defense functions are critically sensitive to cellular cholesterol. Conversely, microorganisms can co-opt the sterol/lipoprotein network in order to facilitate replication and evade immunity. Emerging literature suggests the potential for harnessing these insights towards therapeutic development. Given that >50% of adults in the U.S. have serum cholesterol abnormalities and pneumonia remains a leading cause of death, the potential impact of cholesterol on pulmonary host defense is of tremendous public health significance and warrants further mechanistic and translational investigation. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

  4. THE CORRELATIONS OF RETINOIC ACID RECEPTOR-α AND ESTROGEN RECEPTOR EXPRESSION IN HUMAN BREAST CANCER CELL LINES AND TUMORS

    Institute of Scientific and Technical Information of China (English)

    余黎明; 邵志敏; 蔡三军; 韩企夏; 沈镇宙

    1998-01-01

    Retinoic acid receptor-α(RAR α) plays a major role in the growth inhibitory effect of retinoic acid on human breast cancer ceils, may be it could serve as an indicator to guide the treatment and prevent of breast cancer with retinoic acid in ciiinc. All previous researchs were based on observing the changes ofRAR a mRAN expression. In this study, the expression of RAR a in human breast cell lines was studied by Northern Blot, Western Blot and Immunohistochemistry in mRNA level and protein level. Results showed that RAR a protein expression was correlated with RAR a mRNA expression. RAR α mRNA expression was higher in estrogen receptor (ER)-positive human breast cancer cell lines than in ER-negative ones. So was RAR α protein expression. Both RAR α mRNA amd RAR α protein expression were associated with ER status. The expression of RAR α and the relationship between RAR α and ER status were also determined by immunohistochemistry in 58 human primary breast cancer tumors. 37 (63.8%) tumors were ER-positive and of these 28 (75. 7%) were also RAR α -positive. The coexpression of ER and RAR α was statistleally significant (P<0. 01, by X2 contingency analysis), It was reported that RAR α expression in cultured breast cancer ceils was regulated by estrogen acting via the ER. Our study demonstrated that RAR α expression may be modulated in breast cancer in vivo by estrogen via ER.

  5. Mercaptoacetate blocks fatty acid-induced GLP-1 secretion in male rats by directly antagonizing GPR40 fatty acid receptors.

    Science.gov (United States)

    Li, Ai-Jun; Wang, Qing; Dinh, Thu T; Simasko, Steve M; Ritter, Sue

    2016-04-15

    Mercaptoacetate (MA) is an orexigenic agent reported to block fatty acid (FA) oxidation. Recently, however, we reported evidence from isolated nodose ganglion neurons that MA antagonizes the G protein-coupled long- and medium-chain FA receptor GPR40. GPR40 mediates FA-induced secretion of the satietogenic incretin peptide glucagon-like peptide 1 (GLP-1), by enteroendocrine L cells, as well as FA-induced enhancement of glucose-stimulated insulin secretion. Our results in cultured nodose neurons suggest that MA would also block GPR40 in enteroendocrine cells controlling GLP-1 secretion. If so, this would suggest an alternative mechanism by which MA increases food intake. We tested the hypothesis that MA blocks FA-induced GLP-1 secretion in vitro using cultured STC-1 cells (a murine enteroendocrine cell line) and in vivo in adult male rats. In vitro, MA blocked the increase in both cytosolic Ca(2+)and GLP-1 release stimulated by FAs and also reduced (but less effectively) the response of STC-1 cells to grifolic acid, a partial agonist of the GPR120 FA receptor. In vivo, MA reduced GLP-1 secretion following olive oil gavage while also increasing glucose and decreasing insulin levels. The carnitine palmatoyltransferase 1 antagonist etomoxir did not alter these responses. Results indicate that MA's actions, including its orexigenic effect, are mediated by GPR40 (and possibly GPR120) receptor antagonism and not by blockade of fat oxidation, as previously believed. Analysis of MA's interaction with GPR40 may facilitate understanding of the multiple functions of this receptor and the manner in which FAs participate in the control of hunger and satiety.

  6. Understanding human trafficking in the United States.

    Science.gov (United States)

    Logan, T K; Walker, Robert; Hunt, Gretchen

    2009-01-01

    The topic of modern-day slavery or human trafficking has received increased media and national attention. However, to date there has been limited research on the nature and scope of human trafficking in the United States. This article describes and synthesizes nine reports that assess the U.S. service organizations' legal representative knowledge of, and experience with, human trafficking cases, as well as information from actual cases and media reports. This article has five main goals: (a) to define what human trafficking is, and is not; (b) to describe factors identified as contributing to vulnerability to being trafficked and keeping a person entrapped in the situation; (c) to examine how the crime of human trafficking differs from other kinds of crimes in the United States; (d) to explore how human trafficking victims are identified; and, (e) to provide recommendations to better address human trafficking in the United States.

  7. Selective antagonists at group I metabotropic glutamate receptors: synthesis and molecular pharmacology of 4-aryl-3-isoxazolol amino acids

    DEFF Research Database (Denmark)

    Kromann, Hasse; Sløk, Frank A; Stensbøl, Tine B

    2002-01-01

    Homologation of (S)-glutamic acid (Glu, 1) and Glu analogues has previously provided ligands with activity at metabotropic Glu receptors (mGluRs). The homologue of ibotenic acid (7), 2-amino-3-(3-hydroxy-5-isoxazolyl)propionic acid (HIBO, 8), and the 4-phenyl derivative of 8, compound 9a, are bot...

  8. Synthesis of novel N1-substituted bicyclic pyrazole amino acids and evaluation of their interaction with glutamate receptors

    DEFF Research Database (Denmark)

    Conti, Paola; Grazioso, Giovanni; di Ventimiglia, Samuele Joppolo

    2005-01-01

    N1-substituted bicyclic pyrazole amino acids (S)-9a-9c and (R)-9a-9c, which are conformationally constrained analogues of glutamic acid, were prepared via a strategy based on a 1,3-dipolar cycloaddition. The new amino acids were tested for activity at ionotropic and metabotropic glutamate receptors...

  9. Nuclear trafficking in health and disease.

    Science.gov (United States)

    Mor, Amir; White, Michael A; Fontoura, Beatriz M A

    2014-06-01

    In eukaryotic cells, the cytoplasm and the nucleus are separated by a double-membraned nuclear envelope (NE). Thus, transport of molecules between the nucleus and the cytoplasm occurs via gateways termed the nuclear pore complexes (NPCs), which are the largest intracellular channels in nature. While small molecules can passively translocate through the NPC, large molecules are actively imported into the nucleus by interacting with receptors that bind nuclear pore complex proteins (Nups). Regulatory factors then function in assembly and disassembly of transport complexes. Signaling pathways, cell cycle, pathogens, and other physiopathological conditions regulate various constituents of the nuclear transport machinery. Here, we will discuss several findings related to modulation of nuclear transport during physiological and pathological conditions, including tumorigenesis, viral infection, and congenital syndrome. We will also explore chemical biological approaches that are being used as probes to reveal new mechanisms that regulate nucleocytoplasmic trafficking and that are serving as starting points for drug development.

  10. Ascorbic acid enables reversible dopamine receptor /sup 3/H-agonist binding

    Energy Technology Data Exchange (ETDEWEB)

    Leff, S.; Sibley, D.R.; Hamblin, M.; Creese, I.

    1981-11-16

    The effects of ascorbic acid on dopaminergic /sup 3/H-agonist receptor binding were studied in membrane homogenates of bovine anterior pituitary and caudate, and rat striatum. In all tissues virtually no stereospecific binding (defined using 1uM (+)butaclamol) of the /sup 3/H-agonists N-propylnorapomorphine (NPA), apomorphine, or dopamine could be demonstrated in the absence of ascorbic acid. Although levels of total /sup 3/H-agonist binding were three to five times greater in the absence than in the presence of 0.1% ascorbic acid, the increased binding was entirely non-stereospecific. Greater amounts of dopamine-inhibitable /sup 3/H-NPA binding could be demonstrated in the absence of 0.1% ascorbic acid, but this measure of ''specific binding'' was demonstrated not to represent dopamine receptor binding since several other catecholamines and catechol were equipotent with dopamine and more potent than the dopamine agonist (+/-)amino-6,7-dihydroxy-1,2,3,4-tetrahydronapthalene (ADTN) in inhibiting this binding. High levels of dopamine-displaceable /sup 3/H-agonist binding were detected in fresh and boiled homogenates of cerebellum, an area of brain which receives no dopaminergic innervation, further demonstrating the non-specific nature of /sup 3/H-agonist binding in the absence of ascorbic acid. These studies emphasize that under typical assay conditions ascorbic acid is required in order to demonstrate reversible and specific /sup 3/H-agonist binding to dopamine receptors.

  11. Effect of niflumic acid on electromechanical coupling by tachykinin NK1 receptor activation in rabbit colon.

    Science.gov (United States)

    Patacchini, R; Santicioli, P; Maggi, C A

    1996-05-15

    We have investigated the effect of the Cl- channel blocker, niflumic acid, on the contractile response and electromechanical coupling activated by stimulation of the tachykinin NK1 receptor in the longitudinal muscle of rabbit proximal colon, in the presence of indomethacin (5 microM). The application of submaximal equieffective concentrations of the tachykinin NK1 receptor-selective agonist [Sar9]substance P sulfone (30 nM), of carbachol (300 nM) and KCl (40 mM), produced distinct phasic and tonic components of contraction. Niflumic acid (10-100 microM) preferentially and markedly inhibited the tonic component of the response to [Sar9]substance P sulfone and to carbachol, without affecting the response to KCl. Nifedipine (1 microM) abolished the response to KCl and greatly reduced the response to [Sar9]substance P sulfone and carbachol. The nifedipine-resistant response to [Sar9]substance P sulfone was attenuated by niflumic acid (100 microM), while that to carbachol was unaffected. In sucrose gap experiments, superfusion with niflumic acid (100 microM), in the presence of nifedipine (3 microM), produced membrane hyperpolarization, which was totally blocked by tetraethylammonium (10 mM). Niflumic acid inhibited both depolarization and contraction induced by [Sar9]substance P sulfone, both in the absence or in the presence of tetraethylammonium. The present findings support the idea that a niflumic acid-sensitive mechanism, probably an effect on Cl- channels, takes part in the post-receptorial events activated by tachykinin NK1 receptor stimulation in the longitudinal muscle of rabbit colon, and suggest that this mechanism would be more important for generating the sustained tonic than the phasic component of contraction.

  12. Synthesis of new isoxazoline-based acidic amino acids and investigation of their affinity and selectivity profile at ionotropic glutamate receptors

    DEFF Research Database (Denmark)

    Pinto, Andrea; Conti, Paola; Grazioso, Giovanni;

    2011-01-01

    The synthesis of four new isoxazoline-based amino acids being analogues of previously described glutamate receptor ligands is reported and their affinity for ionotropic glutamate receptors is analyzed in comparison with that of selected model compounds. Molecular modelling investigations have been...

  13. Role of farnesoid X receptor and bile acids in alcoholic liver disease

    Directory of Open Access Journals (Sweden)

    Sharon Manley

    2015-03-01

    Full Text Available Alcoholic liver disease (ALD is one of the major causes of liver morbidity and mortality worldwide. Chronic alcohol consumption leads to development of liver pathogenesis encompassing steatosis, inflammation, fibrosis, cirrhosis, and in extreme cases, hepatocellular carcinoma. Moreover, ALD may also associate with cholestasis. Emerging evidence now suggests that farnesoid X receptor (FXR and bile acids also play important roles in ALD. In this review, we discuss the effects of alcohol consumption on FXR, bile acids and gut microbiome as well as their impacts on ALD. Moreover, we summarize the findings on FXR, FoxO3a (forkhead box-containing protein class O3a and PPARα (peroxisome proliferator-activated receptor alpha in regulation of autophagy-related gene transcription program and liver injury in response to alcohol exposure.

  14. Sex trafficking and the exploitation of adolescents.

    Science.gov (United States)

    McClain, Natalie M; Garrity, Stacy E

    2011-01-01

    Human trafficking affects a surprisingly large number of adolescents around the globe. Women and girls make up the majority of sex trafficking victims. Nurses must be aware of sex trafficking as a form of sexual violence in the adolescent population. Nurses can play a role in identifying, intervening, and advocating for victims of human trafficking as they currently do for patients that are the victims of other types of violent crimes.

  15. Sex trafficking in Nepal: context and process

    OpenAIRE

    Hennink, Monique; Simkhada, Padam

    2004-01-01

    This study has developed a conceptual framework to provide a clearer understanding of the process and context of sex trafficking from Nepal. Quantitative data were analysed from case records of 202 sex-trafficked women at rehabilitation centres in Nepal. In-depth interviews with 42 sex trafficked women, mostly residing at rehabilitation centres in Kathmandu, provide contextual information on the process and circumstances of sex trafficking. The results of this study provide a clearer understa...

  16. Barriers to combating human trafficking in Colombia

    OpenAIRE

    Wilcox, Daniel Joseph

    2015-01-01

    Approved for public release; distribution is unlimited Despite international and domestic policies and programs intended to combat human trafficking, Colombia remains one of the countries with the highest instances of human trafficking in the Western Hemisphere. Factors contributing to human trafficking in Colombia, such as internal violence and displacement, drug trafficking, a weak central government, and widespread corruption, have overpowered what energies the government marshaled agai...

  17. Citric acid cycle intermediates as ligands for orphan G-protein-coupled receptors.

    Science.gov (United States)

    He, Weihai; Miao, Frederick J-P; Lin, Daniel C-H; Schwandner, Ralf T; Wang, Zhulun; Gao, Jinhai; Chen, Jin-Long; Tian, Hui; Ling, Lei

    2004-05-13

    The citric acid cycle is central to the regulation of energy homeostasis and cell metabolism. Mutations in enzymes that catalyse steps in the citric acid cycle result in human diseases with various clinical presentations. The intermediates of the citric acid cycle are present at micromolar concentration in blood and are regulated by respiration, metabolism and renal reabsorption/extrusion. Here we show that GPR91 (ref. 3), a previously orphan G-protein-coupled receptor (GPCR), functions as a receptor for the citric acid cycle intermediate succinate. We also report that GPR99 (ref. 4), a close relative of GPR91, responds to alpha-ketoglutarate, another intermediate in the citric acid cycle. Thus by acting as ligands for GPCRs, succinate and alpha-ketoglutarate are found to have unexpected signalling functions beyond their traditional roles. Furthermore, we show that succinate increases blood pressure in animals. The succinate-induced hypertensive effect involves the renin-angiotensin system and is abolished in GPR91-deficient mice. Our results indicate a possible role for GPR91 in renovascular hypertension, a disease closely linked to atherosclerosis, diabetes and renal failure.

  18. Specific interaction of aurintricarboxylic acid with the human immunodeficiency virus/CD4 cell receptor

    Energy Technology Data Exchange (ETDEWEB)

    Schols, D.; Baba, M.; Pauwels, R.; Desmyter, J.; De Clercq, E. (Katholieke Universiteit Leuven (Belgium))

    1989-05-01

    The triphenylmethane derivative aurintricarboxylic acid (ATA), but not aurin, selectively prevented the binding of OKT4A/Leu-3a monoclonal antibody (mAb) and, to a lesser extent, OKT4 mAb to the CD4 cell receptor for human immunodeficiency virus type 1 (HIV-1). The effect was seen within 1 min at an ATA concentration of 10 {mu}M in various T4{sup +} cells (MT-4, U-937, peripheral blood lymphocytes, and monocytes). It was dose-dependent and reversible. ATA prevented the attachment of radiolabeled HIV-1 particles to MT-4 cells, which could be expected as the result of its specific binding to the HIV/CD4 receptor. Other HIV inhibitors such as suramin, fuchsin acid, azidothymidine, dextran sulfate, heparin, and pentosan polysulfate did not affect OKT4A/Leu-3a mAb binding to the CD4 receptor, although the sulfated polysaccharides suppressed HIV-1 adsorption to the cells at concentrations required for complete protection against HIV-1 cytopathogenicity. Thus, ATA is a selective marker molecule for the CD4 receptor. ATA also interfered with the staining of membrane-associated HIV-1 glycoprotein gp120 by a mAb against it. These unusual properties of a small molecule of nonimmunological origin may have important implications for the study of CD4/HIV/AIDS pathogenesis and possibly treatment.

  19. ISOLATION AND CHARACTERIZATION OF AXOLOTL NPDC-1 AND ITS EFFECTS ON RETINOIC ACID RECEPTOR SIGNALING

    OpenAIRE

    Theodosiou, Maria; Monaghan, James R; Spencer, Michael L; Voss, S. Randal; Noonan, Daniel J.

    2007-01-01

    Retinoic acid, a key morphogen in early vertebrate development and tissue regeneration, mediates its effects through the binding of receptors that act as ligand-induced transcription factors. These binding events function to recruit an array of transcription co-regulatory proteins to specific gene promoters. One such co-regulatory protein, neuronal proliferation and differentiation control-1 (NPDC-1), is broadly expressed during mammalian development and functions as an in vitro repressor of ...

  20. Bioorthogonal click chemistry to assay mu-opioid receptor palmitoylation using 15-hexadecynoic acid and immunoprecipitation

    OpenAIRE

    2014-01-01

    We have developed a modification of bioorthogonal click chemistry to assay the palmitoylation of cellular proteins. This assay utilizes 15-hexadecynoic acid (15-HDYA) as a chemical probe in combination with protein immunoprecipitation using magnetic beads in order to detect S-palmitoylation of proteins of interest. Here we demonstrate the utility of this approach for the mu-opioid receptor (MOR), a GPCR responsible for mediating the analgesic and addictive properties of most clinically releva...

  1. Security Implications of Human-Trafficking Networks

    Science.gov (United States)

    2007-06-15

    to those security concerns. Background How is Human Trafficking Carried Out? While trafficking victims are often found in sweatshops , domestic...labor. This type of trafficking is often found in agricultural labor, the production of goods (typically called sweatshops ) and construction labor

  2. GPR18 undergoes a high degree of constitutive trafficking but is unresponsive to N-Arachidonoyl Glycine

    Directory of Open Access Journals (Sweden)

    David B. Finlay

    2016-03-01

    Full Text Available The orphan receptor GPR18 has become a research target following the discovery of a putative endogenous agonist, N-arachidonoyl glycine (NAGly. Chemical similarity between NAGly and the endocannabinoid anandamide suggested the hypothesis that GPR18 is a third cannabinoid receptor. GPR18-mediated cellular signalling through inhibition of cyclic adenosine monophosphate (cAMP and phosphorylation of extracellular signal-regulated kinase (ERK, in addition to physiological consequences such as regulation of cellular migration and proliferation/apoptosis have been described in response to both NAGly and anandamide. However, discordant findings have also been reported. Here we sought to describe the functional consequences of GPR18 activation in heterologously-expressing HEK cells. GPR18 expression was predominantly intracellular in stably transfected cell lines, but moderate cell surface expression could be achieved in transiently transfected cells which also had higher overall expression. Assays were employed to characterise the ability of NAGly or anandamide to inhibit cAMP or induce ERK phosphorylation through GPR18, or induce receptor trafficking. Positive control experiments, which utilised cells expressing hCB1 receptors (hCB1R, were performed to validate assay design and performance. While these functional pathways in GPR18-expressing cells were not modified on treatment with a panel of putative GPR18 ligands, a constitutive phenotype was discovered for this receptor. Our data reveal that GPR18 undergoes rapid constitutive receptor membrane trafficking—several-fold faster than hCB1R, a highly constitutively active receptor. To enhance the likelihood of detecting agonist-mediated receptor signalling responses, we increased GPR18 protein expression (by tagging with a preprolactin signal sequence and generated a putative constitutively inactive receptor by mutating the hGPR18 gene at amino acid site 108 (alanine to asparagine. This A108N mutant

  3. The Molecular Basis of Ligand Interaction at Free Fatty Acid Receptor 4 (FFA4/GPR120)*

    Science.gov (United States)

    Hudson, Brian D.; Shimpukade, Bharat; Milligan, Graeme; Ulven, Trond

    2014-01-01

    The long-chain fatty acid receptor FFA4 (previously GPR120) is receiving substantial interest as a novel target for the treatment of metabolic and inflammatory disease. This study examines for the first time the detailed mode of binding of both long-chain fatty acid and synthetic agonist ligands at FFA4 by integrating molecular modeling, receptor mutagenesis, and ligand structure-activity relationship approaches in an iterative format. In doing so, residues required for binding of fatty acid and synthetic agonists to FFA4 have been identified. This has allowed for the refinement of a well validated model of the mode of ligand-FFA4 interaction that will be invaluable in the identification of novel ligands and the future development of this receptor as a therapeutic target. The model reliably predicted the effects of substituent variations on agonist potency, and it was also able to predict the qualitative effect of binding site mutations in the majority of cases. PMID:24860101

  4. A gate-latch-lock mechanism for hormone signalling by abscisic acid receptors

    Energy Technology Data Exchange (ETDEWEB)

    Melcher, Karsten; Ng, Ley-Moy; Zhou, X Edward; Soon, Fen-Fen; Xu, Yong; Suino-Powell, Kelly M; Park, Sang-Youl; Weiner, Joshua J; Fujii, Hiroaki; Chinnusamy, Viswanathan; Kovach, Amanda; Li, Jun; Wang, Yonghong; Li, Jiayang; Peterson, Francis C; Jensen, Davin R; Yong, Eu-Leong; Volkman, Brian F; Cutler, Sean R; Zhu, Jian-Kang; Xu, H Eric; (NU Sinapore); (Van Andel); (MCW); (UCR); (Chinese Aca. Sci.)

    2010-01-12

    Abscisic acid (ABA) is a ubiquitous hormone that regulates plant growth, development and responses to environmental stresses. Its action is mediated by the PYR/PYL/RCAR family of START proteins, but it remains unclear how these receptors bind ABA and, in turn, how hormone binding leads to inhibition of the downstream type 2C protein phosphatase (PP2C) effectors. Here we report crystal structures of apo and ABA-bound receptors as well as a ternary PYL2-ABA-PP2C complex. The apo receptors contain an open ligand-binding pocket flanked by a gate that closes in response to ABA by way of conformational changes in two highly conserved β-loops that serve as a gate and latch. Moreover, ABA-induced closure of the gate creates a surface that enables the receptor to dock into and competitively inhibit the PP2C active site. A conserved tryptophan in the PP2C inserts directly between the gate and latch, which functions to further lock the receptor in a closed conformation. Together, our results identify a conserved gate-latch-lock mechanism underlying ABA signalling.

  5. A gate-latch-lock mechanism for hormone signalling by abscisic acid receptors

    KAUST Repository

    Melcher, Karsten

    2009-12-03

    Abscisic acid (ABA) is a ubiquitous hormone that regulates plant growth, development and responses to environmental stresses. Its action is mediated by the PYR/PYL/RCAR family of START proteins, but it remains unclear how these receptors bind ABA and, in turn, how hormone binding leads to inhibition of the downstream type 2C protein phosphatase (PP2C) effectors. Here we report crystal structures of apo and ABA-bound receptors as well as a ternary PYL2-ABA-PP2C complex. The apo receptors contain an open ligand-binding pocket flanked by a gate that closes in response to ABA by way of conformational changes in two highly conserved ?-loops that serve as a gate and latch. Moreover, ABA-induced closure of the gate creates a surface that enables the receptor to dock into and competitively inhibit the PP2C active site. A conserved tryptophan in the PP2C inserts directly between the gate and latch, which functions to further lock the receptor in a closed conformation. Together, our results identify a conserved gate-latch-lock mechanism underlying ABA signalling. © 2009 Macmillan Publishers Limited. All rights reserved.

  6. Aldose Reductase Acts as a Selective Derepressor of PPARγ and the Retinoic Acid Receptor

    Directory of Open Access Journals (Sweden)

    Devi Thiagarajan

    2016-04-01

    Full Text Available Histone deacetylase 3 (HDAC3, a chromatin-modifying enzyme, requires association with the deacetylase-containing domain (DAD of the nuclear receptor corepressors NCOR1 and SMRT for its stability and activity. Here, we show that aldose reductase (AR, the rate-limiting enzyme of the polyol pathway, competes with HDAC3 to bind the NCOR1/SMRT DAD. Increased AR expression leads to HDAC3 degradation followed by increased PPARγ signaling, resulting in lipid accumulation in the heart. AR also downregulates expression of nuclear corepressor complex cofactors including Gps2 and Tblr1, thus affecting activity of the nuclear corepressor complex itself. Though AR reduces HDAC3-corepressor complex formation, it specifically derepresses the retinoic acid receptor (RAR, but not other nuclear receptors such as the thyroid receptor (TR and liver X receptor (LXR. In summary, this work defines a distinct role for AR in lipid and retinoid metabolism through HDAC3 regulation and consequent derepression of PPARγ and RAR.

  7. Structure-based drug design targeting the cell membrane receptor GPBAR1: exploiting the bile acid scaffold towards selective agonism

    Science.gov (United States)

    di Leva, Francesco Saverio; Festa, Carmen; Renga, Barbara; Sepe, Valentina; Novellino, Ettore; Fiorucci, Stefano; Zampella, Angela; Limongelli, Vittorio

    2015-11-01

    Bile acids can regulate nutrient metabolism through the activation of the cell membrane receptor GPBAR1 and the nuclear receptor FXR. Developing an exogenous control over these receptors represents an attractive strategy for the treatment of enterohepatic and metabolic disorders. A number of dual GPBAR1/FXR agonists are known, however their therapeutic use is limited by multiple unwanted effects due to activation of the diverse downstream signals controlled by the two receptors. On the other hand, designing selective GPBAR1 and FXR agonists is challenging since the two proteins share similar structural requisites for ligand binding. Here, taking advantage of our knowledge of the two targets, we have identified through a rational drug design study a series of amine lithocholic acid derivatives as selective GPBAR1 agonists. The presence of the 3α-NH2 group on the steroidal scaffold is responsible for the selectivity over FXR unveiling unprecedented structural insights into bile acid receptors activity modulation.

  8. Oleanolic Acid-Mediated Inhibition of Pregnane X Receptor and Constitutive Androstane Receptor Attenuates Rifampin-Isoniazid Cytotoxicity.

    Science.gov (United States)

    Lin, Yen-Ning; Chen, Chao-Jung; Chang, Hsiao-Yun; Cheng, Wai-Kok; Lee, Ying-Ray; Chen, Jih-Jung; Lim, Yun-Ping

    2017-10-04

    Interactions between transcriptional inducers of cytochrome P450 (CYP450) and pharmacological agents might decrease drug efficacy and induce side effects. Such interactions could be prevented using an antagonist of the pregnane X receptor (PXR) and constitutive androstane receptor (CAR). Here, we aimed to determine the antagonistic effect of oleanolic acid (OA) on PXR and CAR. OA attenuated the promoter activities, expressions, and enzyme catalytic activities of CYP3A4 and CYP2B6 mediated by rifampin (RIF) and CITCO. Moreover, OA displayed species specificity for rodent PXR. Interaction of coregulators with PXR and transcriptional complexes on the CYP3A4 promoter was disrupted by OA. Additionally, OA reversed the cytotoxic effects of isoniazid induced by RIF. These data demonstrate that OA inhibited the transactivation of PXR and CAR, reduced the expression and function of CYP3A4 and CYP2B6, and may therefore serve as an effective agent for reducing probability adverse interactions between transcriptional inducers of CYP450 and therapeutic drugs.

  9. Inhibition of lysophosphatidic acid receptors 1 and 3 attenuates atherosclerosis development in LDL-receptor deficient mice.

    Science.gov (United States)

    Kritikou, Eva; van Puijvelde, Gijs H M; van der Heijden, Thomas; van Santbrink, Peter J; Swart, Maarten; Schaftenaar, Frank H; Kröner, Mara J; Kuiper, Johan; Bot, Ilze

    2016-11-24

    Lysophosphatidic acid (LPA) is a natural lysophospholipid present at high concentrations within lipid-rich atherosclerotic plaques. Upon local accumulation in the damaged vessels, LPA can act as a potent activator for various types of immune cells through its specific membrane receptors LPA1/3. LPA elicits chemotactic, pro-inflammatory and apoptotic effects that lead to atherosclerotic plaque progression. In this study we aimed to inhibit LPA signaling by means of LPA1/3 antagonism using the small molecule Ki16425. We show that LPA1/3 inhibition significantly impaired atherosclerosis progression. Treatment with Ki16425 also resulted in reduced CCL2 production and secretion, which led to less monocyte and neutrophil infiltration. Furthermore, we provide evidence that LPA1/3 blockade enhanced the percentage of non-inflammatory, Ly6C(low) monocytes and CD4(+) CD25(+) FoxP3(+) T-regulatory cells. Finally, we demonstrate that LPA1/3 antagonism mildly reduced plasma LDL cholesterol levels. Therefore, pharmacological inhibition of LPA1/3 receptors may prove a promising approach to diminish atherosclerosis development.

  10. PTH1 receptor is involved in mediating cellular response to long-chain polyunsaturated fatty acids.

    Directory of Open Access Journals (Sweden)

    Jose Candelario

    Full Text Available The molecular pathways by which long chain polyunsaturated fatty acids (LCPUFA influence skeletal health remain elusive. Both LCPUFA and parathyroid hormone type 1 receptor (PTH1R are known to be involved in bone metabolism while any direct link between the two is yet to be established. Here we report that LCPUFA are capable of direct, PTH1R dependent activation of extracellular ligand-regulated kinases (ERK. From a wide range of fatty acids studied, varying in chain length, saturation, and position of double bonds, eicosapentaenoic (EPA and docosahexaenoic fatty acids (DHA caused the highest ERK phosphorylation. Moreover, EPA potentiated the effect of parathyroid hormone (PTH(1-34 in a superagonistic manner. EPA or DHA dependent ERK phosphorylation was inhibited by the PTH1R antagonist and by knockdown of PTH1R. Inhibition of PTH1R downstream signaling molecules, protein kinases A (PKA and C (PKC, reduced EPA and DHA dependent ERK phosphorylation indicating that fatty acids predominantly activate G-protein pathway and not the β-arrestin pathway. Using picosecond time-resolved fluorescence microscopy and a genetically engineered PTH1R sensor (PTH-CC, we detected conformational responses to EPA similar to those caused by PTH(1-34. PTH1R antagonist blocked the EPA induced conformational response of the PTH-CC. Competitive binding studies using fluorescence anisotropy technique showed that EPA and DHA competitively bind to and alter the affinity of PTH1 receptor to PTH(1-34 leading to a superagonistic response. Finally, we showed that EPA stimulates protein kinase B (Akt phosphorylation in a PTH1R-dependent manner and affects the osteoblast survival pathway, by inhibiting glucocorticoid-induced cell death. Our findings demonstrate for the first time that LCPUFAs, EPA and DHA, can activate PTH1R receptor at nanomolar concentrations and consequently provide a putative molecular mechanism for the action of fatty acids in bone.

  11. Human trafficking in domestic legislature

    Directory of Open Access Journals (Sweden)

    Skakavac Zdravko

    2008-01-01

    Full Text Available Human trafficking is an occurrence that, even in our time, is present in alarming proportions, in its actuality and consequences. It is a phenomenon with a long history and has been qualified as a serious international problem and is the object of interest for a large number of international subjects. However, the key international document that defines this phenomenon is the Convention against Transnational Organized Crime from Palermo 2000; specifically its Protocol to Prevent, Suppress and Punish Trafficking in Persons, especially Women and Children. After its adoption, intensive actions were undertaken to regulate the phenomenon on the level of national legislature. It's done so in the local legislature too. According to the criminal law of the republic of Serbia, besides the concrete law against human trafficking, a number of other crimes are connected to human trafficking. This paper deals with the most important ones. The purpose of this paper is to review the legislature on the phenomenon in the domestic law, then the accordance of incrimination with international standards, as well as to indicate the need for further changes in domestic legislature.

  12. Role of the retinoic acid receptor-α in HIV-associated nephropathy.

    Science.gov (United States)

    Ratnam, Krishna K; Feng, Xiaobei; Chuang, Peter Y; Verma, Vikram; Lu, Ting-Chi; Wang, Jinshan; Jin, Yuanmeng; Farias, Eduardo F; Napoli, Joseph L; Chen, Nan; Kaufman, Lewis; Takano, Tomoko; D'Agati, Vivette D; Klotman, Paul E; He, John C

    2011-03-01

    All-trans retinoic acid protects against the development of HIV-associated nephropathy (HIVAN) in HIV-1 transgenic mice (Tg26). In vitro, all-trans retinoic acid inhibits HIV-induced podocyte proliferation and restores podocyte differentiation markers by activating its receptor-α (RARα). Here, we report that Am580, a water-soluble RARα-specific agonist, attenuated proteinuria, glomerosclerosis, and podocyte proliferation, and restored podocyte differentiation markers in kidneys of Tg26 mice. Furthermore, RARα-/- Tg26 mice developed more severe kidney and podocyte injury than did RARα+/- Tg26 mice. Am580 failed to ameliorate kidney injury in RARα-/- Tg26 mice, confirming our hypothesis that Am580 acts through RARα. Although the expression of RARα-target genes was suppressed in the kidneys of Tg26 mice and of patients with HIVAN, the expression of RARα in the kidney was not different between patients with HIVAN and minimal change disease. However, the tissue levels of retinoic acid were reduced in the kidney cortex and isolated glomeruli of Tg26 mice. Consistent with this, the expression of two key enzymes in the retinoic acid synthetic pathway, retinol dehydrogenase type 1 and 9, and the overall enzymatic activity for retinoic acid synthesis were significantly reduced in the glomeruli of Tg26 mice. Thus, a defect in the endogenous synthesis of retinoic acid contributes to loss of the protection by retinoic acid in HIVAN. Hence, RARα agonists may be potential agents for the treatment of HIVAN.

  13. Folic acid mediates activation of the pro-oncogene STAT3 via the Folate Receptor alpha.

    Science.gov (United States)

    Hansen, Mariann F; Greibe, Eva; Skovbjerg, Signe; Rohde, Sarah; Kristensen, Anders C M; Jensen, Trine R; Stentoft, Charlotte; Kjær, Karina H; Kronborg, Camilla S; Martensen, Pia M

    2015-07-01

    The signal transducer and activator of transcription 3 (STAT3) is a well-described pro-oncogene found constitutively activated in several cancer types. Folates are B vitamins that, when taken up by cells through the Reduced Folate Carrier (RFC), are essential for normal cell growth and replication. Many cancer cells overexpress a glycophosphatidylinositol (GPI)-anchored Folate Receptor α (FRα). The function of FRα in cancer cells is still poorly described, and it has been suggested that transport of folate is not its primary function in these cells. We show here that folic acid and folinic acid can activate STAT3 through FRα in a Janus Kinase (JAK)-dependent manner, and we demonstrate that gp130 functions as a transducing receptor for this signalling. Moreover, folic acid can promote dose dependent cell proliferation in FRα-positive HeLa cells, but not in FRα-negative HEK293 cells. After folic acid treatment of HeLa cells, up-regulation of the STAT3 responsive genes Cyclin A2 and Vascular Endothelial Growth Factor (VEGF) were verified by qRT-PCR. The identification of this FRα-STAT3 signal transduction pathway activated by folic and folinic acid contributes to the understanding of the involvement of folic acid in preventing neural tube defects as well as in tumour growth. Previously, the role of folates in these diseases has been attributed to their roles as one-carbon unit donors following endocytosis into the cell. Our finding that folic acid can activate STAT3 via FRα adds complexity to the established roles of B9 vitamins in cancer and neural tube defects.

  14. Salicylic acid receptors activate jasmonic acid signalling through a non-canonical pathway to promote effector-triggered immunity

    Science.gov (United States)

    Liu, Lijing; Sonbol, Fathi-Mohamed; Huot, Bethany; Gu, Yangnan; Withers, John; Mwimba, Musoki; Yao, Jian; He, Sheng Yang; Dong, Xinnian

    2016-01-01

    It is an apparent conundrum how plants evolved effector-triggered immunity (ETI), involving programmed cell death (PCD), as a major defence mechanism against biotrophic pathogens, because ETI-associated PCD could leave them vulnerable to necrotrophic pathogens that thrive on dead host cells. Interestingly, during ETI, the normally antagonistic defence hormones, salicylic acid (SA) and jasmonic acid (JA) associated with defence against biotrophs and necrotrophs respectively, both accumulate to high levels. In this study, we made the surprising finding that JA is a positive regulator of RPS2-mediated ETI. Early induction of JA-responsive genes and de novo JA synthesis following SA accumulation is activated through the SA receptors NPR3 and NPR4, instead of the JA receptor COI1. We provide evidence that NPR3 and NPR4 may mediate this effect by promoting degradation of the JA transcriptional repressor JAZs. This unique interplay between SA and JA offers a possible explanation of how plants can mount defence against a biotrophic pathogen without becoming vulnerable to necrotrophic pathogens. PMID:27725643

  15. Receptor component protein (RCP): a member of a multi-protein complex required for G-protein-coupled signal transduction.

    Science.gov (United States)

    Prado, M A; Evans-Bain, B; Dickerson, I M

    2002-08-01

    The calcitonin-gene-related peptide (CGRP) receptor component protein (RCP) is a 148-amino-acid intracellular protein that is required for G-protein-coupled signal transduction at receptors for the neuropeptide CGRP. RCP works in conjunction with two other proteins to constitute a functional CGRP receptor: calcitonin-receptor-like receptor (CRLR) and receptor-activity-modifying protein 1 (RAMP1). CRLR has the stereotypical seven-transmembrane topology of a G-protein-coupled receptor; it requires RAMP1 for trafficking to the cell surface and for ligand specificity, and requires RCP for coupling to the cellular signal transduction pathway. We have made cell lines that expressed an antisense construct of RCP and determined that CGRP-mediated signal transduction was reduced, while CGRP binding was unaffected. Furthermore, signalling at two other endogenous G-protein-coupled receptors was unaffected, suggesting that RCP was specific for a limited subset of receptors.

  16. Cellular membrane trafficking of mesoporous silica nanoparticles

    Energy Technology Data Exchange (ETDEWEB)

    Fang, I-Ju [Iowa State Univ., Ames, IA (United States)

    2012-01-01

    This dissertation mainly focuses on the investigation of the cellular membrane trafficking of mesoporous silica nanoparticles. We are interested in the study of endocytosis and exocytosis behaviors of mesoporous silica nanoparticles with desired surface functionality. The relationship between mesoporous silica nanoparticles and membrane trafficking of cells, either cancerous cells or normal cells was examined. Since mesoporous silica nanoparticles were applied in many drug delivery cases, the endocytotic efficiency of mesoporous silica nanoparticles needs to be investigated in more details in order to design the cellular drug delivery system in the controlled way. It is well known that cells can engulf some molecules outside of the cells through a receptor-ligand associated endocytosis. We are interested to determine if those biomolecules binding to cell surface receptors can be utilized on mesoporous silica nanoparticle materials to improve the uptake efficiency or govern the mechanism of endocytosis of mesoporous silica nanoparticles. Arginine-glycine-aspartate (RGD) is a small peptide recognized by cell integrin receptors and it was reported that avidin internalization was highly promoted by tumor lectin. Both RGD and avidin were linked to the surface of mesoporous silica nanoparticle materials to investigate the effect of receptor-associated biomolecule on cellular endocytosis efficiency. The effect of ligand types, ligand conformation and ligand density were discussed in Chapter 2 and 3. Furthermore, the exocytosis of mesoporous silica nanoparticles is very attractive for biological applications. The cellular protein sequestration study of mesoporous silica nanoparticles was examined for further information of the intracellular pathway of endocytosed mesoporous silica nanoparticle materials. The surface functionality of mesoporous silica nanoparticle materials demonstrated selectivity among the materials and cancer and normal cell lines. We aimed to determine

  17. ISOLATION AND CHARACTERIZATION OF AXOLOTL NPDC-1 AND ITS EFFECTS ON RETINOIC ACID RECEPTOR SIGNALING

    Science.gov (United States)

    Theodosiou, Maria; Monaghan, James R; Spencer, Michael L; Voss, S Randal; Noonan, Daniel J

    2009-01-01

    Retinoic acid, a key morphogen in early vertebrate development and tissue regeneration, mediates its effects through the binding of receptors that act as ligand-induced transcription factors. These binding events function to recruit an array of transcription co-regulatory proteins to specific gene promoters. One such co-regulatory protein, neuronal proliferation and differentiation control-1 (NPDC-1), is broadly expressed during mammalian development and functions as an in vitro repressor of retinoic acid receptor (RAR)-mediated transcription. To obtain comparative and developmental insights about NPDC-1 function, we cloned the axolotl (Ambystoma mexicanum) orthologue and measured transcript abundances among tissues sampled during the embryonic and juvenile phases of development, and also during spinal cord regeneration. Structurally, the axolotl orthologue of NPDC-1 retained sequence identity to mammalian sequences in all functional domains. Functionally, we observed that axolotl NPDC-1 mRNA expression peaked late in embryogenesis, with highest levels of expression occurring during the time of limb development, a process regulated by retinoic acid signaling. Also similar to what has been observed in mammals, axolotl NPDC-1 directly interacts with axolotl RAR, modulates axolotl RAR DNA binding, and represses cell proliferation and axolotl RAR-mediated gene transcription. These data justify axolotl as a model to further investigate NPDC-1 and its role in regulating retinoic acid signaling. PMID:17331771

  18. Novel retinoic acid receptor alpha agonists for treatment of kidney disease.

    Directory of Open Access Journals (Sweden)

    Yifei Zhong

    Full Text Available Development of pharmacologic agents that protect podocytes from injury is a critical strategy for the treatment of kidney glomerular diseases. Retinoic acid reduces proteinuria and glomerulosclerosis in multiple animal models of kidney diseases. However, clinical studies are limited because of significant side effects of retinoic acid. Animal studies suggest that all trans retinoic acid (ATRA attenuates proteinuria by protecting podocytes from injury. The physiological actions of ATRA are mediated by binding to all three isoforms of the nuclear retinoic acid receptors (RARs: RARα, RARβ, and RARγ. We have previously shown that ATRA exerts its renal protective effects mainly through the agonism of RARα. Here, we designed and synthesized a novel boron-containing derivative of the RARα-specific agonist Am580. This new derivative, BD4, binds to RARα receptor specifically and is predicted to have less toxicity based on its structure. We confirmed experimentally that BD4 binds to RARα with a higher affinity and exhibits less cellular toxicity than Am580 and ATRA. BD4 induces the expression of podocyte differentiation markers (synaptopodin, nephrin, and WT-1 in cultured podocytes. Finally, we confirmed that BD4 reduces proteinuria and improves kidney injury in HIV-1 transgenic mice, a model for HIV-associated nephropathy (HIVAN. Mice treated with BD4 did not develop any obvious toxicity or side effect. Our data suggest that BD4 is a novel RARα agonist, which could be used as a potential therapy for patients with kidney disease such as HIVAN.

  19. Novel retinoic acid receptor alpha agonists for treatment of kidney disease.

    Science.gov (United States)

    Zhong, Yifei; Wu, Yingwei; Liu, Ruijie; Li, Zhengzhe; Chen, Yibang; Evans, Todd; Chuang, Peter; Das, Bhaskar; He, John Cijiang

    2011-01-01

    Development of pharmacologic agents that protect podocytes from injury is a critical strategy for the treatment of kidney glomerular diseases. Retinoic acid reduces proteinuria and glomerulosclerosis in multiple animal models of kidney diseases. However, clinical studies are limited because of significant side effects of retinoic acid. Animal studies suggest that all trans retinoic acid (ATRA) attenuates proteinuria by protecting podocytes from injury. The physiological actions of ATRA are mediated by binding to all three isoforms of the nuclear retinoic acid receptors (RARs): RARα, RARβ, and RARγ. We have previously shown that ATRA exerts its renal protective effects mainly through the agonism of RARα. Here, we designed and synthesized a novel boron-containing derivative of the RARα-specific agonist Am580. This new derivative, BD4, binds to RARα receptor specifically and is predicted to have less toxicity based on its structure. We confirmed experimentally that BD4 binds to RARα with a higher affinity and exhibits less cellular toxicity than Am580 and ATRA. BD4 induces the expression of podocyte differentiation markers (synaptopodin, nephrin, and WT-1) in cultured podocytes. Finally, we confirmed that BD4 reduces proteinuria and improves kidney injury in HIV-1 transgenic mice, a model for HIV-associated nephropathy (HIVAN). Mice treated with BD4 did not develop any obvious toxicity or side effect. Our data suggest that BD4 is a novel RARα agonist, which could be used as a potential therapy for patients with kidney disease such as HIVAN.

  20. Reversible receptor methylation is essential for normal chemotaxis of Escherichia coli in gradients of aspartic acid.

    Science.gov (United States)

    Weis, R M; Koshland, D E

    1988-01-01

    The chemotaxis of wild-type cells of Escherichia coli and double mutants lacking the methyltransferase and the methylesterase activities of the receptor modification system has been compared in spatial gradients of aspartic acid. Previous studies showing that a chemotactic response can be observed for the mutant raised questions about the role of methylation in the bacterial memory. To clarify the role of methylation, the redistribution of bacteria in stabilized defined gradients of aspartic acid was monitored by light scattering. There was no redistribution of the mutant cells in nonsaturating gradients of aspartic acid, but over the same range these mutant bacteria were observed to respond and to adapt during tethering experiments. In large saturating gradients of aspartate, slight movement of the mutant up the gradient was observed. These results show that dynamic receptor methylation is required for the chemotactic response to gentle gradients of aspartic acid and that methylation resets to zero and is part of the normal wild-type memory. There are certain gradients, however, in which the methylation-deficient mutants show chemotactic ability, thus explaining the apparent anomaly. Images PMID:2829179

  1. Effect of salicyclic acid on gluccorticoid receptor in cultured fibroblasts derived from rat carrageenin granuloma.

    Science.gov (United States)

    Koshihara, Y; Yamagishi, M; Murota, S I

    1976-06-23

    The binding activity of [3H]dexamethasone to the specific receptor was studied in the cytoplasmic fraction of a established fibroblast line derived from rat carrageenin granuloma in culture condition. Specific receptor to dexamethasone was demonstrated. Scatchard analysis revealed a single class of binding sites with a dissociation constant for [3H]dexamethasone of 3.64 - 10(-8) M and a concentration of binding sites of 0.825 pmol per mg cytosol protein. The number of cytoplasmic binding sites per cell was calculated at 1.15 - 10(5). Total binding activity to [3H]dexamethasone of the cytoplasmic fraction was enhanced when the cells were cultured in a medium containing salicylic acid was at 37 degrees C. The maximum enhancement was seen at the concentration of 10(-3)M and in 3h treatment of salicylic acid. This enhancement by salicylic acid was lost when cycloheximide was added to the culture medium at the same time. If salicyclic acid was added to the cell free system, it showed no effect on the binding activity. The other non-steroidal anti-inflammatory drugs; phenylbutazone and indomethacin,also enhanced the total binding activity to [3H]dexamethasone of the cytoplasmic fraction at the concentration of 2 - 10(-5) M and 2 - 10(-7) M, respectively.

  2. Fatty Acid Amide Hydrolase (FAAH) Inhibition Enhances Memory Acquisition through Activation of PPAR-alpha Nuclear Receptors

    Science.gov (United States)

    Mazzola, Carmen; Medalie, Julie; Scherma, Maria; Panlilio, Leigh V.; Solinas, Marcello; Tanda, Gianluigi; Drago, Filippo; Cadet, Jean Lud; Goldberg, Steven R.; Yasar, Sevil

    2009-01-01

    Inhibitors of fatty acid amide hydrolase (FAAH) increase endogenous levels of anandamide (a cannabinoid CB[subscript 1]-receptor ligand) and oleoylethanolamide and palmitoylethanolamide (OEA and PEA, ligands for alpha-type peroxisome proliferator-activated nuclear receptors, PPAR-alpha) when and where they are naturally released in the brain.…

  3. Fatty Acid Amide Hydrolase (FAAH) Inhibition Enhances Memory Acquisition through Activation of PPAR-alpha Nuclear Receptors

    Science.gov (United States)

    Mazzola, Carmen; Medalie, Julie; Scherma, Maria; Panlilio, Leigh V.; Solinas, Marcello; Tanda, Gianluigi; Drago, Filippo; Cadet, Jean Lud; Goldberg, Steven R.; Yasar, Sevil

    2009-01-01

    Inhibitors of fatty acid amide hydrolase (FAAH) increase endogenous levels of anandamide (a cannabinoid CB[subscript 1]-receptor ligand) and oleoylethanolamide and palmitoylethanolamide (OEA and PEA, ligands for alpha-type peroxisome proliferator-activated nuclear receptors, PPAR-alpha) when and where they are naturally released in the brain.…

  4. Characteristics of gamma-aminobutyric acid (GABA receptors in the rat central nervous system.

    Directory of Open Access Journals (Sweden)

    Kuroda,Hiroo

    1983-04-01

    Full Text Available Characteristics of gamma-aminobutyric acid (GABA were investigated in the rat central nervous system by radioreceptor assay (RRA. Scatchard analysis revealed that the rat brain had two distinct GABA binding sites with an apparent dissociation constant (Kd of 11.7 nM and 34.7 nM. The highest level of specific [3H]-GABA binding was found in the rat cerebellum. Imidazole acetic acid, a potent GABA agonist, was effective in displacing [3H]-GABA binding but beta-alanine was slightly effective in inhibiting [3H]-GABA binding. Muscimol, the most potent GABA agonist, has been used as a ligand to characterize the postsynaptic GABA receptors. However, the maximal binding capacity (Bmax of muscimol-RRA was about 3 times larger than that of GABA-RRA, suggesting that muscimol might label not only GABA receptors but other unknown receptors as well. An endogenous inhibitor of GABA receptor binding was purified from the P2 fraction of rat brain with 0.05% Triton X-100. The endogenous inhibitor was competitive with GABA on GABA binding sites. The inhibition by the endogenous inhibitor of GABA receptor binding was blocked by the allosteric effect of diazepam. In the presence of diazepam, [3H]-GABA binding with the endogenous inhibitor was larger than that with GABA, whereas there was no difference in the absence of diazepam. This indicated that the endogenous inhibitor was not GABA itself. The molecular weight of the endogenous inhibitor was estimate by gel filtration to be less than 3,000 daltons.

  5. Human trafficking and the healthcare professional.

    Science.gov (United States)

    Barrows, Jeffrey; Finger, Reginald

    2008-05-01

    Despite the legislation passed in the 19th century outlawing human slavery, it is more widespread today than at the conclusion of the civil war. Modern human slavery, termed human trafficking, comes in several forms. The most common type of human trafficking is sex trafficking, the sale of women and children into prostitution. Labor trafficking is the sale of men, women, and children into hard labor for which they receive little or no compensation. Other forms of trafficking include child soldiering, war brides, and organ removal. Healthcare professionals play a critical role in both finding victims of human trafficking while they are still in captivity, as well as caring for their mental and physical needs upon release. Those working in the healthcare profession need to be educated regarding how a trafficking victim may present, as well as their unique healthcare needs.

  6. Pharmacological characterisation of murine α4β1δ GABAA receptors expressed in Xenopus oocytes

    DEFF Research Database (Denmark)

    Villumsen, Inge S; Wellendorph, Petrine; Smart, Trevor G

    2015-01-01

    BACKGROUND: GABAA receptor subunit composition has a profound effect on the receptor's physiological and pharmacological properties. The receptor β subunit is widely recognised for its importance in receptor assembly, trafficking and post-translational modifications, but its influence on extrasyn...

  7. Agonist-dependent endocytosis of γ-aminobutyric acid type A (GABAA) receptors revealed by a γ2(R43Q) epilepsy mutation.

    Science.gov (United States)

    Chaumont, Severine; André, Caroline; Perrais, David; Boué-Grabot, Eric; Taly, Antoine; Garret, Maurice

    2013-09-27

    GABA-gated chloride channels (GABAARs) trafficking is involved in the regulation of fast inhibitory transmission. Here, we took advantage of a γ2(R43Q) subunit mutation linked to epilepsy in humans that considerably reduces the number of GABAARs on the cell surface to better understand the trafficking of GABAARs. Using recombinant expression in cultured rat hippocampal neurons and COS-7 cells, we showed that receptors containing γ2(R43Q) were addressed to the cell membrane but underwent clathrin-mediated dynamin-dependent endocytosis. The γ2(R43Q)-dependent endocytosis was reduced by GABAAR antagonists. These data, in addition to a new homology model, suggested that a conformational change in the extracellular domain of γ2(R43Q)-containing GABAARs increased their internalization. This led us to show that endogenous and recombinant wild-type GABAAR endocytosis in both cultured neurons and COS-7 cells can be amplified by their agonists. These findings revealed not only a direct relationship between endocytosis of GABAARs and a genetic neurological disorder but also that trafficking of these receptors can be modulated by their agonist.

  8. Ginseng pharmacology: a new paradigm based on gintonin-lysophosphatidic acid receptor interactions

    Directory of Open Access Journals (Sweden)

    Seung-Yeol eNah

    2015-10-01

    Full Text Available Ginseng, the root of Panax ginseng, is used as a traditional medicine. Despite the long history of the use of ginseng, there is no specific scientific or clinical rationale for ginseng pharmacology besides its application as a general tonic. The ambiguous description of ginseng pharmacology might be due to the absence of a predominant active ingredient that represents ginseng pharmacology. Recent studies show that ginseng abundantly contains lysophosphatidic acids (LPAs, which are phospholipid-derived growth factor with diverse biological functions including those claimed to be exhibited by ginseng. LPAs in ginseng form a complex with ginseng proteins, which can bind and deliver LPA to its cognate receptors with a high affinity. As a first messenger, gintonin produces second messenger Ca2+ via G protein-coupled LPA receptors. Ca2+ is an intracellular mediator of gintonin and initiates a cascade of amplifications for further intercellular communications by activation of Ca2+-dependent kinases, receptors, gliotransmitter and neurotransmitter release. Ginsenosides, which have been regarded as primary ingredients of ginseng, cannot elicit intracellular [Ca2+]i transients, since they lack specific cell surface receptor. However, ginsenosides exhibit non-specific ion channel and receptor regulations. This is the key characteristic that distinguishes gintonin from ginsenosides. Although the current discourse on ginseng pharmacology is focused on ginsenosides, gintonin can definitely provide a mode of action for ginseng pharmacology that ginsenosides cannot. This review article introduces a novel concept of ginseng ligand-LPA receptor interaction and proposes to establish a paradigm that shifts the focus from ginsenosides to gintonin as a major ingredient representing ginseng pharmacology.

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

    Directory of Open Access Journals (Sweden)

    Nicholas D Holliday

    2012-01-01

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

  10. Anti-inflammatory gallic Acid and wedelolactone are G protein-coupled receptor-35 agonists.

    Science.gov (United States)

    Deng, Huayun; Fang, Ye

    2012-01-01

    G protein-coupled receptor-35 (GPR35) has been shown to be a target of the asthma drugs cromolyn disodium and nedocromil sodium. Gallic acid and caffeic acids are reported to modulate allergic reactions via unknown mode(s) of action. Here we attempt to elucidate whether both phenolic acids share a common mode of action with the two asthma drugs. Label-free dynamic mass redistribution (DMR) assays showed that both phenolic acids triggered robust DMR signals in HT-29 cells, whose characteristics were similar to that of cromolyn disodium. Both phenolic acids resulted in detectable β-arrestin translocation signals in an engineered U2OS cell line stably expressing a C-terminal-modified GPR35, but with lower efficacy than cromolyn disodium. Antiallergic wedelolactone was found to be a potent β-arrestin-biased GPR35 agonist. These results suggest that certain anti-inflammatory phytochemicals including gallic acid and wedelolactone may modulate inflammatory allergic action via their agonism at GPR35. GPR35 may represent a target for the treatment of allergic disorders including asthma.

  11. Ursodeoxycholic Acid Induces Death Receptor-mediated Apoptosis in Prostate Cancer Cells

    Science.gov (United States)

    Lee, Won Sup; Jung, Ji Hyun; Panchanathan, Radha; Yun, Jeong Won; Kim, Dong Hoon; Kim, Hye Jung; Kim, Gon Sup; Ryu, Chung Ho; Shin, Sung Chul; Hong, Soon Chan; Choi, Yung Hyun; Jung, Jin-Myung

    2017-01-01

    Background Bile acids have anti-cancer properties in a certain types of cancers. We determined anticancer activity and its underlying molecular mechanism of ursodeoxycholic acid (UDCA) in human DU145 prostate cancer cells. Methods Cell viability was measured with an MTT assay. UDCA-induced apoptosis was determined with flow cytometric analysis. The expression levels of apoptosis-related signaling proteins were examined with Western blotting. Results UDCA treatment significantly inhibited cell growth of DU145 in a dose-dependent manner. It induced cellular shrinkage and cytoplasmic blebs and accumulated the cells with sub-G1 DNA contents. Moreover, UDCA activated caspase 8, suggesting that UDCA-induced apoptosis is associated with extrinsic pathway. Consistent to this finding, UDCA increased the expressions of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) receptor, death receptor 4 (DR4) and death receptor 5 (DR5), and TRAIL augmented the UDCA-induced cell death in DU145 cells. In addition, UDCA also increased the expressions of Bax and cytochrome c and decreased the expression of Bcl-xL in DU145 cells. This finding suggests that UDCA-induced apoptosis may be involved in intrinsic pathway. Conclusions UDCA induces apoptosis via extrinsic pathway as well as intrinsic pathway in DU145 prostate cancer cells. UDCA may be a promising anti-cancer agent against prostate cancer.

  12. Requirement for sialic acid on the endothelial ligand of a lymphocyte homing receptor.

    Science.gov (United States)

    True, D D; Singer, M S; Lasky, L A; Rosen, S D

    1990-12-01

    The entry of blood-borne lymphocytes into most secondary lymphoid organs is initiated by a highly specific adhesive interaction with the specialized cuboidal endothelial cells of high endothelial venules (HEV). The adhesive receptors on lymphocytes that dictate interactions with HEV in different lymphoid organs are called homing receptors, signifying their critical role in controlling organ-selective lymphocyte migration. Considerable work has established that the mouse peripheral lymph node homing receptor (pnHR), defined by the mAb MEL-14, functions as a lectin-like adhesive protein. We have previously shown that sialidase treatment of peripheral lymph node (PN) HEV abrogates lymphocyte attachment to the HEV both in vivo and in vitro. We extend this evidence by demonstrating that Limax agglutinin (LA), a sialic acid-specific lectin, when reacted with HEV exposed in cryostat-cut tissue sections, blocks lymphocyte attachment to PN HEV and, unexpectedly, to the HEV of Peyer's patches (PP) as well. Using a recombinant form of the pnHR as a histochemical probe for its cognate adhesive site (HEV-ligand) on PN HEV, we demonstrate that both sialidase and Limax agglutinin functionally inactive this ligand. It is concluded that the requirement for sialic acid is at the level of the pnHR interaction with its HEV ligand. A distinct sialyloligosaccharide may encode the recognition determinant of a PP HEV ligand.

  13. Leukocyte Trafficking to the Small Intestine and Colon.

    Science.gov (United States)

    Habtezion, Aida; Nguyen, Linh P; Hadeiba, Husein; Butcher, Eugene C

    2016-02-01

    Leukocyte trafficking to the small and large intestines is tightly controlled to maintain intestinal immune homeostasis, mediate immune responses, and regulate inflammation. A wide array of chemoattractants, chemoattractant receptors, and adhesion molecules expressed by leukocytes, mucosal endothelium, epithelium, and stromal cells controls leukocyte recruitment and microenvironmental localization in intestine and in the gut-associated lymphoid tissues (GALTs). Naive lymphocytes traffic to the gut-draining mesenteric lymph nodes where they undergo antigen-induced activation and priming; these processes determine their memory/effector phenotypes and imprint them with the capacity to migrate via the lymph and blood to the intestines. Mechanisms of T-cell recruitment to GALT and of T cells and plasmablasts to the small intestine are well described. Recent advances include the discovery of an unexpected role for lectin CD22 as a B-cell homing receptor GALT, and identification of the orphan G-protein-coupled receptor 15 (GPR15) as a T-cell chemoattractant/trafficking receptor for the colon. GPR15 decorates distinct subsets of T cells in mice and humans, a difference in species that could affect translation of the results of mouse colitis models to humans. Clinical studies with antibodies to integrin α4β7 and its vascular ligand mucosal vascular addressin cell adhesion molecule 1 are proving the value of lymphocyte trafficking mechanisms as therapeutic targets for inflammatory bowel diseases. In contrast to lymphocytes, cells of the innate immune system express adhesion and chemoattractant receptors that allow them to migrate directly to effector tissue sites during inflammation. We review the mechanisms for innate and adaptive leukocyte localization to the intestinal tract and GALT, and discuss their relevance to human intestinal homeostasis and inflammation.

  14. Dynamics of exosome internalization and trafficking.

    Science.gov (United States)

    Tian, Tian; Zhu, Yan-Liang; Hu, Fei-Hu; Wang, Yuan-Yuan; Huang, Ning-Ping; Xiao, Zhong-Dang

    2013-07-01

    Cells release exosomes into extracellular medium. Although the important roles of exosomes in many physiological and pathological processes are being revealed, the mechanism of exosome-cell interaction remains unclear. In this article, employing real-time fluorescence microscopy, the motion of exosomes on the plasma membrane or in the cytoplasm of recipient PC12 cells was observed directly. In addition, several motion modes of exosomes were revealed by single particle tracking (SPT). The changes between motion modes were also detected, presenting the dynamic courses of exosome attachment onto plasma membrane and exosome uptake. Octadecyl rhodamine B chloride (R18) was found to be useful to distinguish endocytosis from fusion during exosome uptake. Colocalization with organelle markers showed exosomes were sorted to acidic vesicles after internalization. The results provide new sight into the exosome-cell interaction mode and the intercellular trafficking of exosomes. This study will help to understand the roles of exosomes at cell level.

  15. Waking action of ursodeoxycholic acid (UDCA involves histamine and GABAA receptor block.

    Directory of Open Access Journals (Sweden)

    Yevgenij Yanovsky

    Full Text Available Since ancient times ursodeoxycholic acid (UDCA, a constituent of bile, is used against gallstone formation and cholestasis. A neuroprotective action of UDCA was demonstrated recently in models of Alzheimer's disease and retinal degeneration. The mechanisms of UDCA action in the nervous system are poorly understood. We show now that UDCA promotes wakefulness during the active period of the day, lacking this activity in histamine-deficient mice. In cultured hypothalamic neurons UDCA did not affect firing rate but synchronized the firing, an effect abolished by the GABA(AR antagonist gabazine. In histaminergic neurons recorded in slices UDCA reduced amplitude and duration of spontaneous and evoked IPSCs. In acutely isolated histaminergic neurons UDCA inhibited GABA-evoked currents and sIPSCs starting at 10 µM (IC(50 = 70 µM and did not affect NMDA- and AMPA-receptor mediated currents at 100 µM. Recombinant GABA(A receptors composed of α1, β1-3 and γ2L subunits expressed in HEK293 cells displayed a sensitivity to UDCA similar to that of native GABA(A receptors. The mutation α1V256S, known to reduce the inhibitory action of pregnenolone sulphate, reduced the potency of UDCA. The mutation α1Q241L, which abolishes GABA(AR potentiation by several neurosteroids, had no effect on GABA(AR inhibition by UDCA. In conclusion, UDCA enhances alertness through disinhibition, at least partially of the histaminergic system via GABA(A receptors.

  16. Contributions of colonic short-chain fatty acid receptors in energy homeostasis

    Directory of Open Access Journals (Sweden)

    Atsukazu eKuwahara

    2014-09-01

    Full Text Available The gastrointestinal (GI tract is separated from the body’s internal environment by a single layer of epithelial cells, through which nutrients must pass for their absorption into the bloodstream. Besides food and drink, the GI lumen is also exposed to bioactive chemicals and bacterial products including short-chain fatty acids (SCFAs. Therefore, the GI tract has to monitor the composition of its contents continuously to discriminate between necessary and unnecessary compounds. Recent molecular identification of epithelial membrane receptor proteins has revealed the sensory roles of intestinal epithelial cells in the gut chemosensory system. Malfunctioning of these receptors may be responsible for a variety of metabolic dysfunctions associated with obesity and related disorders. Recent studies suggest that SCFAs produced by microbiota fermentation act as signaling molecules and influence the host’s metabolism; uncovering the sensory mechanisms of such bacterial metabolites would help us understand the interactions between the host and microbiota in host energy homeostasis. In this review, the contribution of colonic SCFA receptors in energy metabolism and our recent findings concerning the possible link between SCFA receptors and host energy homeostasis are discussed.

  17. Phylogenetic distribution of [3H]kainic acid receptor binding sites in neuronal tissue.

    Science.gov (United States)

    London, E D; Klemm, N; Coyle, J T

    1980-06-23

    derived from these displacement curves were 1.0 for unlabeled kainic acid but approximately 0.5 for L- and D-glutamic acids and dihydrokainic acid, which is compatible with negative cooperativity. In summary, these studies demonstrated a widespread distribution throughout the animal kingdom of specific binding sites for kainic acid in neural tissue; the characteristics of these receptor sites are remarkably similar from primitive vertebrates to man.

  18. Analysis of ileal sodium/bile acid cotransporter and related nuclear receptor genes in a family with multiple cases of idiopathic bile acid malabsorption

    Institute of Scientific and Technical Information of China (English)

    Marco Montagnani; Anna Abrahamsson; Cecilia G(a)lman; G(o)sta Eggertsen; Hanns-Ulrich Marschall; Elisa Ravaioli; Curt Einarsson; Paul A Dawson

    2006-01-01

    The etiology of most cases of idiopathic bile acid malabsorption (TBAM) is unknown. Tn this study, a Swedish family with bile acid malabsorption in three consecutive generations was screened for mutations in the ileal apical sodium-bile acid cotransporter gene (ASBT; gene symbol, SLC10A2) and in the genes for several of the nuclear receptors known to be important for ASBT expression: the farnesoid X receptor (FXR)and peroxisome proliferator activated receptor alpha (PPARα). The patients presented with a clinical history of idiopathic chronic watery diarrhea, which was responsive to cholestyramine treatment and consistent with IBAM. Bile acid absorption was determined using 75Se-homocholic acid taurine(SeHCAT); bile acid synthesis was estimated by measuring the plasma levels of 7α-hydroxy-4-cholesten-3-one (C4). The ASBT,FXR, and PPARα genes in the affected and unaffected family members were analyzed using single stranded conformation polymorphism (SSCP), denaturing HPLC,and direct sequencing. No ASBT mutations were identified and the ASBT gene did not segregate with the bile acid malabsorption phenotype. Similarly, no mutations or polymorphisms were identified in the FXR or PPARα genes associated with the bile acid malabsorption phenotype. These studies indicate that the intestinal bile acid malabsorption in these patients cannot be attributed to defects in ASBT. In the absence of apparent ileal disease, alternative explanations such as accelerated transit through the small intestine may be responsible for the IBAM.

  19. Glutamate-containing dipeptides do not modulate ligand binding at excitatory amino acid receptors.

    Science.gov (United States)

    Baud, J; Fagg, G E

    1986-10-08

    Dipeptides of the structure X-Glu (e.g. X = Phe, Leu) have been proposed as allosteric modulators of excitatory amino acid receptors in rat brain membranes. Here we report that these dipeptides reduce the binding of L-[3H]Glu (predominantly N-methyl-D-aspartate-sensitive sites) and of [3H]kainate to postsynaptic density preparations isolated from rat brain. However, several observations indicate that the effects of these dipeptides are mediated not by allosteric modulation, but by free L-Glu liberated by the actions of a membrane-associated aminopeptidase. The absolute and relative potencies of the dipeptides are similar at all acidic amino acid binding sites examined to date, suggesting the involvement of a factor with similar activity at each site (e.g. L-Glu). N-Acetyl-Met-Glu is a weak inhibitor of L-Glu and kainate binding, and N-blocked peptides are known to be poor substrates of aminopeptidases. Bestatin, an inhibitor of aminopeptidases, decreases or abolishes the effects of substrate dipeptides on L-Glu and kainate receptor binding, while having no effect itself.

  20. Effect of docosahexaenoic acid on interleukin-2 receptor signaling pathway in lipid rafts

    Institute of Scientific and Technical Information of China (English)

    2006-01-01

    Recent studies have shown that polyunsaturated fatty acids (PUFA) regulated the functions of membrane receptors in T cells and suppressed T cell -mediated immune responses. But the molecular mechanisms of immune regulation are not yet elucidated. Lipid rafts are plasma membrane microdomains, in which many receptors localized. The purpose of this study was to investigate the effect of DHA on IL-2R signaling pathway in lipid rafts. We isolated lipid rafts by discontinuous sucrose density gradient ultracentrifugation, and found that DHA could change the composition of lipid rafts and alter the distribution of key molecules of IL-2R signaling pathway, which transferred from lipid rafts to detergent-soluble membrane fractions. These results revealed that DHA treatment increased the proportion of polyunsaturated fatty acids especially n(3 polyunsaturated fatty acids in lipid rafts and changed the lipid environment of membrane microdomains in T cells. Compared with controls, DHA changed the localization of IL-2R, STAT5a and STAT5b in lipid rafts and suppressed the expression of JAK1, JAK3 and tyrosine phosphotyrosine in soluble membrane fractions. Summarily, this study concluded the effects of DHA on IL-2R signaling pathway in lipid rafts and explained the regulation of PUFAs in T cell-mediated immune responses.

  1. Arginine of retinoic acid receptor beta which coordinates with the carboxyl group of retinoic acid functions independent of the amino acid residues responsible for retinoic acid receptor subtype ligand specificity.

    Science.gov (United States)

    Zhang, Zeng Ping; Hutcheson, Juliet M; Poynton, Helen C; Gabriel, Jerome L; Soprano, Kenneth J; Soprano, Dianne Robert

    2003-01-15

    The biological actions of retinoic acid (RA) are mediated by retinoic acid receptors (RARalpha, RARbeta, and RARgamma) and retinoid X receptors (RXRalpha, RXRbeta, and RXRgamma). Consistent with the X-ray crystal structures of RARalpha and RARgamma, site-directed mutagenesis studies have demonstrated the importance of a conserved Arg residue (alphaArg(276), betaArg(269), and gammaArg(278)) for coordination with the carboxyl group of RA. However, mutation of Arg(269) to Ala in RARbeta causes only a 3- to 6-fold increase in the K(d) for RA and EC(50) in RA-dependent transcriptional transactivation assays while the homologous mutation in either RARalpha or RARgamma causes a 110-fold and a 45-fold increase in EC(50) value, respectively. To further investigate the nature of this difference, we prepared mutant RARs to determine the effect of conversion of betaR269A to a mutant which mimics either RARalpha ligand selectivity (betaA225S/R269A) or RARgamma ligand selectivity (betaI263M/R269A/V338A). Our results demonstrate that in RARbeta mutants that acquire either RARalpha or RARgamma ligand specificity the Arg(269) position responsible for coordination with the carboxyl group of retinoids continued to function like that of RARbeta. Furthermore, three mutant receptors (betaA225S/R269A, betaA225S/F279, and alphaF286A) were found to have a greater than wild-type affinity for the RARalpha-selective ligand Am580. Finally, a homology-based computer model of the ligand binding domain (LBD) of RARbeta and the X-ray crystal structures of the LBD of both RARalpha and RARgamma are used to describe potential mechanisms responsible for the increased affinity of some mutants for Am580 and for the difference in the effect of mutation of Arg(269) in RARbeta compared to its homologous Arg in RARalpha and RARgamma.

  2. Potential role of nuclear receptor ligand all-trans retinoic acids in the treatment of fungal keratitis

    Institute of Scientific and Technical Information of China (English)

    Hong-Yan; Zhou; Wei; Zhong; Hong; Zhang; Miao-Miao; Bi; Shuang; Wang; Wen-Song; Zhang

    2015-01-01

    ·Fungal keratitis(FK) is a worldwide visual impairment disease. This infectious fungus initiates the primary innate immune response and, later the adaptive immune response. The inflammatory process is related to a variety of immune cells, including macrophages, helper T cells, neutrophils, dendritic cells, and Treg cells, and is associated with proinflammatory, chemotactic and regulatory cytokines. All-trans retinoic acids(ATRA)have diverse immunomodulatory actions in a number of inflammatory and autoimmune conditions. These retinoids regulate the transcriptional levels of target genes through the activation of nuclear receptors.Retinoic acid receptor α(RAR α), retinoic acid receptor γ(RAR γ), and retinoid X receptor α(RXR α) are expressed in the cornea and immune cells. This paper summarizes new findings regarding ATRA in immune and inflammatory diseases and analyzes the perspective application of ATRA in FK.

  3. Potential role of nuclear receptor ligand all-trans retinoic acids in the treatment of fungal keratitis

    Directory of Open Access Journals (Sweden)

    Hong-Yan Zhou

    2015-08-01

    Full Text Available Fungal keratitis (FK is a worldwide visual impairment disease. This infectious fungus initiates the primary innate immune response and, later the adaptive immune response. The inflammatory process is related to a variety of immune cells, including macrophages, helper T cells, neutrophils, dendritic cells, and Treg cells, and is associated with proinflammatory, chemotactic and regulatory cytokines. All-trans retinoic acids (ATRA have diverse immunomodulatory actions in a number of inflammatory and autoimmune conditions. These retinoids regulate the transcriptional levels of target genes through the activation of nuclear receptors. Retinoic acid receptor α (RAR α, retinoic acid receptor γ (RAR γ, and retinoid X receptor α (RXR α are expressed in the cornea and immune cells. This paper summarizes new findings regarding ATRA in immune and inflammatory diseases and analyzes the perspective application of ATRA in FK.

  4. Characterization of influenza virus sialic acid receptors in minor poultry species.

    Science.gov (United States)

    Kimble, Brian; Nieto, Gloria Ramirez; Perez, Daniel R

    2010-12-09

    It is commonly accepted that avian influenza viruses (AIVs) bind to terminal α2,3 sialic acid (SA) residues whereas human influenza viruses bind to α2,6 SA residues. By a series of amino acid changes on the HA surface protein, AIVs can switch receptor specificity and recognize α2,6 SA positive cells, including human respiratory epithelial cells. Animal species, like pigs and Japanese quail, that contain both α2,3 and α2,6 SA become ideal environments for receptor switching. Here, we describe the SA patterns and distributions in 6 common minor domestic poultry species: Peking duck, Toulouse geese, Chinese ring-neck pheasant, white midget turkey, bobwhite quail, and pearl guinea fowl. Lectins specific to α2,3 and α2,6 SA (Maakia amurensis agglutinin and Sambuca nigra agglutinin, respectively) were used to detect SA by an alkaline phosphotase-based method and a fluorescent-based method. Differences in SA moieties and their ability to bind influenza viruses were visualized by fluorescent labeling of 4 different H3N2 influenza viruses known to be specific for one receptor or the other. The geese and ducks showed α2,3 SA throughout the respiratory tract and marginal α2,6 SA only in the colon. The four other avian species showed both α2,3 and α2,6 SA in the respiratory tract and the intestines. Furthermore, the turkey respiratory tract showed a positive correlation between age and α2,6 SA levels. The fact that these birds have both avian and human flu receptors, combined with their common presence in backyard farms and live bird markets worldwide, mark them as potential mixing bowl species and necessitates improved surveillance and additional research about the role of these birds in influenza host switching.

  5. Characterization of influenza virus sialic acid receptors in minor poultry species

    Directory of Open Access Journals (Sweden)

    Nieto Gloria

    2010-12-01

    Full Text Available Abstract It is commonly accepted that avian influenza viruses (AIVs bind to terminal α2,3 sialic acid (SA residues whereas human influenza viruses bind to α2,6 SA residues. By a series of amino acid changes on the HA surface protein, AIVs can switch receptor specificity and recognize α2,6 SA positive cells, including human respiratory epithelial cells. Animal species, like pigs and Japanese quail, that contain both α2,3 and α2,6 SA become ideal environments for receptor switching. Here, we describe the SA patterns and distributions in 6 common minor domestic poultry species: Peking duck, Toulouse geese, Chinese ring-neck pheasant, white midget turkey, bobwhite quail, and pearl guinea fowl. Lectins specific to α2,3 and α2,6 SA (Maakia amurensis agglutinin and Sambuca nigra agglutinin, respectively were used to detect SA by an alkaline phosphotase-based method and a fluorescent-based method. Differences in SA moieties and their ability to bind influenza viruses were visualized by fluorescent labeling of 4 different H3N2 influenza viruses known to be specific for one receptor or the other. The geese and ducks showed α2,3 SA throughout the respiratory tract and marginal α2,6 SA only in the colon. The four other avian species showed both α2,3 and α2,6 SA in the respiratory tract and the intestines. Furthermore, the turkey respiratory tract showed a positive correlation between age and α2,6 SA levels. The fact that these birds have both avian and human flu receptors, combined with their common presence in backyard farms and live bird markets worldwide, mark them as potential mixing bowl species and necessitates improved surveillance and additional research about the role of these birds in influenza host switching.

  6. Effects of retinoic acid on growth hormone-releasing hormone receptor, growth hormone secretagogue receptor gene expression and growth hormone secretion in rat anterior pituitary cells.

    Science.gov (United States)

    Maliza, Rita; Fujiwara, Ken; Tsukada, Takehiro; Azuma, Morio; Kikuchi, Motoshi; Yashiro, Takashi

    2016-06-30

    Retinoic acid (RA) is an important signaling molecule in embryonic development and adult tissue. The actions of RA are mediated by the nuclear receptors retinoic acid receptor (RAR) and retinoid X receptor (RXR), which regulate gene expression. RAR and RXR are widely expressed in the anterior pituitary gland. RA was reported to stimulate growth hormone (GH) gene expression in the anterior pituitary cells. However, current evidence is unclear on the role of RA in gene expression of growth hormone-releasing hormone receptor (Ghrh-r), growth hormone secretagogue receptor (Ghs-r) and somatostatin receptors (Sst-rs). Using isolated anterior pituitary cells of rats, we examined the effects of RA on gene expression of these receptors and GH release. Quantitative real-time PCR revealed that treatment with all-trans retinoic acid (ATRA; 10(-6) M) for 24 h increased gene expression levels of Ghrh-r and Ghs-r; however, expressions of Sst-r2 and Sst-r5 were unchanged. Combination treatment with the RAR-agonist Am80 and RXR-agonist PA024 mimicked the effects of ATRA on Ghrh-r and Ghs-r gene expressions. Exposure of isolated pituitary cells to ATRA had no effect on basal GH release. In contrast, ATRA increased growth hormone-releasing hormone (GHRH)- and ghrelin-stimulated GH release from cultured anterior pituitary cells. Our results suggest that expressions of Ghrh-r and Ghs-r are regulated by RA through the RAR-RXR receptor complex and that RA enhances the effects of GHRH and ghrelin on GH release from the anterior pituitary gland.

  7. Biopolitical management, economic calculation and "trafficked women".

    Science.gov (United States)

    Berman, Jacqueline

    2010-01-01

    Narratives surrounding human trafficking, especially trafficking in women for sex work, employ gendered and racialized tropes that have among their effects, a shrouding of women's economic decision-making and state collusion in benefiting from their labour. This paper explores the operation of these narratives in order to understand the ways in which they mask the economics of trafficking by sensationalizing the sexual and criminal aspects of it, which in turn allows the state to pursue political projects under the guise of a benevolent concern for trafficked women and/or protection of its own citizens. This paper will explore one national example: Article 18 of Italian Law 40 (1998). I argue that its passage has led to an increase in cooperation with criminal prosecution of traffickers largely because it approaches trafficked women as capable of making decisions about how and what they themselves want to do. This paper will also consider a more global approach to trafficking embedded in the concept of "migration management", an International Organization for Migration (IOM) framework that is now shaping EU, US and other national immigration laws and policies that impact trafficking. It will also examine the inherent limitations of both the national and global approach as an occasion to unpack how Article 18 and Migration Management function as forms of biopolitical management that participate in the production of "trafficking victims" into a massified population to be managed, rather than engender a more engaged discussion of what constitutes trafficking and how to redress it.

  8. Identification of amino acid residues in PEPHC1 important for binding to the tumor-specific receptor EGFRvIII

    DEFF Research Database (Denmark)

    Hansen, Charlotte Lund; Hansen, Paul Robert; Pedersen, Nina

    2008-01-01

    to identify the amino acid residues important for binding of PEPHC1 to EGFRvIII. The results indicate that the amino acid residues at the N-terminus of PEPHC1 are essential for the binding to the mutated receptor. One analog, [Ala(12)]PEPHC1, showed higher selective binding to EGFRvIII than PEPHC1...

  9. 4-Alkylated homoibotenic acid (HIBO) analogues: versatile pharmacological agents with diverse selectivity profiles towards metabotropic and ionotropic glutamate receptor subtypes

    DEFF Research Database (Denmark)

    Madsen, Ulf; Pickering, Darryl S; Nielsen, Birgitte;

    2005-01-01

    4-Alkylated analogues of homoibotenic acid (HIBO) have previously shown high potency and selectivity at ionotropic and metabotropic glutamic acid receptor (iGluR and mGluR) subtypes. Compounds with different selectivity profiles are valuable pharmacological tools for neuropharmacological studies...

  10. Leptin receptor polymorphisms interact with polyunsaturated fatty acids to augment risk of insulin resistance and metabolic syndrome in adults

    Science.gov (United States)

    The leptin receptor (LEPR) is associated with insulin resistance, a key feature of metabolic syndrome (MetS). Gene-fatty acid interactions may affect MetS risk. The objective was to investigate the relationship among LEPR polymorphisms, insulin resistance, andMetSrisk and whether plasma fatty acids,...

  11. The short-chain fatty acid receptor, FFA2, contributes to gestational glucose homeostasis.

    Science.gov (United States)

    Fuller, Miles; Priyadarshini, Medha; Gibbons, Sean M; Angueira, Anthony R; Brodsky, Michael; Hayes, M Geoffrey; Kovatcheva-Datchary, Petia; Bäckhed, Fredrik; Gilbert, Jack A; Lowe, William L; Layden, Brian T

    2015-11-15

    The structure of the human gastrointestinal microbiota can change during pregnancy, which may influence gestational metabolism; however, a mechanism of action remains unclear. Here we observed that in wild-type (WT) mice the relative abundance of Actinobacteria and Bacteroidetes increased during pregnancy. Along with these changes, short-chain fatty acids (SCFAs), which are mainly produced through gut microbiota fermentation, significantly changed in both the cecum and peripheral blood throughout gestation in these mice. SCFAs are recognized by G protein-coupled receptors (GPCRs) such as free fatty acid receptor-2 (FFA2), and we have previously demonstrated that the fatty acid receptor-2 gene (Ffar2) expression is higher in pancreatic islets during pregnancy. Using female Ffar2-/- mice, we explored the physiological relevance of signaling through this GPCR and found that Ffar2-deficient female mice developed fasting hyperglycemia and impaired glucose tolerance in the setting of impaired insulin secretion compared with WT mice during, but not before, pregnancy. Insulin tolerance tests were similar in Ffar2-/- and WT mice before and during pregnancy. Next, we examined the role of FFA2 in gestational β-cell mass, observing that Ffar2-/- mice had diminished gestational expansion of β-cells during pregnancy. Interestingly, mouse genotype had no significant impact on the composition of the gut microbiome, but did affect the observed SCFA profiles, suggesting a functional difference in the microbiota. Together, these results suggest a potential link between increased Ffar2 expression in islets and the alteration of circulating SCFA levels, possibly explaining how changes in the gut microbiome contribute to gestational glucose homeostasis.

  12. Recessive and dominant mutations in retinoic acid receptor beta in cases with microphthalmia and diaphragmatic hernia.

    Science.gov (United States)

    Srour, Myriam; Chitayat, David; Caron, Véronique; Chassaing, Nicolas; Bitoun, Pierre; Patry, Lysanne; Cordier, Marie-Pierre; Capo-Chichi, José-Mario; Francannet, Christine; Calvas, Patrick; Ragge, Nicola; Dobrzeniecka, Sylvia; Hamdan, Fadi F; Rouleau, Guy A; Tremblay, André; Michaud, Jacques L

    2013-10-01

    Anophthalmia and/or microphthalmia, pulmonary hypoplasia, diaphragmatic hernia, and cardiac defects are the main features of PDAC syndrome. Recessive mutations in STRA6, encoding a membrane receptor for the retinol-binding protein, have been identified in some cases with PDAC syndrome, although many cases have remained unexplained. Using whole-exome sequencing, we found that two PDAC-syndrome-affected siblings, but not their unaffected sibling, were compound heterozygous for nonsense (c.355C>T [p.Arg119(∗)]) and frameshift (c.1201_1202insCT [p.Ile403Serfs(∗)15]) mutations in retinoic acid receptor beta (RARB). Transfection studies showed that p.Arg119(∗) and p.Ile403Serfs(∗)15 altered RARB had no transcriptional activity in response to ligands, confirming that the mutations induced a loss of function. We then sequenced RARB in 15 subjects with anophthalmia and/or microphthalmia and at least one other feature of PDAC syndrome. Surprisingly, three unrelated subjects with microphthalmia and diaphragmatic hernia showed de novo missense mutations affecting the same codon; two of the subjects had the c.1159C>T (Arg387Cys) mutation, whereas the other one carried the c.1159C>A (p.Arg387Ser) mutation. We found that compared to the wild-type receptor, p.Arg387Ser and p.Arg387Cys altered RARB induced a 2- to 3-fold increase in transcriptional activity in response to retinoic acid ligands, suggesting a gain-of-function mechanism. Our study thus suggests that both recessive and dominant mutations in RARB cause anophthalmia and/or microphthalmia and diaphragmatic hernia, providing further evidence of the crucial role of the retinoic acid pathway during eye development and organogenesis.

  13. Intracellular Trafficking of Bacterial Toxins

    OpenAIRE

    Williams, Jeffrey M.; Tsai, Billy

    2016-01-01

    Bacterial toxins often translocate across a cellular membrane to gain access into the host cytosol, modifying cellular components in order to exert their toxic effects. To accomplish this feat, these toxins traffic to a membrane penetration site where they undergo conformational changes essential to eject the toxin’s catalytic subunit into the cytosol. In this brief review, we highlight recent findings that elucidate both the trafficking pathways and membrane translocation mechanisms of toxin...

  14. The stress hormone corticosterone increases synaptic alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors via serum- and glucocorticoid-inducible kinase (SGK) regulation of the GDI-Rab4 complex.

    Science.gov (United States)

    Liu, Wenhua; Yuen, Eunice Y; Yan, Zhen

    2010-02-26

    Corticosterone, the major stress hormone, plays an important role in regulating neuronal functions of the limbic system, although the cellular targets and molecular mechanisms of corticosteroid signaling are largely unknown. Here we show that a short treatment of corticosterone significantly increases alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR)-mediated synaptic transmission and AMPAR membrane trafficking in pyramidal neurons of prefrontal cortex, a key region involved in cognition and emotion. This enhancing effect of corticosterone is through a mechanism dependent on Rab4, the small GTPase-controlling receptor recycling between early endosome and plasma membrane. Guanosine nucleotide dissociation inhibitor (GDI), which regulates the cycle of Rab proteins between membrane and cytosol, forms an increased complex with Rab4 after corticosterone treatment. Corticosterone also triggers an increased GDI phosphorylation at Ser-213 by the serum- and glucocorticoid-inducible kinase (SGK). Moreover, AMPAR synaptic currents and surface expression and their regulation by corticosterone are altered by mutating Ser-213 on GDI. These results suggest that corticosterone, via SGK phosphorylation of GDI at Ser-213, increases the formation of GDI-Rab4 complex, facilitating the functional cycle of Rab4 and Rab4-mediated recycling of AMPARs to the synaptic membrane. It provides a potential mechanism underlying the role of corticosteroid stress hormone in up-regulating excitatory synaptic efficacy in cortical neurons.

  15. Gintonin enhances performance of mice in rotarod test: Involvement of lysophosphatidic acid receptors and catecholamine release.

    Science.gov (United States)

    Lee, Byung-Hwan; Kim, Jisu; Lee, Ra Mi; Choi, Sun-Hye; Kim, Hyeon-Joong; Hwang, Sung-Hee; Lee, Myung Koo; Bae, Chun-Sik; Kim, Hyoung-Chun; Rhim, Hyewon; Lim, Kiwon; Nah, Seung-Yeol

    2016-01-26

    Ginseng has a long history of use as a tonic for restoration of vigor. One example of ginseng-derived tonic effect is that it can improve physical stamina under conditions of stress. However, the active ingredient and the underlying molecular mechanism responsible for the ergogenic effect are unknown. Recent studies show that ginseng contains a novel ingredient, gintonin, which consists of a unique class of herbal-medicine lysophosphatidic acids (LPAs). Gintonin activates G protein-coupled LPA receptors to produce a transient [Ca(2+)]i signal, which is coupled to diverse intra- and inter-cellular signal transduction pathways that stimulate hormone or neurotransmitter release. However, relatively little is known about how gintonin-mediated cellular modulation is linked to physical endurance. In the present study, systemic administration of gintonin, but not ginsenosides, in fasted mice increased blood glucose concentrations in a dose-dependent manner. Gintonin treatment elevated blood glucose to a maximum level after 30min. This elevation in blood glucose level could be abrogated by the LPA1/3 receptor antagonist, Ki16425, or the β-adrenergic receptor antagonist, propranolol. Furthermore, gintonin-dependent enhanced performance of fasted mice in rotarod test was likewise abrogated by Ki16425. Gintonin also elevated plasma epinephrine and norepinephrine concentrations. The present study shows that gintonin mediates catecholamine release through activation of the LPA receptor and that activation of the β-adrenergic receptor is coupled to liver glycogenolysis, thereby increasing the supply of glucose and enhancing performance in the rotarod test. Thus, gintonin acts via the LPA-catecholamine-glycogenolysis axis, representing a candidate mechanism that can explain how ginseng treatment enhances physical stamina.

  16. Retinoic Acid-Related Orphan Receptors (RORs: Regulatory Functions in Immunity, Development, Circadian Rhythm, and Metabolism

    Directory of Open Access Journals (Sweden)

    Donald N. Cook

    2015-12-01

    Full Text Available In this overview, we provide an update on recent progress made in understanding the mechanisms of action, physiological functions, and roles in disease of retinoic acid related orphan receptors (RORs. We are particularly focusing on their roles in the regulation of adaptive and innate immunity, brain function, retinal development, cancer, glucose and lipid metabolism, circadian rhythm, metabolic and inflammatory diseases and neuropsychiatric disorders. We also summarize the current status of ROR agonists and inverse agonists, including their regulation of ROR activity and their therapeutic potential for management of various diseases in which RORs have been implicated.

  17. Effect of propofol on the reactivity of acetylcholinesterase, N-methyl-D-aspartate receptors, and gamma-aminobutyric acid receptors in the hippocampus of aged rats after chronic cerebral ischemia

    Institute of Scientific and Technical Information of China (English)

    Gang Chen; Jiangbei Cao; Weidong Mi

    2011-01-01

    We induced ischemic brain injury in aging rats to examine the effects of varying doses of propofol on hippocampal activities of acetylcholinesterase, N-methyl-D-aspartate receptors, and γ-aminobutyric acid receptors. Propofol exhibited no obvious impact on acetylcholinesterase activity, but directly activated the γ-aminobutyric acid receptor. The neuroprotective function of propofol on the hippocampus of aging rats following cerebral ischemic injury may be related to altered activities of γ-aminobutyric acid receptors and N-methyl-D-aspartate receptors.

  18. A conserved aspartic acid is important for agonist (VUAA1 and odorant/tuning receptor-dependent activation of the insect odorant co-receptor (Orco.

    Directory of Open Access Journals (Sweden)

    Brijesh N Kumar

    Full Text Available Insect odorant receptors function as heteromeric odorant-gated cation channels comprising a conventional odorant-sensitive tuning receptor, and a conserved co-receptor (Orco. An Orco agonist, VUAA1, is able to activate both heteromeric and homomeric Orco-containing channels. Very little is known about specific residues in Orco that contribute to cation permeability and gating. We investigated the importance of two conserved Asp residues, one in each of transmembrane domains 5 and 7, for channel function by mutagenesis. Drosophila melanogaster Orco and its substitution mutants were expressed in HEK cells and VUAA1-stimulated channel activity was determined by Ca(2+ influx and whole-cell patch clamp electrophysiology. Substitution of D466 in transmembrane 7 with amino acids other than glutamic acid resulted in a substantial reduction in channel activity. The D466E Orco substitution mutant was ~2 times more sensitive to VUAA1. The permeability of the D466E Orco mutant to cations was unchanged relative to wild-type Orco. When D466E Orco is co-expressed with a conventional tuning odorant receptor, the heteromeric complex also shows increased sensitivity to an odorant. Thus, the effect of the D466E mutation is not specific to VUAA1 agonism or dependent on homomeric Orco assembly. We suggest the gain-of-activation characteristic of the D466E mutant identifies an amino acid that is likely to be important for activation of both heteromeric and homomeric insect odorant receptor channels.

  19. A Conserved Aspartic Acid Is Important for Agonist (VUAA1) and Odorant/Tuning Receptor-Dependent Activation of the Insect Odorant Co-Receptor (Orco)

    Science.gov (United States)

    Kumar, Brijesh N.; Taylor, Robert W.; Pask, Gregory M.; Zwiebel, Laurence J.; Newcomb, Richard D.; Christie, David L.

    2013-01-01

    Insect odorant receptors function as heteromeric odorant-gated cation channels comprising a conventional odorant-sensitive tuning receptor, and a conserved co-receptor (Orco). An Orco agonist, VUAA1, is able to activate both heteromeric and homomeric Orco-containing channels. Very little is known about specific residues in Orco that contribute to cation permeability and gating. We investigated the importance of two conserved Asp residues, one in each of transmembrane domains 5 and 7, for channel function by mutagenesis. Drosophila melanogaster Orco and its substitution mutants were expressed in HEK cells and VUAA1-stimulated channel activity was determined by Ca2+ influx and whole-cell patch clamp electrophysiology. Substitution of D466 in transmembrane 7 with amino acids other than glutamic acid resulted in a substantial reduction in channel activity. The D466E Orco substitution mutant was ∼2 times more sensitive to VUAA1. The permeability of the D466E Orco mutant to cations was unchanged relative to wild-type Orco. When D466E Orco is co-expressed with a conventional tuning odorant receptor, the heteromeric complex also shows increased sensitivity to an odorant. Thus, the effect of the D466E mutation is not specific to VUAA1 agonism or dependent on homomeric Orco assembly. We suggest the gain-of-activation characteristic of the D466E mutant identifies an amino acid that is likely to be important for activation of both heteromeric and homomeric insect odorant receptor channels. PMID:23894621

  20. [Role of the fatty acids in ovarian functions: involvement of peroxisome proliferator-activated receptors (PPAR) and adipokines].

    Science.gov (United States)

    Dupont, J; Froment, P; Ramé, C; Pierre, P; Coyral-Castel, S; Chabrolle, C

    2008-12-01

    The impact of nutrition and energy reserves on the reproductive functions is known for a very long time. However, the metabolic factors involved in the interactions between nutrition and reproduction are still poorly understood. These factors may be hormones or nutrients (glucose, protein and fatty acids). However, it remains to determine whether these factors act directly or indirectly on the reproductive tissues. In this issue, we briefly summarize the impact of fatty acids on the development of ovarian follicles, oocyte and embryo. We then discuss the current hypotheses about the mechanisms of action of these fatty acids on the ovarian functions. We describe more particularly the role of some receptors of fatty acids, Peroxisome Proliferator-Activated Receptors (PPAR) and Liver X Receptors (LXR) and two adipokines, leptin and adiponectin on ovarian cells.

  1. Characterization of Leptin Intracellular Trafficking

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    E Walum

    2009-12-01

    Full Text Available Leptin is produced by adipose tissue, and its concentration in plasma is related to the amount of fat in the body. The leptin receptor (OBR is a member of the class I cytokine receptor family and several different isoforms, produced by alternative mRNA splicing are found in many tissues, including the hypothalamus. The two predominant isoforms includes a long form (OBRl with an intracellular domain of 303 amino acids and a shorter form (OBRs with an intracellular domain of 34 amino acids. Since OBRl is mainly expressed in the hypotalamus, it has been suggested to be the main signalling form. The peripheral production of leptin by adipocyte tissue and its effects as a signal of satiety in the central nervous system imply that leptin gains access to regions of the brain regulating in energy balance by crossing the blood-brain barrier. In an attempt to characterize the intracellular transport of leptin, we have followed binding internalization and degradation of leptin in HEK293 cells. We have also monitored the intracellular transport pathway of fluorescent conjugated leptin in HEK293 cells. Phenylarsine oxide, a general inhibitor of endocytosis, as well as incubation at mild hypertonic conditions, prevented the uptake of leptin, confirming a receptor-mediated internalization process. When internalized, 125I-leptin was rapidly accumulated inside the cells and reached a maximum after 10 min. After 70 minutes about 40-50% of total counts in each time point were found in the medium as TCA-soluble material. Leptin sorting, at the level of early endosomes, did not seem to involve recycling endosomes, since FITC-leptin was sorted from Cy3- transferrin containing compartments at 37°C. At 45 minutes of continuos internalization, FITC-leptin appeared mainly accumulated in late endocytic structures colocalizing with internalized rhodamine coupled epidermial growth factor (EGF and the lysosomal marker protein lamp-1. The transport of leptin was also shown

  2. A novel hydroxyfuroic acid compound as an insulin receptor activator – structure and activity relationship of a prenylindole moiety to insulin receptor activation

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    Tsai Henry J

    2009-07-01

    Full Text Available Abstract Background Diabetes Mellitus is a chronic disease and many patients of which require frequent subcutaneous insulin injection to maintain proper blood glucose levels. Due to the inconvenience of insulin administration, an orally active insulin replacement has long been a prime target for many pharmaceutical companies. Demethylasterriquinone (DMAQ B1, extracted from tropical fungus, Pseudomassaria sp., has been reported to be an orally effective agent at lowering circulating glucose levels in diabetic (db/db mice; however, the cytotoxicity associated with the quinone moiety has not been addressed thus far. Methods A series of hydroxyfuroic acid compounds were synthesized and tested for their efficacies at activating human insulin receptor. Cytotoxicity to Chinese hamster ovary cells, selectivities over insulin-like growth factor-1 (IGF-1, epidermal growth factor (EGF, and fibroblast growth factor (FGF receptors were examined in this study. Result and Conclusion This study reports a new non-quinone DMAQ B1 derivative, a hydroxyfuroic acid compound (D-410639, which is 128 fold less cytotoxic as DMAQ B1 and as potent as compound 2, a DMAQ B1 synthetic derivative from Merck, at activating human insulin receptor. D-410639 has little activation potential on IGF-1 receptor but is a moderate inhibitor to EGF receptor. Structure and activity relationship of the prenylindole moiety to insulin receptor activation is discussed.

  3. Probing the orthosteric binding site of GABAA receptors with heterocyclic GABA carboxylic acid bioisosteres

    DEFF Research Database (Denmark)

    Petersen, Jette G; Bergmann, Rikke; Krogsgaard-Larsen, Povl;

    2013-01-01

    selective and potent GABAAR agonists. This review investigates the use of heterocyclic carboxylic acid bioisosteres within the GABAAR area. Several heterocycles including 3-hydroxyisoxazole, 3-hydroxyisoxazoline, 3-hydroxyisothiazole, and the 1- and 3-hydroxypyrazole rings have been employed in order to map...... the orthosteric binding site. The physicochemical properties of the heterocyclic moieties making them suitable for bioisosteric replacement of the carboxylic acid in the molecule of GABA are discussed. A variety of synthetic strategies for synthesis of the heterocyclic scaffolds are available. Likewise, methods...... for introduction of substituents into specific positions of the heterocyclic scaffolds facilitate the investigation of different regions in the orthosteric binding pocket in close vicinity of the core scaffolds of muscimol/GABA. The development of structural models, from pharmacophore models to receptor homology...

  4. Identification of amino acids involved in histamine potentiation of GABA(A receptors

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    Ulrike eThiel

    2015-05-01

    Full Text Available Histamine is a neurotransmitter involved in a number of physiological and neuronal functions. In mammals, such as humans and rodents, the histaminergic neurons found in the tuberomamillary nucleus (TMN project widely throughout the central nervous system (CNS. Histamine acts as positive modulator of GABA(A receptors (GABA(ARs and, in high concentrations (10 mM, as negative modulator of the strychnine-sensitive glycine receptor. However, the exact molecular mechanisms by which histamine acts on GABA(ARs are unknown. In our study, we aimed to identify amino acids potentially involved in the modulatory effect of histamine on GABA(ARs. We expressed GABA(ARs with 12 different point mutations in Xenopus laevis oocytes and characterized the effect of histamine on GABA-induced currents using the two-electrode voltage clamp technique. Our data demonstrate that the amino acid residues ß2(N265 and ß2(M286, which are important for modulation by propofol, are not involved in the action of histamine. However, we found that histamine modulation is dependent on the amino acid residues alpha1(R120, ß2(Y157, ß3(D163, ß3(V175 and ß3(Q185. We showed that the amino acid residues ß2(Y157 and ß3(Q185 mediate the positive modulatory effect of histamine on GABA-induced currents, whereas alpha1(R120 and ß2(D163 form a potential histamine interaction site in GABA(ARs.

  5. Effects of bile acids and the bile acid receptor FXR agonist on the respiratory rhythm in the in vitro brainstem medulla slice of neonatal Sprague-Dawley rats.

    Directory of Open Access Journals (Sweden)

    Cong Zhao

    Full Text Available Intrahepatic cholestasis of pregnancy is always accompanied by adverse fetal outcomes such as malfunctions of respiration. Farnesoid X receptor (FXR plays a critical role in the homeostasis of bile acids. Thus, we are determined to explore the effects of farnesoid X receptor (FXR and five bile acids on respiratory rhythm generation and modulation of neonatal rats. Spontaneous periodic respiratory-related rhythmical discharge activity (RRDA was recorded from hypoglossal nerves during the perfusion of modified Krebs solution. Group 1-6 was each given GW4064 and five bile acids of chenodeoxycholic acid (CDCA, deoxycholic acid (DCA, lithocholic acid (LCA, cholic acid (CA as well as ursodeoxycholic acid (UDCA at different concentrations to identify their specific functions on respiratory rhythm modulations. Group 7 was applied to receive FXR blocker Z-guggulsterone and Z-guggulsterone with the above bile acids separately to explore the role of FXR in the respiratory rhythm modulation. Group 8 was given dimethyl sulfoxide (DMSO as controls. Apart from UDCA, CDCA, DCA LCA and CA all exerted effects on RRDA recorded from hypoglossal nerves in a concentration-dependent manner. Respiratory cycle (RC, Inspiratory time (TI, Expiratory Time (TE and Integral Amplitude (IA were influenced and such effects could be reversed by Z-guggulsterone. FXR may contribute to the effects on the modulation of respiratory rhythm exerted by bile acids.

  6. Structure-activity relationships of fatty acid amide ligands in activating and desensitizing G protein-coupled receptor 119.

    Science.gov (United States)

    Kumar, Pritesh; Kumar, Akhilesh; Song, Zhao-Hui

    2014-01-15

    The purpose of the current study was to apply a high throughput assay to investigate the structure-activity relationships of fatty acid amides for activating and desensitizing G protein-coupled receptor 119, a promising therapeutic target for both type 2 diabetes and obesity. A cell-based, homogenous time resolved fluorescence (HTRF) method for measuring G protein-coupled receptor 119-mediated increase of cyclic adenosine monophosphate (cAMP) levels was validated and applied in this study. Using novel fatty acid amides and detailed potency and efficacy analyses, we have demonstrated that degree of saturation in acyl chain and charged head groups of fatty acid amides have profound effects on the ability of these compounds to activate G protein-coupled receptor 119. In addition, we have demonstrated for the first time that pretreatments with G protein-coupled receptor 119 agonists desensitize the receptor and the degrees of desensitization caused by fatty acid amides correlate well with their structure-activity relationships in activating the receptor.

  7. Barriers to Combating Human Trafficking in Colombia

    Science.gov (United States)

    2015-03-01

    COMBATING HUMAN TRAFFICKING IN COLOMBIA by Daniel Joseph Wilcox March 2015 Thesis Advisor: Rodrigo Nieto-Gómez Second Reader: Maiah Jaskoski...Master ’s Thesis 4. TITLE AND SUBTITLE BARRIERS TO COMBATING HUMAN TRAFFICKING IN COLOMBIA 6. AUTHOR(S) Daniel Joseph Wilcox 7. PERFORMING...maximum 200 words) 12b. DISTRIBUTION CODE Despite intema.tional and domestic policies and programs intended to combat human trafficking , Colombia

  8. Decreasing Human Trafficking through Sex Work Decriminalization.

    Science.gov (United States)

    Albright, Erin; D'Adamo, Kate

    2017-01-01

    In order to decrease human trafficking, health care workers should support the full decriminalization of prostitution. Similar to trafficking in other forms of labor, preventing trafficking in the sex trade requires addressing the different forms of marginalization that create vulnerable communities. By removing punitive laws that prevent reporting of exploitation and abuse, decriminalization allows sex workers to work more safely, thereby reducing marginalization and vulnerability. Decriminalization can also help destigmatize sex work and help resist political, social, and cultural marginalization of sex workers.

  9. The political aspects of human trafficking

    Directory of Open Access Journals (Sweden)

    N. M. Lukach

    2014-01-01

    The negative international results of human trafficking are researched by the author. Namely, problems of governance organization that are created by powerful criminal groups of human traffickers and smugglers, mass stay of a significant number of non­citizens in the country; formation of the negative international image of the origin, destination or transit country as the state which is unable to counter effectively illegal migration and human trafficking.

  10. Bacterial d-amino acids suppress sinonasal innate immunity through sweet taste receptors in solitary chemosensory cells.

    Science.gov (United States)

    Lee, Robert J; Hariri, Benjamin M; McMahon, Derek B; Chen, Bei; Doghramji, Laurel; Adappa, Nithin D; Palmer, James N; Kennedy, David W; Jiang, Peihua; Margolskee, Robert F; Cohen, Noam A

    2017-09-05

    In the upper respiratory epithelium, bitter and sweet taste receptors present in solitary chemosensory cells influence antimicrobial innate immune defense responses. Whereas activation of bitter taste receptors (T2Rs) stimulates surrounding epithelial cells to release antimicrobial peptides, activation of the sweet taste receptor (T1R) in the same cells inhibits this response. This mechanism is thought to control the magnitude of antimicrobial peptide release based on the sugar content of airway surface liquid. We hypothesized that d-amino acids, which are produced by various bacteria and activate T1R in taste receptor cells in the mouth, may also activate T1R in the airway. We showed that both the T1R2 and T1R3 subunits of the sweet taste receptor (T1R2/3) were present in the same chemosensory cells of primary human sinonasal epithelial cultures. Respiratory isolates of Staphylococcus species, but not Pseudomonas aeruginosa, produced at least two d-amino acids that activate the sweet taste receptor. In addition to inhibiting P. aeruginosa biofilm formation, d-amino acids derived from Staphylococcus inhibited T2R-mediated signaling and defensin secretion in sinonasal cells by activating T1R2/3. d-Amino acid-mediated activation of T1R2/3 also enhanced epithelial cell death during challenge with Staphylococcus aureus in the presence of the bitter receptor-activating compound denatonium benzoate. These data establish a potential mechanism for interkingdom signaling in the airway mediated by bacterial d-amino acids and the mammalian sweet taste receptor in airway chemosensory cells. Copyright © 2017 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.

  11. NR4A orphan nuclear receptors influence retinoic acid and docosahexaenoic acid signaling via up-regulation of fatty acid binding protein 5

    Energy Technology Data Exchange (ETDEWEB)

    Volakakis, Nikolaos; Joodmardi, Eliza [Ludwig Institute for Cancer Research Ltd., Box 240, S-17177 Stockholm (Sweden); Perlmann, Thomas, E-mail: thomas.perlmann@licr.ki.se [Ludwig Institute for Cancer Research Ltd., Box 240, S-17177 Stockholm (Sweden); The Department of Cell and Molecular Biology, Karolinska Institute, S-17177 Stockholm (Sweden)

    2009-12-25

    The orphan nuclear receptor (NR) Nurr1 is expressed in the developing and adult nervous system and is also induced as an immediate early gene in a variety of cell types. In silico analysis of human promoters identified fatty acid binding protein 5 (FABP5), a protein shown to enhance retinoic acid-mediated PPAR{beta}/{delta} signaling, as a potential Nurr1 target gene. Nurr1 has previously been implicated in retinoid signaling via its heterodimerization partner RXR. Since NRs are commonly involved in cross-regulatory control we decided to further investigate the regulatory relationship between Nurr1 and FABP5. FABP5 expression was up-regulated by Nurr1 and other NR4A NRs in HEK293 cells, and Nurr1 was shown to activate and bind to the FABP5 promoter, supporting that FABP5 is a direct downstream target of NR4A NRs. We also show that the RXR ligand docosahexaenoic acid (DHA) can induce nuclear translocation of FABP5. Moreover, via up-regulation of FABP5 Nurr1 can enhance retinoic acid-induced signaling of PPAR{beta}/{delta} and DHA-induced activation of RXR. We also found that other members of the NR4A orphan NRs can up-regulate FABP5. Thus, our findings suggest that NR4A orphan NRs can influence signaling events of other NRs via control of FABP5 expression levels.

  12. Salvianolic Acid A, as a Novel ETA Receptor Antagonist, Shows Inhibitory Effects on Tumor in Vitro.

    Science.gov (United States)

    Zhang, Qiao; Wang, Shifeng; Yu, Yangyang; Sun, Shengnan; Zhang, Yuxin; Zhang, Yanling; Yang, Wei; Li, Shiyou; Qiao, Yanjiang

    2016-08-02

    Endothelin-1 (ET-1) autocrine and paracrine signaling modulate cell proliferation of tumor cells by activating its receptors, endothelin A receptor (ETAR) and endothelin B receptor (ETBR). Dysregulation of ETAR activation promotes tumor development and progression. The potential of ETAR antagonists and the dual-ETAR and ETBR antagonists as therapeutic approaches are under preclinical and clinical studies. Salvianolic acid A (Sal A) is a hydrophilic polyphenolic derivative isolated from Salvia miltiorrhiza Bunge (Danshen), which has been reported as an anti-cancer and cardio-protective herbal medicine. In this study, we demonstrate that Sal A inhibits ETAR activation induced by ET-1 in both recombinant and endogenous ETAR expression cell lines. The IC50 values were determined as 5.7 µM in the HEK293/ETAR cell line and 3.14 µM in HeLa cells, respectively. Furthermore, our results showed that Sal A suppressed cell proliferation and extended the doubling times of multiple cancer cells, including HeLa, DU145, H1975, and A549 cell lines. In addition, Sal A inhibited proliferation of DU145 cell lines stimulated by exogenous ET-1 treatment. Moreover, the cytotoxicity and cardio-toxicity of Sal A were assessed in human umbilical vein endothelial cells (HUVEC) and Human-induced pluripotent stem cell-derived cardiomyocytes (hiPS-CMs), which proved that Sal A demonstrates no cytotoxicity or cardiotoxicity. Collectively, our findings indicate that Sal A is a novel anti-cancer candidate through targeting ETAR.

  13. The Arabidopsis NPR1 Protein Is a Receptor for the Plant Defense Hormone Salicylic Acid

    Directory of Open Access Journals (Sweden)

    Yue Wu

    2012-06-01

    Full Text Available Salicylic acid (SA is an essential hormone in plant immunity, but its receptor has remained elusive for decades. The transcriptional coregulator NPR1 is central to the activation of SA-dependent defense genes, and we previously found that Cys521 and Cys529 of Arabidopsis NPR1's transactivation domain are critical for coactivator function. Here, we demonstrate that NPR1 directly binds SA, but not inactive structural analogs, with an affinity similar to that of other hormone-receptor interactions and consistent with in vivo Arabidopsis SA concentrations. Binding of SA occurs through Cys521/529 via the transition metal copper. Mechanistically, our results suggest that binding of SA causes a conformational change in NPR1 that is accompanied by the release of the C-terminal transactivation domain from the N-terminal autoinhibitory BTB/POZ domain. While NPR1 is already known as a link between the SA signaling molecule and defense-gene activation, we now show that NPR1 is the receptor for SA.

  14. Acid-responsive PEGylated doxorubicin prodrug nanoparticles for neuropilin-1 receptor-mediated targeted drug delivery.

    Science.gov (United States)

    Song, Huijuan; Zhang, Ju; Wang, Weiwei; Huang, Pingsheng; Zhang, Yumin; Liu, Jianfeng; Li, Chen; Kong, Deling

    2015-12-01

    Self-assembled prodrug nanoparticles have demonstrated great promise in cancer chemotherapy. In the present study, we developed a new kind of prodrug nanoparticles for targeted drug delivery. PEGylated doxorubicin conjugate with an acid-cleavable cis-aconityl spacer was prepared. Then it was functionalized with a tumor-penetrating peptide, Cys-Arg-Gly-Asp-Lys (CRGDK), providing the prodrug nanoparticles with the specific binding ability to neurophilin-1 receptor. In acid mediums, doxorubicin could be released from the prodrug nanoparticles with an accumulative release around 60% through the acid-triggered hydrolysis of cis-aconityl bond and nanoparticle disassembly. Whereas, drug release was slow under a neutral pH and the accumulative drug release was less than 16%. In the cell culture tests, our prodrug nanoparticles showed enhanced endocytosis and cytotoxicity in cancer cells including HepG2, MCF-7 and MDA-MB-231 cells, but lower cytotoxicity in human cardiomyocyte H2C9. In the animal experiments, the prodrug nanoparticles were intravenously injected into Balb/c nude mice bearing MDA-MB-231 tumors. Enhanced drug penetration and accumulation in tumors, accompanying with a rapid early tumor-binding behavior, was observed after intravenous injection of the peptide modified prodrug nanoparticles. These data suggests that the acid-sensitive and tumor-targeting PEGylated doxorubicin prodrug nanoparticle may be an efficient drug delivery system for cancer chemotherapy.

  15. Acidic Tumor Microenvironment and pH-Sensing G protein-Coupled Receptors

    Directory of Open Access Journals (Sweden)

    Calvin R. Justus

    2013-12-01

    Full Text Available The tumor microenvironment is acidic due to glycolytic cancer cell metabolism, hypoxia, and deficient blood perfusion. It is proposed that acidosis in the tumor microenvironment is an important stress factor and selection force for cancer cell somatic evolution. Acidic pH has pleiotropic effects on the proliferation, migration, invasion, metastasis and therapeutic response of cancer cells and the function of immune cells, vascular cells, and other stromal cells. However, the molecular mechanisms by which cancer cells and stromal cells sense and respond to acidic pH in the tumor microenvironment are poorly understood. In this article the role of a family of pH-sensing G protein-coupled receptors (GPCRs in tumor biology is reviewed. Recent studies show that the pH-sensing GPCRs, including GPR4, GPR65 (TDAG8, GPR68 (OGR1, and GPR132 (G2A, regulate cancer cell metastasis and proliferation, immune cell function, inflammation, and blood vessel formation. Activation of the proton-sensing GPCRs by acidosis transduces multiple downstream G protein signaling pathways. Since GPCRs are major drug targets, small molecule modulators of the pH-sensing GPCRs are being actively developed and evaluated. Research on the pH-sensing GPCRs will continue to provide important insights into the molecular interaction between tumor and its acidic microenvironment and may identify new targets for cancer therapy and chemoprevention.

  16. Human Trafficking in the Emergency Department

    Science.gov (United States)

    Patel, Ronak B.; Ahn, Roy; Burke, Thomas F.

    2010-01-01

    Human trafficking continues to persist, affecting up to 200 million people worldwide. As clinicians in emergency departments commonly encounter victims of intimate partner violence, some of these encounters will be with trafficking victims. These encounters provide a rare opportunity for healthcare providers to intervene and help. This case report of a human trafficking patient from a teaching hospital illustrates the complexity in identifying these victims. Clinicians can better identify potential trafficking cases by increasing their awareness of this phenomenon, using qualified interpreters, isolating potential victims by providing privacy and using simple clear reassuring statements ensuring security. A multidisciplinary approach can then be mobilized to help these patients. PMID:21293753

  17. Discrimination between sialic acid-containing receptors and pseudoreceptors regulates polyomavirus spread in the mouse.

    Science.gov (United States)

    Bauer, P H; Cui, C; Liu, W R; Stehle, T; Harrison, S C; DeCaprio, J A; Benjamin, T L

    1999-07-01

    Variations in the polyomavirus major capsid protein VP1 underlie important biological differences between highly pathogenic large-plaque and relatively nonpathogenic small-plaque strains. These polymorphisms constitute major determinants of virus spread in mice and also dictate previously recognized strain differences in sialyloligosaccharide binding. X-ray crystallographic studies have shown that these determinants affect binding to the sialic acids. Here we report results of further experiments designed to test the importance of specific contacts between VP1 and the carbohydrate moieties of the receptor. With minor exceptions, substitutions at positions predicted from crystallography to be important in binding the terminal alpha-2,3-linked sialic acid or the penultimate sugar (galactose) destroyed the ability of the virus to replicate in cell culture. Substitutions that prevented binding to a branched disialyloligosaccharide were found to result in viruses that were both viable in culture and tumorigenic in the mouse. Conversely, substitutions that allowed recognition and binding of the branched carbohydrate chain inhibited spread in the mouse, though the viruses remained viable in culture. Mice of five different inbred strains, all highly susceptible to large-plaque virus, showed resistance to the spread of polyomavirus strains bearing the VP1 type which binds the branched-chain receptor. We suggest that glycoproteins bearing the appropriate O-linked branched sialyloligosaccharide chains are effective pseudoreceptors in the host and that they block the spread of potentially tumorigenic or virulent virus strains.

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

    Directory of Open Access Journals (Sweden)

    Tony Velkov

    2013-01-01

    Full Text Available 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- FABP displays a high binding affinity for PPAR subtype selective drugs. NMR chemical shift perturbation mapping and proteolytic protection experiments show that the binding of the PPAR subtype selective drugs produces conformational changes that stabilize the portal region of L-FABP. NMR chemical shift perturbation studies also revealed that L-FABP can form a complex with the PPAR ligand binding domain (LBD of PPARα. This protein-protein interaction may represent a mechanism for facilitating the activation of PPAR transcriptional activity via the direct channeling of ligands between the binding pocket of L-FABP and the PPARαLBD. The role of L-FABP in the delivery of ligands directly to PPARα via this channeling mechanism has important implications for regulatory pathways that mediate xenobiotic responses and host protection in tissues such as the small intestine and the liver where L-FABP is highly expressed.

  19. Nuclear and extra-nuclear effects of retinoid acid receptors: how they are interconnected.

    Science.gov (United States)

    Piskunov, Aleksandr; Al Tanoury, Ziad; Rochette-Egly, Cécile

    2014-01-01

    The nuclear retinoic acid receptors (RAR α, β and γ) and their isoforms are ligand-dependent regulators of transcription Transcription , which mediate the effects of all-trans retinoic acid (RA), the active endogenous metabolite of Vitamin A. They heterodimerize with Retinoid X Receptors (RXRs α, β and γ), and regulate the expression of a battery of target genes Target genes involved in cell growth and differentiation Differentiation . During the two last decades, the description of the crystallographic structures of RARs, the characterization of the polymorphic response elements of their target genes Target genes , and the identification of the multiprotein complexes involved in their transcriptional activity have provided a wealth of information on their pleiotropic effects. However, the regulatory scenario became even more complicated once it was discovered that RARs are phosphoproteins and that RA can activate kinase signaling cascades via a pool of RARs present in membrane lipid rafts. Now it is known that these RA-activated kinases Kinases translocate to the nucleus where they phosphorylate RARs and other retinoid signaling factors. The phosphorylation Phosphorylation state of the RARs dictates whether the transcriptional programs which are known to be induced by RA are facilitated and/or switched on. Thus, kinase signaling pathways appear to be crucial for fine-tuning the appropriate physiological activity of RARs.

  20. Activation of transmembrane bile acid receptor TGR5 stimulates insulin secretion in pancreatic {beta} cells

    Energy Technology Data Exchange (ETDEWEB)

    Kumar, Divya P.; Rajagopal, Senthilkumar; Mahavadi, Sunila [Department of Physiology and Biophysics, Virginia Commonwealth University School of Medicine, Richmond, VA (United States); Mirshahi, Faridoddin [Division of Gastroenterology, Hepatology and Nutrition, Department of Internal Medicine, Virginia Commonwealth University School of Medicine, Richmond, VA (United States); Grider, John R. [Department of Physiology and Biophysics, Virginia Commonwealth University School of Medicine, Richmond, VA (United States); Murthy, Karnam S., E-mail: skarnam@vcu.edu [Department of Physiology and Biophysics, Virginia Commonwealth University School of Medicine, Richmond, VA (United States); Sanyal, Arun J., E-mail: asanyal@mcvh-vcu.edu [Division of Gastroenterology, Hepatology and Nutrition, Department of Internal Medicine, Virginia Commonwealth University School of Medicine, Richmond, VA (United States)

    2012-10-26

    Highlights: Black-Right-Pointing-Pointer G protein coupled receptor TGR5 is expressed in mouse and human islets. Black-Right-Pointing-Pointer TGR5 is coupled to activation of Gs and Ca{sup 2+} release via cAMP/Epac/PLC-{epsilon} pathway. Black-Right-Pointing-Pointer Activation of TGR5 by bile salts and selective ligands causes insulin secretion. Black-Right-Pointing-Pointer TGR5 could be a potential therapeutic target to treat diabetes. -- Abstract: Bile acids act as signaling molecules and stimulate the G protein coupled receptor, TGR5, in addition to nuclear farnesoid X receptor to regulate lipid, glucose and energy metabolism. Bile acid induced activation of TGR5 in the enteroendocrine cells promotes glucagon like peptide-1 (GLP-1) release, which has insulinotropic effect in the pancreatic {beta} cells. In the present study, we have identified the expression of TGR5 in pancreatic {beta} cell line MIN6 and also in mouse and human pancreatic islets. TGR5 selective ligands, oleanolic acid (OA) and INT-777 selectively activated G{alpha}{sub s} and caused an increase in intracellular cAMP and Ca{sup 2+}. OA and INT-777 also increased phosphoinositide (PI) hydrolysis and the increase was blocked by NF449 (a selective G{alpha}{sub s} inhibitor) or (U73122) (PI hydrolysis inhibitor). OA, INT-777 and lithocholic acid increased insulin release in MIN6 and human islets and the increase was inhibited by treatment with NF449, (U73122) or BAPTA-AM (chelator of calcium), but not with myristoylated PKI (PKA inhibitor), suggesting that the release is dependent on G{sub s}/cAMP/Ca{sup 2+} pathway. 8-pCPT-2 Prime -O-Me-cAMP, a cAMP analog, which activates Epac, but not PKA also stimulated PI hydrolysis. In conclusion, our study demonstrates that the TGR5 expressed in the pancreatic {beta} cells regulates insulin secretion and highlights the importance of ongoing therapeutic strategies targeting TGR5 in the control of glucose homeostasis.

  1. Selective agonists of retinoic acid receptors: comparative toxicokinetics and embryonic exposure.

    Science.gov (United States)

    Arafa, H M; Elmazar, M M; Hamada, F M; Reichert, U; Shroot, B; Nau, H

    2000-01-01

    Three biologically active synthetic retinoids were investigated that bind selectively to retinoic acid receptors RARs (alpha, beta and gamma). The retinoids were previously demonstrated to have different teratogenic effects in the mouse in terms of potency and regioselectivity. The teratogenic potency rank order (alpha >beta >gamma) was found to be more or less compatible with the receptor binding affinities and transactivation potencies of the retinoid ligands to their respective receptors. The RARalpha agonist (Am580; CD336) induced a wide spectrum of malformations; CD2019 (RARbeta agonist) and especially CD437 (RARgamma agonist) produced more restricted defects. In the current study we tried to address whether the differences in teratogenic effects are solely related to binding affinity and transactivation differences or also due to differences in embryonic exposure. Therefore, transplacental kinetics of the ligands were assessed following administration of a single oral dose of 15 mg/kg of either retinoid given to NMRI mice on day 11 of gestation. Am580 was rapidly transferred to the embryo resulting in the highest embryonic exposure [embryo to maternal plasma area under the time vs concentration curve (AUC)(0-24 h )ratio (E/M) was 1.7], in accordance with its highest teratogenic potency. The low placental transfer of CD2019 (E/M of 0.3) was compatible with its lower teratogenic potential. Of major interest was the finding that the CD437, though being least teratogenic, exhibited considerable embryonic exposure (E/M of 0.6). These findings suggest that both the embryonic exposure and receptor binding transactivation selectivity are crucial determinants of the teratogenicity of these retinoid ligands.

  2. N-Methyl D-Aspartic Acid (NMDA Receptors and Depression

    Directory of Open Access Journals (Sweden)

    Enver Yusuf Sivrioglu

    2009-06-01

    Full Text Available The monoaminergic hypothesis of depression has provided the basis for extensive research into the pathophysiology of mood disorders and has been of great significance for the development of effective antidepressants. Current antidepressant treatments not only increase serotonin and/or noradrenaline bioavailability but also originate adaptive changes increasing synaptic plasticity. Novel approaches to depression and to antidepressant therapy are now focused on intracellular targets that regulate neuroplasticity and cell survival. Accumulating evidence indicates that there is an anatomical substrate for such a devastating neuropsychiatric disease as major depression. Loss of synaptic plasticity and hippocampal atrophy appear to be prominent features of this highly prevalent disorder. A combination of genetic susceptibility and environmental factors make hippocampal neurons more vulnerable to stress. Abundant experimental evidence indicates that stress causes neuronal damage in brain regions, notably in hippocampal subfields. Stress-induced activation of glutamatergic transmission may induce neuronal cell death through excessive stimulation of N-methyl-D-aspartic acid (NMDA receptors. Recent studies mention that the increase of nitric oxide synthesis and inflammation in major depression may contribute to neurotoxicity through NMDA receptor. Both standard antidepressants and NMDA receptor antagonists are able to prevent stress-induced neuronal damage. NMDA antagonists are effective in widely used animal models of depression and some of them appear to be effective also in the few clinical trials performed to date. We are still far from understanding the complex cellular and molecular events involved in mood disorders. There appears to be an emerging role for glutamate neurotransmission in the search for the pathogenesis of major depression. Attenuation of NMDA receptor function mechanism appears to be a promising target in the search for a more

  3. Modulation of NMDA receptor function by inhibition of D-amino acid oxidase in rodent brain.

    Science.gov (United States)

    Strick, Christine A; Li, Cheryl; Scott, Liam; Harvey, Brian; Hajós, Mihály; Steyn, Stefanus J; Piotrowski, Mary A; James, Larry C; Downs, James T; Rago, Brian; Becker, Stacey L; El-Kattan, Ayman; Xu, Youfen; Ganong, Alan H; Tingley, F David; Ramirez, Andres D; Seymour, Patricia A; Guanowsky, Victor; Majchrzak, Mark J; Fox, Carol B; Schmidt, Christopher J; Duplantier, Allen J

    2011-01-01

    Observations that N-Methyl-D-Aspartate (NMDA) antagonists produce symptoms in humans that are similar to those seen in schizophrenia have led to the current hypothesis that schizophrenia might result from NMDA receptor hypofunction. Inhibition of D-amino acid oxidase (DAAO), the enzyme responsible for degradation of D-serine, should lead to increased levels of this co-agonist at the NMDA receptor, and thereby provide a therapeutic approach to schizophrenia. We have profiled some of the preclinical biochemical, electrophysiological, and behavioral consequences of administering potent and selective inhibitors of DAAO to rodents to begin to test this hypothesis. Inhibition of DAAO activity resulted in a significant dose and time dependent increase in D-serine only in the cerebellum, although a time delay was observed between peak plasma or brain drug concentration and cerebellum D-serine response. Pharmacokinetic/pharmacodynamic (PK/PD) modeling employing a mechanism-based indirect response model was used to characterize the correlation between free brain drug concentration and D-serine accumulation. DAAO inhibitors had little or no activity in rodent models considered predictive for antipsychotic activity. The inhibitors did, however, affect cortical activity in the Mescaline-Induced Scratching model, produced a modest but significant increase in NMDA receptor-mediated synaptic currents in primary neuronal cultures from rat hippocampus, and resulted in a significant increase in evoked hippocampal theta rhythm, an in vivo electrophysiological model of hippocampal activity. These findings demonstrate that although DAAO inhibition did not cause a measurable increase in D-serine in forebrain, it did affect hippocampal and cortical activity, possibly through augmentation of NMDA receptor-mediated currents. Copyright © 2011 Elsevier Ltd. All rights reserved.

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

    Science.gov (United States)

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

    2017-06-30

    The bile acid-activated receptors, nuclear farnesoid X receptor (FXR) and the membrane Takeda G-protein receptor 5 (TGR5), are known to improve glucose and insulin sensitivity in obese and diabetic mice. However, the metabolic roles of these two receptors and the underlying mechanisms are incompletely understood. Here, we studied the effects of the dual FXR and TGR5 agonist INT-767 on hepatic bile acid synthesis and intestinal secretion of glucagon-like peptide-1 (GLP-1) in wild-type, Fxr(-/-), and Tgr5(-/-) mice. INT-767 efficaciously stimulated intracellular Ca(2+) levels, cAMP activity, and GLP-1 secretion and improved glucose and lipid metabolism more than did the FXR-selective obeticholic acid and TGR5-selective INT-777 agonists. Interestingly, INT-767 reduced expression of the genes in the classic bile acid synthesis pathway but induced those in the alternative pathway, which is consistent with decreased taurocholic acid and increased tauromuricholic acids in bile. Furthermore, FXR activation induced expression of FXR target genes, including fibroblast growth factor 15, and unexpectedly Tgr5 and prohormone convertase 1/3 gene expression in the ileum. We identified an FXR-responsive element on the Tgr5 gene promoter. Fxr(-/-) and Tgr5(-/-) mice exhibited reduced GLP-1 secretion, which was stimulated by INT-767 in the Tgr5(-/-) mice but not in the Fxr(-/-) mice. Our findings uncovered a novel mechanism in which INT-767 activation of FXR induces Tgr5 gene expression and increases Ca(2+) levels and cAMP activity to stimulate GLP-1 secretion and improve hepatic glucose and lipid metabolism in high-fat diet-induced obese mice. Activation of both FXR and TGR5 may therefore represent an effective therapy for managing hepatic steatosis, obesity, and diabetes. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  5. Phosphorylation-dependent Trafficking of Plasma Membrane Proteins in Animal and Plant Cells

    Institute of Scientific and Technical Information of China (English)

    Remko Offringa; and Fang Huang

    2013-01-01

    In both unicellular and multicellular organisms, transmembrane (TM) proteins are sorted to and retained at specific membrane domains by endomembrane trafficking mechanisms that recognize sorting signals in the these proteins. The trafficking and distribution of plasma membrane (PM)-localized TM proteins (PM proteins), especially of those PM proteins that show an asymmetric distribution over the PM, has received much attention, as their proper PM localization is crucial for elementary signaling and transport processes, and defects in their localization often lead to severe disease symptoms or developmental defects. The subcellular localization of PM proteins is dynamically regulated by post-translational modifications, such as phosphorylation and ubiquitination. These modificaitons mostly occur on sorting signals that are located in the larger cytosolic domains of the cargo proteins. Here we review the effects of phosphorylation of PM proteins on their trafficking, and present the key examples from the animal field that have been subject to studies for already several decades, such as that of aquaporin 2 and the epidermal growth factor receptor. Our knowledge on cargo trafficking in plants is largely based on studies of the family of PIN FORMED (PIN) carriers that mediate the efflux of the plant hormone auxin. We will review what is known on the subcellular distribution and trafficking of PIN proteins, with a focus on how this is modulated by phosphorylation, and identify and discuss analogies and differences in trafficking with the well-studied animal examples.

  6. Phosphorylation-dependent trafficking of plasma membrane proteins in animal and plant cells.

    Science.gov (United States)

    Offringa, Remko; Huang, Fang

    2013-09-01

    In both unicellular and multicellular organisms, transmembrane (TM) proteins are sorted to and retained at specific membrane domains by endomembrane trafficking mechanisms that recognize sorting signals in the these proteins. The trafficking and distribution of plasma membrane (PM)-localized TM proteins (PM proteins), especially of those PM proteins that show an asymmetric distribution over the PM, has received much attention, as their proper PM localization is crucial for elementary signaling and transport processes, and defects in their localization often lead to severe disease symptoms or developmental defects. The subcellular localization of PM proteins is dynamically regulated by post-translational modifications, such as phosphorylation and ubiquitination. These modificaitons mostly occur on sorting signals that are located in the larger cytosolic domains of the cargo proteins. Here we review the effects of phosphorylation of PM proteins on their trafficking, and present the key examples from the animal field that have been subject to studies for already several decades, such as that of aquaporin 2 and the epidermal growth factor receptor. Our knowledge on cargo trafficking in plants is largely based on studies of the family of PIN FORMED (PIN) carriers that mediate the efflux of the plant hormone auxin. We will review what is known on the subcellular distribution and trafficking of PIN proteins, with a focus on how this is modulated by phosphorylation, and identify and discuss analogies and differences in trafficking with the well-studied animal examples.

  7. TUDCA: An Agonist of the Bile Acid Receptor GPBAR1/TGR5 With Anti-Inflammatory Effects in Microglial Cells.

    Science.gov (United States)

    Yanguas-Casás, Natalia; Barreda-Manso, M Asunción; Nieto-Sampedro, Manuel; Romero-Ramírez, Lorenzo

    2017-08-01

    Bile acids are steroid acids found in the bile of mammals. The bile acid conjugate tauroursodeoxycholic acid (TUDCA) is neuroprotective in different animal models of stroke and neurological diseases. We have previously shown that TUDCA has anti-inflammatory effects on glial cell cultures and in a mouse model of acute neuroinflammation. We show now that microglial cells (central nervous system resident macrophages) express the G protein-coupled bile acid receptor 1/Takeda G protein-coupled receptor 5 (GPBAR1/TGR5) in vivo and in vitro. TUDCA binding to GPBAR1/TGR5 caused an increase in intracellular cAMP levels in microglia that induced anti-inflammatory markers, while reducing pro-inflammatory ones. This anti-inflammatory effect of TUDCA was inhibited by small interference RNA for GPBAR1/TGR5 receptor, as well as by treatment with a protein kinase A (PKA) inhibitor. In the mouse model of acute neuroinflammation, treating the animals with TUDCA was clearly anti-inflammatory. TUDCA biased the microglial phenotype in vivo and in vitro toward the anti-inflammatory. The bile acid receptor GPBAR1/TGR5 could be a new therapeutic target for pathologies coursing with neuroinflammation and microglia activation, such as traumatic brain injuries, stroke, or neurodegenerative diseases. TUDCA and other GPBAR1/TGR5 agonists need to be further investigated, to determine their potential in attenuating the neuropathologies associated with microglia activation. J. Cell. Physiol. 232: 2231-2245, 2017. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  8. Allosteric Regulation in the Ligand Binding Domain of Retinoic Acid Receptorγ

    Science.gov (United States)

    Amal, Ismail; Lutzing, Régis; Stote, Roland H.; Rochette-Egly, Cécile; Rochel, Natacha; Dejaegere, Annick

    2017-01-01

    Retinoic acid (RA) plays key roles in cell differentiation and growth arrest through nuclear retinoic acid receptors (RARs), which are ligand-dependent transcription factors. While the main trigger of RAR activation is the binding of RA, phosphorylation of the receptors has also emerged as an important regulatory signal. Phosphorylation of the RARγ N-terminal domain (NTD) is known to play a functional role in neuronal differentiation. In this work, we investigated the phosphorylation of RARγ ligand binding domain (LBD), and present evidence that the phosphorylation status of the LBD affects the phosphorylation of the NTD region. We solved the X-ray structure of a phospho-mimetic mutant of the LBD (RARγ S371E), which we used in molecular dynamics simulations to characterize the consequences of the S371E mutation on the RARγ structural dynamics. Combined with simulations of the wild-type LBD, we show that the conformational equilibria of LBD salt bridges (notably R387-D340) are affected by the S371E mutation, which likely affects the recruitment of the kinase complex that phosphorylates the NTD. The molecular dynamics simulations also showed that a conservative mutation in this salt bridge (R387K) affects the dynamics of the LBD without inducing large conformational changes. Finally, cellular assays showed that the phosphorylation of the NTD of RARγ is differentially regulated by retinoic acid in RARγWT and in the S371N, S371E and R387K mutants. This multidisciplinary work highlights an allosteric coupling between phosphorylations of the LBD and the NTD of RARγ and supports the importance of structural dynamics involving electrostatic interactions in the regulation of RARs activity. PMID:28125680

  9. γ-Aminobutyric Acid B Receptor Mediated Inhibition of Gonadotropin-Releasing Hormone Neurons Is Suppressed by Kisspeptin-G Protein-Coupled Receptor 54 Signaling

    Science.gov (United States)

    Zhang, Chunguang; Bosch, Martha A.; Rønnekleiv, Oline K.; Kelly, Martin J.

    2009-01-01

    γ-Aminobutyric acid (GABA) is one of the most important neurotransmitters that regulate the excitability of GnRH neurons. Numerous studies have shown that GABA activates Cl− currents in GnRH neurons, and these effects are antagonized by GABAA receptor antagonists. The GABAB receptor is a heterodimer composed of GABAB R1 and R2, and although both subunits have been localized in GnRH neurons, nothing is known about the cellular signaling of this Gαi,o-coupled receptor in GnRH neurons. Using whole-cell recordings from mouse enhanced green fluorescent protein-GnRH neurons, we found that the GABAB receptor agonist baclofen hyperpolarized GnRH neurons through activation of an inwardly rectifying K+ current in a concentration-dependent manner. The effects of baclofen were antagonized by the selective GABAB receptor antagonist CGP 52432 with a Ki (inhibitory constant) of 85 nm. Furthermore, in the presence of the GABAA receptor antagonist picrotoxin, GABA hyperpolarized GnRH neurons in a similar manner. Treatment with 17β-estradiol as compared with oil vehicle did not significantly alter either the EC50 for the baclofen-induced response (0.8 ± 0.1 vs. 1.0 ± 0.1 μm, respectively) or the maximal outward current (10.8 ± 1.7 pA vs. 11.4 ± 0.6 pA, respectively) in GnRH neurons. However, the outward current (and membrane hyperpolarization) was abrogated by submaximal concentrations of the G protein-coupled receptor 54 (GPR54) agonist kisspeptin-10 in both groups, indicating that Gαq-coupled (GPR54) can desensitize the GABAB receptor-mediated response. Therefore, the activation of GABAB receptors in GnRH neurons may provide increased inhibitory tone during estrogen-negative feedback states that is attenuated by kisspeptin during positive feedback. PMID:19164470

  10. MEMBRANE BILE ACID RECEPTOR TGR5 - A NEW TARGET IN THE STUDY OF METABOLIC, INFLAMMATORY AND NEOPLASTIC DISEASES

    Directory of Open Access Journals (Sweden)

    O. M. Drapkina

    2016-01-01

    Full Text Available TGR5 are G-protein-linked, membrane bile acids receptors that widely express in tissues of animals and humans. Namely tissue localization of TGR5 determines biological effects of activation of these receptors. This review focuses on the role of TGR5 as a new pharmacological target for the treatment of patients with metabolic syndrome, diabetes, obesity, atherosclerosis, liver disease and cancer processes.

  11. Eicosanoid regulation of hematopoiesis and hematopoietic stem and progenitor trafficking.

    Science.gov (United States)

    Hoggatt, J; Pelus, L M

    2010-12-01

    Hematopoietic stem cell (HSC) transplantation is a potentially curative treatment for numerous hematological malignancies. The transplant procedure as performed today takes advantage of HSC trafficking; either egress of HSC from the bone marrow to the peripheral blood, that is, mobilization, for acquisition of the hematopoietic graft, and/or trafficking of HSC from the peripheral blood to bone marrow niches in the recipient patient, that is HSC homing. Numerous studies, many of which are reviewed herein, have defined hematopoietic regulatory mechanisms mediated by the 20-carbon lipid family of eicosanoids, and recent evidence strongly supports a role for eicosanoids in regulation of hematopoietic trafficking, adding a new role whereby eicosanoids regulate hematopoiesis. Short-term exposure of HSC to the eicosanoid prostaglandin E(2) increases CXCR4 receptor expression, migration and in vivo homing of HSC. In contrast, cannabinoids reduce hematopoietic progenitor cell (HPC) CXCR4 expression and induce HPC mobilization when administered in vivo. Leukotrienes have been shown to alter CD34(+) cell adhesion, migration and regulate HSC proliferation, suggesting that eicosanoids have both opposing and complimentary roles in the regulation of hematopoiesis. As numerous FDA approved compounds regulate eicosanoid signaling or biosynthesis, the utility of eicosanoid-based therapeutic strategies to improve hematopoietic transplantation can be rapidly evaluated.

  12. Negative regulation of gamma-aminobutyric acid type A receptor on free calcium ion levels following facial nerve injury

    Institute of Scientific and Technical Information of China (English)

    Fugao Zhu; Dawei Sun; Yanqing Wang; Rui Zhou; Junfeng Wen; Xiuming Wan; Yanjun Wang; Banghua Liu

    2010-01-01

    Previous studies have demonstrated that muscarinic, and nicotinic receptors increase free Ca2+ levels in the facial nerve nucleus via various channels following facial nerve injury. However, intracellular Ca2+ overload can trigger either necrotic or apoptotic cell death. Gamma-aminobutyric acid (GABA), an important inhibitory neurotransmitter in the central nervous system, exists in the facial nerve nucleus. It is assumed that GABA negatively regulates free Ca2+ levels in the facial nerve nucleus. The present study investigated GABA type A (GABAA) receptor expression in the facial nerve nucleus in a rat model of facial nerve injury using immunohistochemistry and laser confocal microscopy, as well as the regulatory effects of GABAA receptor on nicotinic receptor response following facial nerve injury. Subunits α1, α3, α5, β1, β2, δ, and γ3 of GABAA receptors were expressed in the facial nerve nucleus following facial nerve injury. In addition, GABAA receptor expression significantly inhibited the increase in nicotinic receptor-mediated free Ca2+ levels in the facial nerve nucleus following facial nerve injury in a concentration-dependent fashion. These results suggest that GABAA receptors exhibit negative effects on nicotinic receptor responses following facial nerve injury.

  13. Fatty acids modulate Toll-like receptor 4 activation through regulation of receptor dimerization and recruitment into lipid rafts in a reactive oxygen species-dependent manner.

    Science.gov (United States)

    Wong, Scott W; Kwon, Myung-Ja; Choi, Augustine M K; Kim, Hong-Pyo; Nakahira, Kiichi; Hwang, Daniel H

    2009-10-02

    The saturated fatty acids acylated on Lipid A of lipopolysaccharide (LPS) or bacterial lipoproteins play critical roles in ligand recognition and receptor activation for Toll-like Receptor 4 (TLR4) and TLR2. The results from our previous studies demonstrated that saturated and polyunsaturated fatty acids reciprocally modulate the activation of TLR4. However, the underlying mechanism has not been understood. Here, we report for the first time that the saturated fatty acid lauric acid induced dimerization and recruitment of TLR4 into lipid rafts, however, dimerization was not observed in non-lipid raft fractions. Similarly, LPS and lauric acid enhanced the association of TLR4 with MD-2 and downstream adaptor molecules, TRIF and MyD88, into lipid rafts leading to the activation of downstream signaling pathways and target gene expression. However, docosahexaenoic acid (DHA), an n-3 polyunsaturated fatty acid, inhibited LPS- or lauric acid-induced dimerization and recruitment of TLR4 into lipid raft fractions. Together, these results demonstrate that lauric acid and DHA reciprocally modulate TLR4 activation by regulation of the dimerization and recruitment of TLR4 into lipid rafts. In addition, we showed that TLR4 recruitment to lipid rafts and dimerization were coupled events mediated at least in part by NADPH oxidase-dependent reactive oxygen species generation. These results provide a new insight in understanding the mechanism by which fatty acids differentially modulate TLR4-mediated signaling pathway and consequent inflammatory responses which are implicated in the development and progression of many chronic diseases.

  14. Lipids: architects and regulators of membrane dynamics and trafficking.

    Science.gov (United States)

    Moreau, Patrick

    2007-05-01

    We have recently shown that an inhibition of sterol synthesis by fenpropimorph leads to an accumulation of sterol precursors, hydroxypalmitic acid-containing glucosylceramides and detergent resistant membranes in the Golgi bodies instead of the plasma membrane, suggesting that the individual molecules or the microdomains were blocked in the Golgi. These results and others from several eukaryotic models link lipid metabolism with membrane morphodynamics that are involved in membrane trafficking. Focus has been expanded to other lipid families, and numerous evidences are given showing lipids and lipid-modifying enzymes as key regulators of membrane homeostasis which can strongly regulate membrane morphodynamics and therefore trafficking. Beside protein-based machineries, lipid-based machineries are also shown as crucial regulatory forces involved in protein transport and sorting.

  15. Metabotropic glutamate receptors are involved in the detection of IMP and L-amino acids by mouse taste sensory cells.

    Science.gov (United States)

    Pal Choudhuri, S; Delay, R J; Delay, E R

    2016-03-01

    G-protein-coupled receptors are thought to be involved in the detection of umami and L-amino acid taste. These include the heterodimer taste receptor type 1 member 1 (T1r1)+taste receptor type 1 member 3 (T1r3), taste and brain variants of mGluR4 and mGluR1, and calcium sensors. While several studies suggest T1r1+T1r3 is a broadly tuned lLamino acid receptor, little is known about the function of metabotropic glutamate receptors (mGluRs) in L-amino acid taste transduction. Calcium imaging of isolated taste sensory cells (TSCs) of T1r3-GFP and T1r3 knock-out (T1r3 KO) mice was performed using the ratiometric dye Fura 2 AM to investigate the role of different mGluRs in detecting various L-amino acids and inosine 5' monophosphate (IMP). Using agonists selective for various mGluRs such as (RS)-3,5-dihydroxyphenylglycine (DHPG) (an mGluR1 agonist) and L-(+)-2-amino-4-phosphonobutyric acid (l-AP4) (an mGluR4 agonist), we evaluated TSCs to determine if they might respond to these agonists, IMP, and three L-amino acids (monopotassium L-glutamate, L-serine and L-arginine). Additionally, we used selective antagonists against different mGluRs such as (RS)-L-aminoindan-1,5-dicarboxylic acid (AIDA) (an mGluR1 antagonist), and (RS)-α-methylserine-O-phosphate (MSOP) (an mGluR4 antagonist) to determine if they can block responses elicited by these L-amino acids and IMP. We found that L-amino acid- and IMP-responsive cells also responded to each agonist. Antagonists for mGluR4 and mGluR1 significantly blocked the responses elicited by IMP and each of the L-amino acids. Collectively, these data provide evidence for the involvement of taste and brain variants of mGluR1 and mGluR4 in L-amino acid and IMP taste responses in mice, and support the concept that multiple receptors contribute to IMP and L-amino acid taste.

  16. Multilayered proteomics reveals molecular switches dictating ligand-dependent EGFR trafficking

    DEFF Research Database (Denmark)

    Francavilla, Chiara; Papetti, Moreno; Rigbolt, Kristoffer T G

    2016-01-01

    identified RAB7 phosphorylation and RCP recruitment to EGFR as switches for EGF and TGF-α outputs, controlling receptor trafficking, signaling duration, proliferation, and migration. By manipulating RCP levels or phosphorylation of RAB7 in EGFR-positive cancer cells, we were able to switch a TGF...

  17. Agonist-directed signaling of serotonin 5-HT2C receptors: differences between serotonin and lysergic acid diethylamide (LSD).

    Science.gov (United States)

    Backstrom, J R; Chang, M S; Chu, H; Niswender, C M; Sanders-Bush, E

    1999-08-01

    For more than 40 years the hallucinogen lysergic acid diethylamide (LSD) has been known to modify serotonin neurotransmission. With the advent of molecular and cellular techniques, we are beginning to understand the complexity of LSD's actions at the serotonin 5-HT2 family of receptors. Here, we discuss evidence that signaling of LSD at 5-HT2C receptors differs from the endogenous agonist serotonin. In addition, RNA editing of the 5-HT2C receptor dramatically alters the ability of LSD to stimulate phosphatidylinositol signaling. These findings provide a unique opportunity to understand the mechanism(s) of partial agonism.

  18. The role of γ-aminobutyric acid and its receptors in the nucleus of basal optic root in pigeons

    Institute of Scientific and Technical Information of China (English)

    付煜西; 高宏峰; 王书荣; Stephen A.George

    1997-01-01

    The effects of γ-aminobutyric acid (GABA) and its antagonists bicuculline and 2-hydroxysaclofen on neuronal firings in the nucleus of basal optic root (nBOR) in pigeons were studied by using extracellular recording and microiontophoretic techniques. The results suggest that GABA may be an inhibitory neurotransmitter or modulator within nBOR, functioning by means of main mediation of GABAA receptors and of minor mediation of GABAB receptors. Furthermore, GABA and its GABAA receptors are involved in the modulation of directional selectivity in part of nBOR neurons.

  19. Preliminary Validation of the Sex Trafficking Attitudes Scale.

    Science.gov (United States)

    Houston-Kolnik, Jaclyn D; Todd, Nathan R; Wilson, Midge

    2016-09-01

    This study presents the Sex Trafficking Attitudes Scale (STAS), assessing cognitive, behavioral, and affective attitudes toward the sex trafficking of women and girls. Across two studies, exploratory and confirmatory factor analyses revealed and confirmed six subscales: (a) Knowledge About Sex Trafficking, (b) Awareness of Sex Trafficking, (c) Attitudes Toward Ability to Leave Sex Trafficking, (d) Attitudes Toward Helping Survivors, (e) Empathic Reactions Toward Sex Trafficking, and (f) Efficacy to Reduce Sex Trafficking. Results showed support for convergent validity as the subscales were associated with related measures. The STAS holds promise to expand research and inform efforts to support trafficking survivors.

  20. (+)Lysergic acid diethylamide, but not its nonhallucinogenic congeners, is a potent serotonin 5HT1C receptor agonist

    Energy Technology Data Exchange (ETDEWEB)

    Burris, K.D.; Breeding, M.; Sanders-Bush, E. (Department of Pharmacology, Vanderbilt University School of Medicine, Nashville, TN (United States))

    1991-09-01

    Activation of central serotonin 5HT2 receptors is believed to be the primary mechanism whereby lysergic acid diethylamide (LSD) and other hallucinogens induce psychoactive effects. This hypothesis is based on extensive radioligand binding and electrophysiological and behavioral studies in laboratory animals. However, the pharmacological profiles of 5HT2 and 5HT1C receptors are similar, making it difficult to distinguish between effects due to activation of one or the other receptor. For this reason, it was of interest to investigate the interaction of LSD with 5HT1C receptors. Agonist-stimulated phosphoinositide hydrolysis in rat choroid plexus was used as a direct measure of 5HT1C receptor activation. (+)LSD potently stimulated phosphoinositide hydrolysis in intact choroid plexus and in cultures of choroid plexus epithelial cells, with EC50 values of 9 and 26 nM, respectively. The effect of (+)LSD in both systems was blocked by 5HT receptor antagonists with an order of activity consistent with interaction at 5HT1C receptors. Neither (+)-2-bromo-LSD nor lisuride, two nonhallucinogenic congeners of LSD, were able to stimulate 5HT1C receptors in cultured cells or intact choroid plexus. In contrast, lisuride, like (+)LSD, is a partial agonist at 5HT2 receptors in cerebral cortex slices and in NIH 3T3 cells transfected with 5HT2 receptor cDNA. The present finding that (+)LSD, but not its nonhallucinogenic congeners, is a 5HT1C receptor agonist suggests a possible role for these receptors in mediating the psychoactive effects of LSD.

  1. Trafficking in Persons Report 10th Edition

    Science.gov (United States)

    2010-06-01

    society , including NGOs, schools, civic associations, and community leaders, to identify the needs of the community, plan effective interventions...increase identification of forced labor trafficking. the City of vienna’s specialist center for unaccompanied minors accommodated 121 children in 2009...trafficked in Belgium are unaccompanied , vulnerable asylum seekers and refugees . according to the Belgian immigration office, the government identified

  2. A Comprehensive Approach to Combating Illicit Trafficking

    Science.gov (United States)

    2010-06-01

    espanol /cpo_trata�asp Trafficking Point of Contact Fernando García-Robles OAS 1889 F St� NW Washington, DC 20006 USA Tel: 202 458 3202 Email: Mgarcia...Research Materials http://www�oas�org/dsp/ espanol /cpo_armas�asp Trafficking Points of Contact Alison August Treppel OAS 1889 F St� NW Washington, DC

  3. Human Trafficking. Ministering to The 'Invisible' Victim.

    Science.gov (United States)

    Scanlon, Colleen; Krausa, Laura

    2016-07-01

    Human trafficking is modern-day slavery - an insidious, criminal industry that gener- ates billions of dollars in labor trafficking alone. It knows no boundary of continent, country, race or class; it is a shattering, impartial predator that robs individuals of their basic human dignity.

  4. TRACE-ing human trafficking : Project Findings

    NARCIS (Netherlands)

    Rijken, Conny; Pijnenburg, Annick

    2016-01-01

    Human trafficking is one of the largest criminal enterprises in the world. It is a multi-billion-dollar crime of global scale. This is because human trafficking as a criminal enterprise continues to evolve as a high profit-low risk business for perpetrators and challenges policy makers, law enforcem

  5. L-Amino Acids Elicit Diverse Response Patterns in Taste Sensory Cells: A Role for Multiple Receptors.

    Directory of Open Access Journals (Sweden)

    Shreoshi Pal Choudhuri

    Full Text Available Umami, the fifth basic taste, is elicited by the L-amino acid, glutamate. A unique characteristic of umami taste is the response potentiation by 5' ribonucleotide monophosphates, which are also capable of eliciting an umami taste. Initial reports using human embryonic kidney (HEK cells suggested that there is one broadly tuned receptor heterodimer, T1r1+T1r3, which detects L-glutamate and all other L-amino acids. However, there is growing evidence that multiple receptors detect glutamate in the oral cavity. While much is understood about glutamate transduction, the mechanisms for detecting the tastes of other L-amino acids are less well understood. We used calcium imaging of isolated taste sensory cells and taste cell clusters from the circumvallate and foliate papillae of C57BL/6J and T1r3 knockout mice to determine if other receptors might also be involved in detection of L-amino acids. Ratiometric imaging with Fura-2 was used to study calcium responses to monopotassium L-glutamate, L-serine, L-arginine, and L-glutamine, with and without inosine 5' monophosphate (IMP. The results of these experiments showed that the response patterns elicited by L-amino acids varied significantly across taste sensory cells. L-amino acids other than glutamate also elicited synergistic responses in a subset of taste sensory cells. Along with its role in synergism, IMP alone elicited a response in a large number of taste sensory cells. Our data indicate that synergistic and non-synergistic responses to L-amino acids and IMP are mediated by multiple receptors or possibly a receptor complex.

  6. The role of G-protein-coupled receptor 120 in fatty acids sensing in chicken oral tissues.

    Science.gov (United States)

    Sawamura, Ryo; Kawabata, Yuko; Kawabata, Fuminori; Nishimura, Shotaro; Tabata, Shoji

    2015-03-01

    Clarification of the mechanism of chickens' taste sense will provide meaningful information for creating and improving new feedstuff for chickens, because the character of taste receptors in oral tissues affects feeding behavior in animals. Although fatty acids are partly recognized via G-protein coupled receptor 120 (GPR120) for fat taste in mammalian oral tissues, the fat taste receptor of chickens has not been elucidated. Here we cloned chicken GPR120 (cGPR120) from the chicken palate, which contains taste buds. By using Ca(2+) imaging methods, we identified oleic acid and linoleic acid as cGPR120 agonists. Interestingly, in a behavioral study the chickens preferred corn oil-rich feed over mineral oil (control oil)-rich feed. Because corn oil contains high amounts of oleic acid and linoleic acid, this result was thought to be reasonable. Taken together, the present results suggest that cGPR120 is one of the functional fat taste receptors in chickens.

  7. Depletion of retinoic acid receptors initiates a novel positive feedback mechanism that promotes teratogenic increases in retinoic acid.

    Directory of Open Access Journals (Sweden)

    Enrico D'Aniello

    Full Text Available Normal embryonic development and tissue homeostasis require precise levels of retinoic acid (RA signaling. Despite the importance of appropriate embryonic RA signaling levels, the mechanisms underlying congenital defects due to perturbations of RA signaling are not completely understood. Here, we report that zebrafish embryos deficient for RA receptor αb1 (RARαb1, a conserved RAR splice variant, have enlarged hearts with increased cardiomyocyte (CM specification, which are surprisingly the consequence of increased RA signaling. Importantly, depletion of RARαb2 or concurrent depletion of RARαb1 and RARαb2 also results in increased RA signaling, suggesting this effect is a broader consequence of RAR depletion. Concurrent depletion of RARαb1 and Cyp26a1, an enzyme that facilitates degradation of RA, and employment of a novel transgenic RA sensor line support the hypothesis that the increases in RA signaling in RAR deficient embryos are the result of increased embryonic RA coupled with compensatory RAR expression. Our results support an intriguing novel mechanism by which depletion of RARs elicits a previously unrecognized positive feedback loop that can result in developmental defects due to teratogenic increases in embryonic RA.

  8. Is the anti-trafficking framework really for the 'victims'? : reflections on Burmese victims of human trafficking and non-trafficked migrants in Thailand

    OpenAIRE

    Yamada, Miwa

    2011-01-01

    Since the year 2000 when the Protocol to Prevent, Suppress and Punish Trafficking in Persons, Especially Women and Children, human trafficking has been regarded as one of the egregious violations of human rights, and global efforts have been made to eradicate it. The anti-trafficking framework has multiple dimensions, and the way the anti-trafficking framework is constructed influences its impact on the victims and non-trafficked migrants. This paper will analyze the impact of the anti-traffi...

  9. Is the anti-trafficking framework really for the 'victims'? : reflections on Burmese victims of human trafficking and non-trafficked migrants in Thailand

    OpenAIRE

    Yamada, Miwa

    2011-01-01

    Since the year 2000 when the Protocol to Prevent, Suppress and Punish Trafficking in Persons, Especially Women and Children, human trafficking has been regarded as one of the egregious violations of human rights, and global efforts have been made to eradicate it. The anti-trafficking framework has multiple dimensions, and the way the anti-trafficking framework is constructed influences its impact on the victims and non-trafficked migrants. This paper will analyze the impact of the anti-traffi...

  10. Was Trafficking in Persons Really Criminalised?

    Directory of Open Access Journals (Sweden)

    Kristiina Kangaspunta

    2015-04-01

    Full Text Available This paper examines the successes and setbacks in the criminal justice response to trafficking in persons. While today, the majority of countries have passed specific legislation criminalising human trafficking in response to the United Nations Protocol to Prevent, Suppress and Punish Trafficking in Persons, Especially Women and Children, there are still very few convictions of trafficking. Using currently available knowledge, this paper discusses four possible reasons for low conviction rates. Further, the paper suggests that due to the heavy dependency on victim testimonies when prosecuting trafficking in persons crimes, members of criminal organisations that are easily identifiable by victims may face criminal charges more frequently than other members of the criminal group, particularly those in positions of greater responsibility who profit the most from the criminal activities. In this context, the exceptionally high number of women among convicted offenders is explored.

  11. Examining the Risk of Nuclear Trafficking

    Energy Technology Data Exchange (ETDEWEB)

    Balatsky, Galya [Los Alamos National Laboratory; Severe, William R [Los Alamos National Laboratory; Schoeneck, Jeffery [DHS

    2009-01-01

    The need to stop illicit trafficking of nuclear and radioactive materials around the world is undeniable and urgent. This issue is particularly evident due to the highly dangerous consequences of the risks involved, the known interest of terrorist groups in acquiring such materials and the vulnerability of theft and diversion of such materials. Yet the phenomenon of nuclear trafficking remains a subject where the unknown dominates what is known on the subject. The trafficking panel at the Institute for Nuclear Materials Management (INMM) Workshop on Reducing the Risk of Radioactive and Nuclear Materials that took place in Albuquerque, New Mexico, March 10-11, 2009, dealt with some of the issues associated with nuclear trafficking. Different points of view on how to better address trafficking and thwart perpetrator efforts were discussed. This paper presents some of these views and addresses practical measures that should be considered to improve the situation.

  12. Activation of peroxisome proliferator-activated receptor-{alpha} enhances fatty acid oxidation in human adipocytes

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Joo-Young; Hashizaki, Hikari; Goto, Tsuyoshi; Sakamoto, Tomoya; Takahashi, Nobuyuki [Laboratory of Molecular Function of Food, Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Uji, Kyoto 611-0011 (Japan); Kawada, Teruo, E-mail: fat@kais.kyoto-u.ac.jp [Laboratory of Molecular Function of Food, Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Uji, Kyoto 611-0011 (Japan)

    2011-04-22

    Highlights: {yields} PPAR{alpha} activation increased mRNA expression levels of adipocyte differentiation marker genes and GPDH activity in human adipocytes. {yields} PPAR{alpha} activation also increased insulin-dependent glucose uptake in human adipocytes. {yields} PPAR{alpha} activation did not affect lipid accumulation in human adipocytes. {yields} PPAR{alpha} activation increased fatty acid oxidation through induction of fatty acid oxidation-related genes in human adipocytes. -- Abstract: Peroxisome proliferator-activated receptor-{alpha} (PPAR{alpha}) is a key regulator for maintaining whole-body energy balance. However, the physiological functions of PPAR{alpha} in adipocytes have been unclarified. We examined the functions of PPAR{alpha} using human multipotent adipose tissue-derived stem cells as a human adipocyte model. Activation of PPAR{alpha} by GW7647, a potent PPAR{alpha} agonist, increased the mRNA expression levels of adipocyte differentiation marker genes such as PPAR{gamma}, adipocyte-specific fatty acid-binding protein, and lipoprotein lipase and increased both GPDH activity and insulin-dependent glucose uptake level. The findings indicate that PPAR{alpha} activation stimulates adipocyte differentiation. However, lipid accumulation was not changed, which is usually observed when PPAR{gamma} is activated. On the other hand, PPAR{alpha} activation by GW7647 treatment induced the mRNA expression of fatty acid oxidation-related genes such as CPT-1B and AOX in a PPAR{alpha}-dependent manner. Moreover, PPAR{alpha} activation increased the production of CO{sub 2} and acid soluble metabolites, which are products of fatty acid oxidation, and increased oxygen consumption rate in human adipocytes. The data indicate that activation of PPAR{alpha} stimulates both adipocyte differentiation and fatty acid oxidation in human adipocytes, suggesting that PPAR{alpha} agonists could improve insulin resistance without lipid accumulation in adipocytes. The expected

  13. Chemically engineering ligand selectivity at the free fatty acid receptor 2 based on pharmacological variation between species orthologs

    Science.gov (United States)

    Hudson, Brian D.; Christiansen, Elisabeth; Tikhonova, Irina G.; Grundmann, Manuel; Kostenis, Evi; Adams, David R.; Ulven, Trond; Milligan, Graeme

    2012-01-01

    When it is difficult to develop selective ligands within a family of related G-protein-coupled receptors (GPCRs), chemically engineered receptors activated solely by synthetic ligands (RASSLs) are useful alternatives for probing receptor function. In the present work, we explored whether a RASSL of the free fatty acid receptor 2 (FFA2) could be developed on the basis of pharmacological variation between species orthologs. For this, bovine FFA2 was characterized, revealing distinct ligand selectivity compared with human FFA2. Homology modeling and mutational analysis demonstrated a single mutation in human FFA2 of C4.57G resulted in a human FFA2 receptor with ligand selectivity similar to the bovine receptor. This was exploited to generate human FFA2-RASSL by the addition of a second mutation at a known orthosteric ligand interaction site, H6.55Q. The resulting FFA2-RASSL displayed a >100-fold loss of activity to endogenous ligands, while responding to the distinct ligand sorbic acid with pEC50 values for inhibition of cAMP, 5.83 ± 0.11; Ca2+ mobilization, 4.63 ± 0.05; ERK phosphorylation, 5.61 ± 0.06; and dynamic mass redistribution, 5.35 ± 0.06. This FFA2-RASSL will be useful in future studies on this receptor and demonstrates that exploitation of pharmacological variation between species orthologs is a powerful method to generate novel chemically engineered GPCRs.—Hudson, B. D., Christiansen, E., Tikhonova, I. G., Grundmann, M., Kostenis, E., Adams, D. R., Ulven, T., Milligan, G. Chemically engineering ligand selectivity at the free fatty acid receptor 2 based on pharmacological variation between species orthologs. PMID:22919070

  14. Lysophosphatidic Acid Receptor Is a Functional Marker of Adult Hippocampal Precursor Cells

    Directory of Open Access Journals (Sweden)

    Tara L. Walker

    2016-04-01

    Full Text Available Here, we show that the lysophosphatidic acid receptor 1 (LPA1 is expressed by a defined population of type 1 stem cells and type 2a precursor cells in the adult mouse dentate gyrus. LPA1, in contrast to Nestin, also marks the quiescent stem cell population. Combining LPA1-GFP with EGFR and prominin-1 expression, we have enabled the prospective separation of both proliferative and non-proliferative precursor cell populations. Transcriptional profiling of the isolated proliferative precursor cells suggested immune mechanisms and cytokine signaling as molecular regulators of adult hippocampal precursor cell proliferation. In addition to LPA1 being a marker of this important stem cell population, we also show that the corresponding ligand LPA is directly involved in the regulation of adult hippocampal precursor cell proliferation and neurogenesis, an effect that can be attributed to LPA signaling via the AKT and MAPK pathways.

  15. Salvianolic Acid A, as a Novel ETA Receptor Antagonist, Shows Inhibitory Effects on Tumor in Vitro

    Directory of Open Access Journals (Sweden)

    Qiao Zhang

    2016-08-01

    Full Text Available Endothelin-1 (ET-1 autocrine and paracrine signaling modulate cell proliferation of tumor cells by activating its receptors, endothelin A receptor (ETAR and endothelin B receptor (ETBR. Dysregulation of ETAR activation promotes tumor development and progression. The potential of ETAR antagonists and the dual-ETAR and ETBR antagonists as therapeutic approaches are under preclinical and clinical studies. Salvianolic acid A (Sal A is a hydrophilic polyphenolic derivative isolated from Salvia miltiorrhiza Bunge (Danshen, which has been reported as an anti-cancer and cardio-protective herbal medicine. In this study, we demonstrate that Sal A inhibits ETAR activation induced by ET-1 in both recombinant and endogenous ETAR expression cell lines. The IC50 values were determined as 5.7 µM in the HEK293/ETAR cell line and 3.14 µM in HeLa cells, respectively. Furthermore, our results showed that Sal A suppressed cell proliferation and extended the doubling times of multiple cancer cells, including HeLa, DU145, H1975, and A549 cell lines. In addition, Sal A inhibited proliferation of DU145 cell lines stimulated by exogenous ET-1 treatment. Moreover, the cytotoxicity and cardio-toxicity of Sal A were assessed in human umbilical vein endothelial cells (HUVEC and Human-induced pluripotent stem cell-derived cardiomyocytes (hiPS-CMs, which proved that Sal A demonstrates no cytotoxicity or cardiotoxicity. Collectively, our findings indicate that Sal A is a novel anti-cancer candidate through targeting ETAR.

  16. Identification of a chemoreceptor for tricarboxylic acid cycle intermediates: differential chemotactic response towards receptor ligands.

    Science.gov (United States)

    Lacal, Jesús; Alfonso, Carlos; Liu, Xianxian; Parales, Rebecca E; Morel, Bertrand; Conejero-Lara, Francisco; Rivas, Germán; Duque, Estrella; Ramos, Juan L; Krell, Tino

    2010-07-23

    We report the identification of McpS as the specific chemoreceptor for 6 tricarboxylic acid (TCA) cycle intermediates and butyrate in Pseudomonas putida. The analysis of the bacterial mutant deficient in mcpS and complementation assays demonstrate that McpS is the only chemoreceptor of TCA cycle intermediates in the strain under study. TCA cycle intermediates are abundantly present in root exudates, and taxis toward these compounds is proposed to facilitate the access to carbon sources. McpS has an unusually large ligand-binding domain (LBD) that is un-annotated in InterPro and is predicted to contain 6 helices. The ligand profile of McpS was determined by isothermal titration calorimetry of purified recombinant LBD (McpS-LBD). McpS recognizes TCA cycle intermediates but does not bind very close structural homologues and derivatives like maleate, aspartate, or tricarballylate. This implies that functional similarity of ligands, such as being part of the same pathway, and not structural similarity is the primary element, which has driven the evolution of receptor specificity. The magnitude of chemotactic responses toward these 7 chemoattractants, as determined by qualitative and quantitative chemotaxis assays, differed largely. Ligands that cause a strong chemotactic response (malate, succinate, and fumarate) were found by differential scanning calorimetry to increase significantly the midpoint of protein unfolding (T(m)) and unfolding enthalpy (DeltaH) of McpS-LBD. Equilibrium sedimentation studies show that malate, the chemoattractant that causes the strongest chemotactic response, stabilizes the dimeric state of McpS-LBD. In this respect clear parallels exist to the Tar receptor and other eukaryotic receptors, which are discussed.

  17. Evolutionary diversification of retinoic acid receptor ligand-binding pocket structure by molecular tinkering

    Science.gov (United States)

    Gutierrez-Mazariegos, Juliana; Nadendla, Eswar Kumar; Studer, Romain A.; Alvarez, Susana; de Lera, Angel R.; Kuraku, Shigehiro; Bourguet, William; Laudet, Vincent

    2016-01-01

    Whole genome duplications (WGDs) have been classically associated with the origin of evolutionary novelties and the so-called duplication–degeneration–complementation model describes the possible fates of genes after duplication. However, how sequence divergence effectively allows functional changes between gene duplicates is still unclear. In the vertebrate lineage, two rounds of WGDs took place, giving rise to paralogous gene copies observed for many gene families. For the retinoic acid receptors (RARs), for example, which are members of the nuclear hormone receptor (NR) superfamily, a unique ancestral gene has been duplicated resulting in three vertebrate paralogues: RARα, RARβ and RARγ. It has previously been shown that this single ancestral RAR was neofunctionalized to give rise to a larger substrate specificity range in the RARs of extant jawed vertebrates (also called gnathostomes). To understand RAR diversification, the members of the cyclostomes (lamprey and hagfish), jawless vertebrates representing the extant sister group of gnathostomes, provide an intermediate situation and thus allow the characterization of the evolutionary steps that shaped RAR ligand-binding properties following the WGDs. In this study, we assessed the ligand-binding specificity of cyclostome RARs and found that their ligand-binding pockets resemble those of gnathostome RARα and RARβ. In contrast, none of the cyclostome receptors studied showed any RARγ-like specificity. Together, our results suggest that cyclostome RARs cover only a portion of the specificity repertoire of the ancestral gnathostome RARs and indicate that the establishment of ligand-binding specificity was a stepwise event. This iterative process thus provides a rare example for the diversification of receptor–ligand interactions of NRs following WGDs. PMID:27069642

  18. Lysophosphatidic acid receptors (LPARs): Potential targets for the treatment of neuropathic pain.

    Science.gov (United States)

    Velasco, María; O'Sullivan, Catherine; Sheridan, Graham K

    2017-02-01

    Neuropathic pain can arise from lesions to peripheral or central nerve fibres leading to spontaneous action potential generation and a lowering of the nociceptive threshold. Clinically, neuropathic pain can manifest in many chronic disease states such as cancer, diabetes or multiple sclerosis (MS). The bioactive lipid, lysophosphatidic acid (LPA), via activation of its receptors (LPARs), is thought to play a central role in both triggering and maintaining neuropathic pain. In particular, following an acute nerve injury, the excitatory neurotransmitters glutamate and substance P are released from primary afferent neurons leading to upregulated synthesis of lysophosphatidylcholine (LPC), the precursor for LPA production. LPC is converted to LPA by autotaxin (ATX), which can then activate macrophages/microglia and modulate neuronal functioning. A ubiquitous feature of animal models of neuropathic pain is demyelination of damaged nerves. It is thought that LPA contributes to demyelination through several different mechanisms. Firstly, high levels of LPA are produced following macrophage/microglial activation that triggers a self-sustaining feed-forward loop of de novo LPA synthesis. Secondly, macrophage/microglial activation contributes to inflammation-mediated demyelination of axons, thus initiating neuropathic pain. Therefore, targeting LPA production and/or the family of LPA-activated G protein-coupled receptors (GPCRs) may prove to be fruitful clinical approaches to treating demyelination and the accompanying neuropathic pain. This review discusses our current understanding of the role of LPA/LPAR signalling in the initiation of neuropathic pain and suggests potential targeted strategies for its treatment. This article is part of the Special Issue entitled 'Lipid Sensing G Protein-Coupled Receptors in the CNS'. Copyright © 2016 Elsevier Ltd. All rights reserved.

  19. Selective activation of liver X receptors by acanthoic acid-related diterpenes.

    Science.gov (United States)

    Traves, Paqui G; Hortelano, Sonsoles; Zeini, Miriam; Chao, Ta-Hsiang; Lam, Thanh; Neuteboom, Saskia T; Theodorakis, Emmanuel A; Palladino, Michael A; Castrillo, Antonio; Bosca, Lisardo

    2007-06-01

    Terpenoids constitute a large family of natural steroids that are widely distributed in plants and insects. We investigated the effects of a series of diterpenes structurally related to acanthoic acid in macrophage functions. We found that diterpenes with different substitutions at the C4 position in ring A are potent activators of liver X receptors (LXRalpha and LXRbeta) in both macrophage cell lines from human and mouse origin and primary murine macrophages. Activation of LXR by these diterpenes was evaluated in transient transfection assays and gene