WorldWideScience

Sample records for receptor signaling complexes

  1. Assembly of Oligomeric Death Domain Complexes during Toll Receptor Signaling*

    OpenAIRE

    Moncrieffe, Martin C.; Grossmann, J. Günter; Gay, Nicholas J.

    2008-01-01

    The Drosophila Toll receptor is activated by the endogenous protein ligand Spätzle in response to microbial stimuli in immunity and spatial cues during embryonic development. Downstream signaling is mediated by the adaptor proteins Tube, the kinase Pelle, and the Drosophila homologue of myeloid differentiation primary response protein (dMyD88). Here we have characterized heterodimeric (dMyD88-Tube) and heterotrimeric (dMyD88-Tube-Pelle) death domain complexes. We show ...

  2. Assembly of Oligomeric Death Domain Complexes during Toll Receptor Signaling*

    Science.gov (United States)

    Moncrieffe, Martin C.; Grossmann, J. Günter; Gay, Nicholas J.

    2008-01-01

    The Drosophila Toll receptor is activated by the endogenous protein ligand Spätzle in response to microbial stimuli in immunity and spatial cues during embryonic development. Downstream signaling is mediated by the adaptor proteins Tube, the kinase Pelle, and the Drosophila homologue of myeloid differentiation primary response protein (dMyD88). Here we have characterized heterodimeric (dMyD88-Tube) and heterotrimeric (dMyD88-Tube-Pelle) death domain complexes. We show that both the heterodimeric and heterotrimeric complexes form kidney-shaped structures and that Tube is bivalent and has separate high affinity binding sites for dMyD88 and Pelle. Additionally we found no interaction between the isolated death domains of Pelle and dMyD88. These results indicate that the mode of assembly of the heterotrimeric dMyD88-Tube-Pelle complex downstream of the activated Toll receptor is unique. The measured dissociation constants for the interaction between the death domains of dMyD88 and Tube and of Pelle and a preformed dMyD88-Tube complex are used to propose a model of the early postreceptor events in Drosophila Toll receptor signaling. PMID:18829464

  3. Assembly of oligomeric death domain complexes during Toll receptor signaling.

    Science.gov (United States)

    Moncrieffe, Martin C; Grossmann, J Günter; Gay, Nicholas J

    2008-11-28

    The Drosophila Toll receptor is activated by the endogenous protein ligand Spätzle in response to microbial stimuli in immunity and spatial cues during embryonic development. Downstream signaling is mediated by the adaptor proteins Tube, the kinase Pelle, and the Drosophila homologue of myeloid differentiation primary response protein (dMyD88). Here we have characterized heterodimeric (dMyD88-Tube) and heterotrimeric (dMyD88-Tube-Pelle) death domain complexes. We show that both the heterodimeric and heterotrimeric complexes form kidney-shaped structures and that Tube is bivalent and has separate high affinity binding sites for dMyD88 and Pelle. Additionally we found no interaction between the isolated death domains of Pelle and dMyD88. These results indicate that the mode of assembly of the heterotrimeric dMyD88-Tube-Pelle complex downstream of the activated Toll receptor is unique. The measured dissociation constants for the interaction between the death domains of dMyD88 and Tube and of Pelle and a preformed dMyD88-Tube complex are used to propose a model of the early postreceptor events in Drosophila Toll receptor signaling.

  4. Design principles of nuclear receptor signaling: how complex networking improves signal transduction

    Science.gov (United States)

    Kolodkin, Alexey N; Bruggeman, Frank J; Plant, Nick; Moné, Martijn J; Bakker, Barbara M; Campbell, Moray J; van Leeuwen, Johannes P T M; Carlberg, Carsten; Snoep, Jacky L; Westerhoff, Hans V

    2010-01-01

    The topology of nuclear receptor (NR) signaling is captured in a systems biological graphical notation. This enables us to identify a number of ‘design' aspects of the topology of these networks that might appear unnecessarily complex or even functionally paradoxical. In realistic kinetic models of increasing complexity, calculations show how these features correspond to potentially important design principles, e.g.: (i) cytosolic ‘nuclear' receptor may shuttle signal molecules to the nucleus, (ii) the active export of NRs may ensure that there is sufficient receptor protein to capture ligand at the cytoplasmic membrane, (iii) a three conveyor belts design dissipating GTP-free energy, greatly aids response, (iv) the active export of importins may prevent sequestration of NRs by importins in the nucleus and (v) the unspecific nature of the nuclear pore may ensure signal-flux robustness. In addition, the models developed are suitable for implementation in specific cases of NR-mediated signaling, to predict individual receptor functions and differential sensitivity toward physiological and pharmacological ligands. PMID:21179018

  5. Cooperative ethylene receptor signaling

    OpenAIRE

    Liu, Qian; Wen, Chi-Kuang

    2012-01-01

    The gaseous plant hormone ethylene is perceived by a family of five ethylene receptor members in the dicotyledonous model plant Arabidopsis. Genetic and biochemical studies suggest that the ethylene response is suppressed by ethylene receptor complexes, but the biochemical nature of the receptor signal is unknown. Without appropriate biochemical measures to trace the ethylene receptor signal and quantify the signal strength, the biological significance of the modulation of ethylene responses ...

  6. Ethylene Regulates Levels of Ethylene Receptor/CTR1 Signaling Complexes in Arabidopsis thaliana*

    Science.gov (United States)

    Shakeel, Samina N.; Gao, Zhiyong; Amir, Madiha; Chen, Yi-Feng; Rai, Muneeza Iqbal; Haq, Noor Ul; Schaller, G. Eric

    2015-01-01

    The plant hormone ethylene is perceived by a five-member family of receptors in Arabidopsis thaliana. The receptors function in conjunction with the Raf-like kinase CTR1 to negatively regulate ethylene signal transduction. CTR1 interacts with multiple members of the receptor family based on co-purification analysis, interacting more strongly with receptors containing a receiver domain. Levels of membrane-associated CTR1 vary in response to ethylene, doing so in a post-transcriptional manner that correlates with ethylene-mediated changes in levels of the ethylene receptors ERS1, ERS2, EIN4, and ETR2. Interactions between CTR1 and the receptor ETR1 protect ETR1 from ethylene-induced turnover. Kinetic and dose-response analyses support a model in which two opposing factors control levels of the ethylene receptor/CTR1 complexes. Ethylene stimulates the production of new complexes largely through transcriptional induction of the receptors. However, ethylene also induces turnover of receptors, such that levels of ethylene receptor/CTR1 complexes decrease at higher ethylene concentrations. Implications of this model for ethylene signaling are discussed. PMID:25814663

  7. Receptor density balances signal stimulation and attenuation in membrane-assembled complexes of bacterial chemotaxis signaling proteins

    Science.gov (United States)

    Besschetnova, Tatiana Y.; Montefusco, David J.; Asinas, Abdalin E.; Shrout, Anthony L.; Antommattei, Frances M.; Weis, Robert M.

    2008-01-01

    All cells possess transmembrane signaling systems that function in the environment of the lipid bilayer. In the Escherichia coli chemotaxis pathway, the binding of attractants to a two-dimensional array of receptors and signaling proteins simultaneously inhibits an associated kinase and stimulates receptor methylation—a slower process that restores kinase activity. These two opposing effects lead to robust adaptation toward stimuli through a physical mechanism that is not understood. Here, we provide evidence of a counterbalancing influence exerted by receptor density on kinase stimulation and receptor methylation. Receptor signaling complexes were reconstituted over a range of defined surface concentrations by using a template-directed assembly method, and the kinase and receptor methylation activities were measured. Kinase activity and methylation rates were both found to vary significantly with surface concentration—yet in opposite ways: samples prepared at high surface densities stimulated kinase activity more effectively than low-density samples, whereas lower surface densities produced greater methylation rates than higher densities. FRET experiments demonstrated that the cooperative change in kinase activity coincided with a change in the arrangement of the membrane-associated receptor domains. The counterbalancing influence of density on receptor methylation and kinase stimulation leads naturally to a model for signal regulation that is compatible with the known logic of the E. coli pathway. Density-dependent mechanisms are likely to be general and may operate when two or more membrane-related processes are influenced differently by the two-dimensional concentration of pathway elements. PMID:18711126

  8. Binding of the Ras activator son of sevenless to insulin receptor substrate-1 signaling complexes.

    Science.gov (United States)

    Baltensperger, K; Kozma, L M; Cherniack, A D; Klarlund, J K; Chawla, A; Banerjee, U; Czech, M P

    1993-06-25

    Signal transmission by insulin involves tyrosine phosphorylation of a major insulin receptor substrate (IRS-1) and exchange of Ras-bound guanosine diphosphate for guanosine triphosphate. Proteins containing Src homology 2 and 3 (SH2 and SH3) domains, such as the p85 regulatory subunit of phosphatidylinositol-3 kinase and growth factor receptor-bound protein 2 (GRB2), bind tyrosine phosphate sites on IRS-1 through their SH2 regions. Such complexes in COS cells were found to contain the heterologously expressed putative guanine nucleotide exchange factor encoded by the Drosophila son of sevenless gene (dSos). Thus, GRB2, p85, or other proteins with SH2-SH3 adapter sequences may link Sos proteins to IRS-1 signaling complexes as part of the mechanism by which insulin activates Ras.

  9. Cocaine Disrupts Histamine H3 Receptor Modulation of Dopamine D1 Receptor Signaling: σ1-D1-H3 Receptor Complexes as Key Targets for Reducing Cocaine's Effects

    Science.gov (United States)

    Moreno, Estefanía; Moreno-Delgado, David; Navarro, Gemma; Hoffmann, Hanne M.; Fuentes, Silvia; Rosell-Vilar, Santi; Gasperini, Paola; Rodríguez-Ruiz, Mar; Medrano, Mireia; Mallol, Josefa; Cortés, Antoni; Casadó, Vicent; Lluís, Carme; Ferré, Sergi; Ortiz, Jordi; Canela, Enric

    2014-01-01

    The general effects of cocaine are not well understood at the molecular level. What is known is that the dopamine D1 receptor plays an important role. Here we show that a key mechanism may be cocaine's blockade of the histamine H3 receptor-mediated inhibition of D1 receptor function. This blockade requires the σ1 receptor and occurs upon cocaine binding to σ1-D1-H3 receptor complexes. The cocaine-mediated disruption leaves an uninhibited D1 receptor that activates Gs, freely recruits β-arrestin, increases p-ERK 1/2 levels, and induces cell death when over activated. Using in vitro assays with transfected cells and in ex vivo experiments using both rats acutely treated or self-administered with cocaine along with mice depleted of σ1 receptor, we show that blockade of σ1 receptor by an antagonist restores the protective H3 receptor-mediated brake on D1 receptor signaling and prevents the cell death from elevated D1 receptor signaling. These findings suggest that a combination therapy of σ1R antagonists with H3 receptor agonists could serve to reduce some effects of cocaine. PMID:24599455

  10. CD163: a signal receptor scavenging haptoglobin-hemoglobin complexes from plasma

    DEFF Research Database (Denmark)

    Graversen, Jonas Heilskov; Madsen, Mette; Moestrup, Søren K

    2002-01-01

    as the endocytic receptor binding hemoglobin (Hb) in complex with the plasma protein haptoglobin (Hp). This specific receptor-ligand interaction leading to removal from plasma of the Hp-Hb complex-but not free Hp or Hb-now explains the depletion of circulating Hp in individuals with increased intravascular...

  11. Entamoeba histolytica: a beta 1 integrin-like fibronectin receptor assembles a signaling complex similar to those of mammalian cells.

    Science.gov (United States)

    Flores-Robles, Donaciano; Rosales, Carlos; Rosales-Encina, José Luis; Talamás-Rohana, Patricia

    2003-01-01

    During tissue invasion, Entamoeba histolytica trophozoites interact with endothelial cells and extracellular matrix (ECM) proteins such as fibronectin (FN), collagen, and laminin. It has been demonstrated that trophozoites interact with FN through a beta1 integrin-like FN receptor (beta 1EhFNR), activating tyrosine kinases. In order to characterize the signaling process triggered by the amoebic receptor, activation, and association of tyrosine kinases and structural proteins were determined. As a result of FN binding by the beta 1EhFNR, the receptor itself, FAK, and paxillin were phosphorylated in tyrosine. Co-immunoprecipitation experiments showed that a multimolecular signaling complex was formed by the amoebic FN receptor, FAK, paxillin, and vinculin. These results strongly suggest that a signaling pathway, similar to the one used in mammalian cells, is activated when E. histolytica trophozoites adhere to FN.

  12. Unraveling the Complexities of Androgen Receptor Signaling in Prostate Cancer Cells

    OpenAIRE

    Heemers, Hannelore V.; Tindall, Donald J.

    2009-01-01

    Androgen signaling is critical for proliferation of prostate cancer cells but cannot be fully inhibited by current androgen deprivation therapies. A study by Xu et al. in this issue of Cancer Cell provides insights into the complexities of androgen signaling in prostate cancer and suggests avenues to target a subset of androgen-sensitive genes.

  13. Biogenesis of the mitochondrial TOM complex: Mim1 promotes insertion and assembly of signal-anchored receptors.

    Science.gov (United States)

    Becker, Thomas; Pfannschmidt, Sylvia; Guiard, Bernard; Stojanovski, Diana; Milenkovic, Dusanka; Kutik, Stephan; Pfanner, Nikolaus; Meisinger, Chris; Wiedemann, Nils

    2008-01-04

    The translocase of the outer membrane (TOM complex) is the central entry gate for nuclear-encoded mitochondrial precursor proteins. All Tom proteins are also encoded by nuclear genes and synthesized as precursors in the cytosol. The channel-forming beta-barrel protein Tom40 is targeted to mitochondria via Tom receptors and inserted into the outer membrane by the sorting and assembly machinery (SAM complex). A further outer membrane protein, Mim1, plays a less defined role in assembly of Tom40 into the TOM complex. The three receptors Tom20, Tom22, and Tom70 are anchored in the outer membrane by a single transmembrane alpha-helix, located at the N terminus in the case of Tom20 and Tom70 (signal-anchored) or in the C-terminal portion in the case of Tom22 (tail-anchored). Insertion of the precursor of Tom22 into the outer membrane requires pre-existing Tom receptors while the import pathway of the precursors of Tom20 and Tom70 is only poorly understood. We report that Mim1 is required for efficient membrane insertion and assembly of Tom20 and Tom70, but not Tom22. We show that Mim1 associates with SAM(core) components to a large SAM complex, explaining its role in late steps of the assembly pathway of Tom40. We conclude that Mim1 is not only required for biogenesis of the beta-barrel protein Tom40 but also for membrane insertion and assembly of signal-anchored Tom receptors. Thus, Mim1 plays an important role in the efficient assembly of the mitochondrial TOM complex.

  14. CYLD Limits Lys63- and Met1-Linked Ubiquitin at Receptor Complexes to Regulate Innate Immune Signaling

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    Matous Hrdinka

    2016-03-01

    Full Text Available Innate immune signaling relies on the deposition of non-degradative polyubiquitin at receptor-signaling complexes, but how these ubiquitin modifications are regulated by deubiquitinases remains incompletely understood. Met1-linked ubiquitin (Met1-Ub is assembled by the linear ubiquitin assembly complex (LUBAC, and this is counteracted by the Met1-Ub-specific deubiquitinase OTULIN, which binds to the catalytic LUBAC subunit HOIP. In this study, we report that HOIP also interacts with the deubiquitinase CYLD but that CYLD does not regulate ubiquitination of LUBAC components. Instead, CYLD limits extension of Lys63-Ub and Met1-Ub conjugated to RIPK2 to restrict signaling and cytokine production. Accordingly, Met1-Ub and Lys63-Ub were individually required for productive NOD2 signaling. Our study thus suggests that LUBAC, through its associated deubiquitinases, coordinates the deposition of not only Met1-Ub but also Lys63-Ub to ensure an appropriate response to innate immune receptor activation.

  15. Orphan receptor GPR179 forms macromolecular complexes with components of metabotropic signaling cascade in retina ON-bipolar neurons.

    Science.gov (United States)

    Orlandi, Cesare; Cao, Yan; Martemyanov, Kirill A

    2013-10-29

    In the mammalian retina, synaptic transmission between light-excited rod photoreceptors and downstream ON-bipolar neurons is indispensable for dim vision, and disruption of this process leads to congenital stationary night blindness in human patients. The ON-bipolar neurons use the metabotropic signaling cascade, initiated by the mGluR6 receptor, to generate depolarizing responses to light-induced changes in neurotransmitter glutamate release from the photoreceptor axonal terminals. Evidence for the identity of the components involved in transducing these signals is growing rapidly. Recently, the orphan receptor, GPR179, a member of the G protein-coupled receptor (GPCR) superfamily, has been shown to be indispensable for the synaptic responses of ON-bipolar cells. In our study, we investigated the interaction of GPR179 with principle components of the signal transduction cascade. We used immunoprecipitation and proximity ligation assays in transfected cells and native retinas to characterize the protein-protein interactions involving GPR179. The influence of cascade components on GPR179 localization was examined through immunohistochemical staining of the retinas from genetic mouse models. We demonstrated that, in mouse retinas, GPR179 forms physical complexes with the main components of the metabotropic cascade, recruiting mGluR6, TRPM1, and the RGS proteins. Elimination of mGluR6 or RGS proteins, but not TRPM1, detrimentally affects postsynaptic targeting or GPR179 expression. These observations suggest that the mGluR6 signaling cascade is scaffolded as a macromolecular complex in which the interactions between the components ensure the optimal spatiotemporal characteristics of signal transduction.

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

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

    2014-01-17

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

  17. Maize and Arabidopsis ARGOS Proteins Interact with Ethylene Receptor Signaling Complex, Supporting a Regulatory Role for ARGOS in Ethylene Signal Transduction[OPEN

    Science.gov (United States)

    Shi, Jinrui; Wang, Hongyu; Habben, Jeffrey E.

    2016-01-01

    The phytohormone ethylene regulates plant growth and development as well as plant response to environmental cues. ARGOS genes reduce plant sensitivity to ethylene when overexpressed in transgenic Arabidopsis (Arabidopsis thaliana) and maize (Zea mays). A previous genetic study suggested that the endoplasmic reticulum and Golgi-localized maize ARGOS1 targets the ethylene signal transduction components at or upstream of CONSTITUTIVE TRIPLE RESPONSE1, but the mechanism of ARGOS modulating ethylene signaling is unknown. Here, we demonstrate in Arabidopsis that ZmARGOS1, as well as the Arabidopsis ARGOS homolog ORGAN SIZE RELATED1, physically interacts with Arabidopsis REVERSION-TO-ETHYLENE SENSITIVITY1 (RTE1), an ethylene receptor interacting protein that regulates the activity of ETHYLENE RESPONSE1. The protein-protein interaction was also detected with the yeast split-ubiquitin two-hybrid system. Using the same yeast assay, we found that maize RTE1 homolog REVERSION-TO-ETHYLENE SENSITIVITY1 LIKE4 (ZmRTL4) and ZmRTL2 also interact with maize and Arabidopsis ARGOS proteins. Like AtRTE1 in Arabidopsis, ZmRTL4 and ZmRTL2 reduce ethylene responses when overexpressed in maize, indicating a similar mechanism for ARGOS regulating ethylene signaling in maize. A polypeptide fragment derived from ZmARGOS8, consisting of a Pro-rich motif flanked by two transmembrane helices that are conserved among members of the ARGOS family, can interact with AtRTE1 and maize RTL proteins in Arabidopsis. The conserved domain is necessary and sufficient to reduce ethylene sensitivity in Arabidopsis and maize. Overall, these results suggest a physical association between ARGOS and the ethylene receptor signaling complex via AtRTE1 and maize RTL proteins, supporting a role for ARGOS in regulating ethylene perception and the early steps of signal transduction in Arabidopsis and maize. PMID:27268962

  18. IL-7 Induces an Epitope Masking of γc Protein in IL-7 Receptor Signaling Complex

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    Tae Sik Goh

    2017-01-01

    Full Text Available IL-7 signaling via IL-7Rα and common γ-chain (γc is necessary for the development and homeostasis of T cells. Although the delicate mechanism in which IL-7Rα downregulation allows the homeostasis of T cell with limited IL-7 has been well known, the exact mechanism behind the interaction between IL-7Rα and γc in the absence or presence of IL-7 remains unclear. Additionally, we are still uncertain as to how only IL-7Rα is separately downregulated by the binding of IL-7 from the IL-7Rα/γc complex. We demonstrate here that 4G3, TUGm2, and 3E12 epitope masking of γc protein are induced in the presence of IL-7, indicating that the epitope alteration is induced by IL-7 binding to the preassembled receptor core. Moreover, the epitope masking of γc protein is inversely correlated with the expression of IL-7Rα upon IL-7 binding, implying that the structural alteration of γc might be involved in the regulation of IL-7Rα expression. The conformational change in γc upon IL-7 binding may contribute not only to forming the functional IL-7 signaling complex but also to optimally regulating the expression of IL-7Rα.

  19. IL-7 Induces an Epitope Masking of γc Protein in IL-7 Receptor Signaling Complex

    Science.gov (United States)

    Goh, Tae Sik; Jo, Yuna; Lee, Byunghyuk; Kim, Geona; Hwang, Hyunju; Ko, Eunhee; Kang, Seung Wan; Oh, Sae-Ock; Baek, Sun-Yong; Yoon, Sik; Lee, Jung Sub

    2017-01-01

    IL-7 signaling via IL-7Rα and common γ-chain (γc) is necessary for the development and homeostasis of T cells. Although the delicate mechanism in which IL-7Rα downregulation allows the homeostasis of T cell with limited IL-7 has been well known, the exact mechanism behind the interaction between IL-7Rα and γc in the absence or presence of IL-7 remains unclear. Additionally, we are still uncertain as to how only IL-7Rα is separately downregulated by the binding of IL-7 from the IL-7Rα/γc complex. We demonstrate here that 4G3, TUGm2, and 3E12 epitope masking of γc protein are induced in the presence of IL-7, indicating that the epitope alteration is induced by IL-7 binding to the preassembled receptor core. Moreover, the epitope masking of γc protein is inversely correlated with the expression of IL-7Rα upon IL-7 binding, implying that the structural alteration of γc might be involved in the regulation of IL-7Rα expression. The conformational change in γc upon IL-7 binding may contribute not only to forming the functional IL-7 signaling complex but also to optimally regulating the expression of IL-7Rα. PMID:28127156

  20. Hsp90α forms a stable complex at the cilium neck for the interaction of signalling molecules in IGF-1 receptor signalling.

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    Wang, Hongzhong; Zou, Xinle; Wei, Zhuang; Wu, Yuan; Li, Rongxia; Zeng, Rong; Chen, Zhengjun; Liao, Kan

    2015-01-01

    The primary cilium is composed of an axoneme that protrudes from the cell surface, a basal body beneath the membrane and a transition neck in between. It is a sensory organelle on the plasma membrane, involved in mediating extracellular signals. In the transition neck region of the cilium, the microtubules change from triplet to doublet microtubules. This region also contains the transition fibres that crosslink the axoneme with the membrane and the necklace proteins that regulate molecules being transported into and out of the cilium. In this protein-enriched, complex area it is important to maintain the correct assembly of all of these proteins. Here, through immunofluorescent staining and protein isolation, we identify the molecular chaperone Hsp90α clustered at the periciliary base. At the transition neck region, phosphorylated Hsp90α forms a stable ring around the axoneme. Heat shock treatment causes Hsp90α to dissipate and induces resorption of cilia. We further identify that Hsp90α at the transition neck region represents a signalling platform on which IRS-1 interacts with intracellular downstream signalling molecules involved in IGF-1 receptor signalling. © 2015. Published by The Company of Biologists Ltd.

  1. Wnt-5a/Frizzled9 Receptor Signaling through the Gαo-Gβγ Complex Regulates Dendritic Spine Formation*

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    Ramírez, Valerie T.; Ramos-Fernández, Eva; Henríquez, Juan Pablo; Lorenzo, Alfredo; Inestrosa, Nibaldo C.

    2016-01-01

    Wnt ligands play crucial roles in the development and regulation of synapse structure and function. Specifically, Wnt-5a acts as a secreted growth factor that regulates dendritic spine formation in rodent hippocampal neurons, resulting in postsynaptic development that promotes the clustering of the PSD-95 (postsynaptic density protein 95). Here, we focused on the early events occurring after the interaction between Wnt-5a and its Frizzled receptor at the neuronal cell surface. Additionally, we studied the role of heterotrimeric G proteins in Wnt-5a-dependent synaptic development. We report that FZD9 (Frizzled9), a Wnt receptor related to Williams syndrome, is localized in the postsynaptic region, where it interacts with Wnt-5a. Functionally, FZD9 is required for the Wnt-5a-mediated increase in dendritic spine density. FZD9 forms a precoupled complex with Gαo under basal conditions that dissociates after Wnt-5a stimulation. Accordingly, we found that G protein inhibition abrogates the Wnt-5a-dependent pathway in hippocampal neurons. In particular, the activation of Gαo appears to be a key factor controlling the Wnt-5a-induced dendritic spine density. In addition, we found that Gβγ is required for the Wnt-5a-mediated increase in cytosolic calcium levels and spinogenesis. Our findings reveal that FZD9 and heterotrimeric G proteins regulate Wnt-5a signaling and dendritic spines in cultured hippocampal neurons. PMID:27402827

  2. Peptides derived from specific interaction sites of the fibroblast growth factor 2 - FGF receptor complexes induce receptor activation and signaling

    DEFF Research Database (Denmark)

    Manfè, Valentina; Kochoyan, Artur; Bock, Elisabeth

    2010-01-01

    J. Neurochem. (2010) 10.1111/j.1471-4159.2010.06718.x Abstract Basic fibroblast growth factor (FGF2, bFGF) is the most extensively studied member of the FGF family and is involved in neurogenesis, differentiation, neuroprotection, and synaptic plasticity in the CNS. FGF2 executes its pleiotropic...... biologic actions by binding, dimerizing, and activating FGF receptors (FGFRs). The present study reports the physiologic impact of various FGF2-FGFR1 contact sites employing three different synthetic peptides, termed canofins, designed based on structural analysis of the interactions between FGF2 and FGFR1...

  3. Dynamic complexity: plant receptor complexes at the plasma membrane.

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    Burkart, Rebecca C; Stahl, Yvonne

    2017-12-01

    Plant receptor complexes at the cell surface perceive many different external and internal signalling molecules and relay these signals into the cell to regulate development, growth and immunity. Recent progress in the analyses of receptor complexes using different live cell imaging approaches have shown that receptor complex formation and composition are dynamic and take place at specific microdomains at the plasma membrane. In this review we focus on three prominent examples of Arabidopsis thaliana receptor complexes and how their dynamic spatio-temporal distribution at the PM has been studied recently. We will elaborate on the newly emerging concept of plasma membrane microdomains as potential hubs for specific receptor complex assembly and signalling outputs. Copyright © 2017 Elsevier Ltd. All rights reserved.

  4. Formation and biochemical characterization of tube/pelle death domain complexes: critical regulators of postreceptor signaling by the Drosophila toll receptor.

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    Schiffmann, D A; White, J H; Cooper, A; Nutley, M A; Harding, S E; Jumel, K; Solari, R; Ray, K P; Gay, N J

    1999-09-07

    In Drosophila, the Toll receptor signaling pathway is required for embryonic dorso-ventral patterning and at later developmental stages for innate immune responses. It is thought that dimerization of the receptor by binding of the ligand spätzle causes the formation of a postreceptor activation complex at the cytoplasmic surface of the membrane. Two components of this complex are the adaptor tube and protein kinase pelle. These proteins both have "death domains", protein interaction motifs found in a number of signaling pathways, particularly those involved in apoptotic cell death. It is thought that pelle is bound by tube during formation of the activation complexes, and that this interaction is mediated by the death domains. In this paper, we show using the yeast two-hybrid system that the wild-type tube and pelle death domains bind together. Mutant tube proteins which do not support signaling in the embryo are also unable to bind pelle in the 2-hybrid assay. We have purified proteins corresponding to the death domains of tube and pelle and show that these form corresponding heterodimeric complexes in vitro. Partial proteolysis reveals a smaller core consisting of the minimal death domain sequences. We have studied the tube/pelle interaction with the techniques of surface plasmon resonance, analytical ultracentrifugation and isothermal titration calorimetry. These measurements produce a value of K(d) for the complex of about 0.5 microM.

  5. Prostaglandin Receptor Signaling in Disease

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    Toshiyuki Matsuoka

    2007-01-01

    Full Text Available Prostanoids, consisting of the prostaglandins (PGs and the thromboxanes (TXs, are a group of lipid mediators formed in response to various stimuli. They include PGD2, PGE2, PGF2α, PGI2, and TXA2. They are released outside of the cells immediately after synthesis, and exert their actions by binding to a G-protein coupled rhodopsin-type receptor on the surface of target cells. There are eight types of the prostanoid receptors conserved in mammals from mouse to human. They are the PGD receptor (DP, four subtypes of the PGE receptor (EP1, EP2, EP3, and EP4, the PGF receptor (FP, PGI receptor (IP, and TXA receptor (TP. Recently, mice deficient in each of these prostanoid receptors were generated and subjected to various experimental models of disease. These studies have revealed the roles of PG receptor signaling in various pathological conditions, and suggest that selective manipulation of the prostanoid receptors may be beneficial in treatment of the pathological conditions. Here we review these recent findings of roles of prostanoid receptor signaling and their therapeutic implications.

  6. Nuclear Receptor Signaling Atlas (NURSA)

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    U.S. Department of Health & Human Services — The Nuclear Receptor Signaling Atlas (NURSA) is designed to foster the development of a comprehensive understanding of the structure, function, and role in disease...

  7. Conformational coupling between receptor and kinase binding sites through a conserved salt bridge in a signaling complex scaffold protein.

    Directory of Open Access Journals (Sweden)

    Davi R Ortega

    Full Text Available Bacterial chemotaxis is one of the best studied signal transduction pathways. CheW is a scaffold protein that mediates the association of the chemoreceptors and the CheA kinase in a ternary signaling complex. The effects of replacing conserved Arg62 of CheW with other residues suggested that the scaffold protein plays a more complex role than simply binding its partner proteins. Although R62A CheW had essentially the same affinity for chemoreceptors and CheA, cells expressing the mutant protein are impaired in chemotaxis. Using a combination of molecular dynamics simulations (MD, NMR spectroscopy, and circular dichroism (CD, we addressed the role of Arg62. Here we show that Arg62 forms a salt bridge with another highly conserved residue, Glu38. Although this interaction is unimportant for overall protein stability, it is essential to maintain the correct alignment of the chemoreceptor and kinase binding sites of CheW. Computational and experimental data suggest that the role of the salt bridge in maintaining the alignment of the two partner binding sites is fundamental to the function of the signaling complex but not to its assembly. We conclude that a key feature of CheW is to maintain the specific geometry between the two interaction sites required for its function as a scaffold.

  8. Glucocorticoid receptor signaling in health and disease

    Science.gov (United States)

    Kadmiel, Mahita; Cidlowski, John A.

    2013-01-01

    Glucocorticoids are steroid hormones regulated in a circadian and stres-associated manner to maintain various metabolic and homeostatic functions that are necessary for life. Synthetic glucocorticoids are widely prescribed drugs for many conditions including asthma, chronic obstructive pulmonary disease (COPD), and inflammatory disorders of the eye. Research in the last few years has begun to unravel the profound complexity of glucocorticoid signaling and has contributed remarkably to improved therapeutic strategies. Glucocorticoids signal through the glucocorticoid receptor, a member of the superfamily of nuclear receptors, in both genomic and non-genomic ways in almost every tissue in the human body. In this review, we will provide an update on glucocorticoid receptor signaling and highlight the role of GR signaling in physiological and pathophysiological conditions in the major organ systems in the human body. PMID:23953592

  9. Role of a transductional-transcriptional processor complex involving MyD88 and IRF-7 in Toll-like receptor signaling

    Science.gov (United States)

    Honda, Kenya; Yanai, Hideyuki; Mizutani, Tatsuaki; Negishi, Hideo; Shimada, Naoya; Suzuki, Nobutaka; Ohba, Yusuke; Takaoka, Akinori; Yeh, Wen-Chen; Taniguchi, Tadatsugu

    2004-01-01

    Toll-like receptor (TLR) activation is central to immunity, wherein the activation of the TLR9 subfamily members TLR9 and TLR7 results in the robust induction of type I IFNs (IFN-α/β) by means of the MyD88 adaptor protein. However, it remains unknown how the TLR signal “input” can be processed through MyD88 to “output” the induction of the IFN genes. Here, we demonstrate that the transcription factor IRF-7 interacts with MyD88 to form a complex in the cytoplasm. We provide evidence that this complex also involves IRAK4 and TRAF6 and provides the foundation for the TLR9-dependent activation of the IFN genes. The complex defined in this study represents an example of how the coupling of the signaling adaptor and effector kinase molecules together with the transcription factor regulate the processing of an extracellular signal to evoke its versatile downstream transcriptional events in a cell. Thus, we propose that this molecular complex may function as a cytoplasmic transductional-transcriptional processor. PMID:15492225

  10. Adaptor Protein Complex-2 (AP-2) and Epsin-1 Mediate Protease-activated Receptor-1 Internalization via Phosphorylation- and Ubiquitination-dependent Sorting Signals*

    Science.gov (United States)

    Chen, Buxin; Dores, Michael R.; Grimsey, Neil; Canto, Isabel; Barker, Breann L.; Trejo, JoAnn

    2011-01-01

    Signaling by protease-activated receptor-1 (PAR1), a G protein-coupled receptor (GPCR) for thrombin, is regulated by desensitization and internalization. PAR1 desensitization is mediated by β-arrestins, like most classic GPCRs. In contrast, internalization of PAR1 occurs through a clathrin- and dynamin-dependent pathway independent of β-arrestins. PAR1 displays two modes of internalization. Constitutive internalization of unactivated PAR1 is mediated by the clathrin adaptor protein complex-2 (AP-2), where the μ2-adaptin subunit binds directly to a tyrosine-based motif localized within the receptor C-tail domain. However, AP-2 depletion only partially inhibits agonist-induced internalization of PAR1, suggesting a function for other clathrin adaptors in this process. Here, we now report that AP-2 and epsin-1 are both critical mediators of agonist-stimulated PAR1 internalization. We show that ubiquitination of PAR1 and the ubiquitin-interacting motifs of epsin-1 are required for epsin-1-dependent internalization of activated PAR1. In addition, activation of PAR1 promotes epsin-1 de-ubiquitination, which may increase its endocytic adaptor activity to facilitate receptor internalization. AP-2 also regulates activated PAR1 internalization via recognition of distal C-tail phosphorylation sites rather than the canonical tyrosine-based motif. Thus, AP-2 and epsin-1 are both required to promote efficient internalization of activated PAR1 and recognize discrete receptor sorting signals. This study defines a new pathway for internalization of mammalian GPCRs. PMID:21965661

  11. Membrane Trafficking of Death Receptors: Implications on Signalling

    Directory of Open Access Journals (Sweden)

    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.

  12. TAM receptor signaling in development.

    Science.gov (United States)

    Burstyn-Cohen, Tal

    2017-01-01

    TYRO3, AXL and MERTK comprise the TAM family of receptor protein tyrosine kinases. Activated by their ligands, protein S (PROS1) and growth-arrest-specific 6 (GAS6), they mediate numerous cellular functions throughout development and adulthood. Expressed by a myriad of cell types and tissues, they have been implicated in homeostatic regulation of the immune, nervous, vascular, bone and reproductive systems. The loss-of-function of TAM signaling in adult tissues culminates in the destruction of tissue homeostasis and diseased states, while TAM gain-of-function in various tumors promotes cancer phenotypes. Combinatorial ligand-receptor interactions may elicit different molecular and cellular responses. Many of the TAM regulatory functions are essentially developmental, taking place both during embryogenesis and postnatally. This review highlights current knowledge on the role of TAM receptors and their ligands during these developmental processes in the immune, nervous, vascular and reproductive systems.

  13. Chansporter complexes in cell signaling.

    Science.gov (United States)

    Abbott, Geoffrey W

    2017-09-01

    Ion channels facilitate diffusion of ions across cell membranes for such diverse purposes as neuronal signaling, muscular contraction, and fluid homeostasis. Solute transporters often utilize ionic gradients to move aqueous solutes up their concentration gradient, also fulfilling a wide variety of tasks. Recently, an increasing number of ion channel-transporter ('chansporter') complexes have been discovered. Chansporter complex formation may overcome what could otherwise be considerable spatial barriers to rapid signal integration and feedback between channels and transporters, the ions and other substrates they transport, and environmental factors to which they must respond. Here, current knowledge in this field is summarized, covering both heterologous expression structure/function findings and potential mechanisms by which chansporter complexes fulfill contrasting roles in cell signaling in vivo. © 2017 Federation of European Biochemical Societies.

  14. Androgen Receptor Signaling in Bladder Cancer

    OpenAIRE

    Li, Peng; Chen, Jinbo; Miyamoto, Hiroshi

    2017-01-01

    Emerging preclinical findings have indicated that steroid hormone receptor signaling plays an important role in bladder cancer outgrowth. In particular, androgen-mediated androgen receptor signals have been shown to correlate with the promotion of tumor development and progression, which may clearly explain some sex-specific differences in bladder cancer. This review summarizes and discusses the available data, suggesting the involvement of androgens and/or the androgen receptor pathways in u...

  15. The interleukin-4 receptor: signal transduction by a hematopoietin receptor.

    Science.gov (United States)

    Keegan, A D; Pierce, J H

    1994-02-01

    Over the last several years, the receptors for numerous cytokines have been molecularly characterized. Analysis of their amino acid sequences shows that some of these receptors bear certain motifs in their extracellular domains that define a family of receptors called the Hematopoietin receptor superfamily. Significant advances in characterizing the structure, function, and mechanisms of signal transduction have been made for several members of this family. The purpose of this review is to discuss the recent advances made for one of the family members, the interleukin (IL) 4 receptor. Other receptor systems have recently been reviewed elsewhere. The IL-4 receptor consists of, at the minimum, the cloned 140 kDa IL-4-binding chain with the potential for associating with other chains. The IL-4 receptor transduces its signal by activating a tyrosine kinase that phosphorylates cellular substrates, including the receptor itself, and the 170 kDa substrate called 4PS. Phosphorylated 4PS interacts with the SH2 domain of the enzyme PI-3'-kinase and increases its enzymatic activity. These early events in the IL-4 receptor initiated signaling pathway may trigger a series of signals that will ultimately lead to an IL-4 specific biologic outcome.

  16. Proliferative signaling initiated in ACTH receptors

    Directory of Open Access Journals (Sweden)

    C.F.P. Lotfi

    2000-10-01

    Full Text Available This article reviews recent results of studies aiming to elucidate modes of integrating signals initiated in ACTH receptors and FGF2 receptors, within the network system of signal transduction found in Y1 adrenocortical cells. These modes of signal integration should be central to the mechanisms underlying the regulation of the G0->G1->S transition in the adrenal cell cycle. FGF2 elicits a strong mitogenic response in G0/G1-arrested Y1 adrenocortical cells, that includes a rapid and transient activation of extracellular signal-regulated kinases-mitogen-activated protein kinases (ERK-MAPK (2 to 10 min, b transcription activation of c-fos, c-jun and c-myc genes (10 to 30 min, c induction of c-Fos and c-Myc proteins by 1 h and cyclin D1 protein by 5 h, and d onset of DNA synthesis stimulation within 8 h. ACTH, itself a weak mitogen, interacts with FGF2 in a complex manner, blocking the FGF2 mitogenic response during the early and middle G1 phase, keeping ERK-MAPK activation and c-Fos and cyclin D1 induction at maximal levels, but post-transcriptionally inhibiting c-Myc expression. c-Fos and c-Jun proteins are mediators in both the strong and the weak mitogenic responses respectively triggered by FGF2 and ACTH. Induction of c-Fos and stimulation of DNA synthesis by ACTH are independent of PKA and are inhibited by the PKC inhibitor GF109203X. In addition, ACTH is a poor activator of ERK-MAPK, but c-Fos induction and DNA synthesis stimulation by ACTH are strongly inhibited by the inhibitor of MEK1 PD98059.

  17. Cocaine Inhibits Dopamine D2 Receptor Signaling via Sigma-1-D2 Receptor Heteromers

    Science.gov (United States)

    Navarro, Gemma; Moreno, Estefania; Bonaventura, Jordi; Brugarolas, Marc; Farré, Daniel; Aguinaga, David; Mallol, Josefa; Cortés, Antoni; Casadó, Vicent; Lluís, Carmen; Ferre, Sergi

    2013-01-01

    Under normal conditions the brain maintains a delicate balance between inputs of reward seeking controlled by neurons containing the D1-like family of dopamine receptors and inputs of aversion coming from neurons containing the D2-like family of dopamine receptors. Cocaine is able to subvert these balanced inputs by altering the cell signaling of these two pathways such that D1 reward seeking pathway dominates. Here, we provide an explanation at the cellular and biochemical level how cocaine may achieve this. Exploring the effect of cocaine on dopamine D2 receptors function, we present evidence of σ1 receptor molecular and functional interaction with dopamine D2 receptors. Using biophysical, biochemical, and cell biology approaches, we discovered that D2 receptors (the long isoform of the D2 receptor) can complex with σ1 receptors, a result that is specific to D2 receptors, as D3 and D4 receptors did not form heteromers. We demonstrate that the σ1-D2 receptor heteromers consist of higher order oligomers, are found in mouse striatum and that cocaine, by binding to σ1 -D2 receptor heteromers, inhibits downstream signaling in both cultured cells and in mouse striatum. In contrast, in striatum from σ1 knockout animals these complexes are not found and this inhibition is not seen. Taken together, these data illuminate the mechanism by which the initial exposure to cocaine can inhibit signaling via D2 receptor containing neurons, destabilizing the delicate signaling balance influencing drug seeking that emanates from the D1 and D2 receptor containing neurons in the brain. PMID:23637801

  18. FSHR and LHR Expression and Signaling as Well as Maturation and Apoptosis of Cumulus-Oocyte Complexes Following Treatment with FSH Receptor Binding Inhibitor in Sheep

    Directory of Open Access Journals (Sweden)

    Suocheng Wei

    2017-09-01

    Full Text Available Background/Aims: Currently, it remains unknown whether FSH receptor binding inhibitor (FRBI influences follicular development and reproduction functions in humans and animals. The present study aimed to investigate FRBI effects on in vitro maturation (IVM and apoptosis of cumulus-oocyte complexes (COCs of sheep, to determine the effect of FRBI on mRNA and protein levels of FSHR and LHR in COCs, and to elucidate the signal pathway of FRBI effects. Methods: COCs were in vitro cultured for 24h in the IVM media supplemented with varying concentrations of FRBI (0, 10, 20, 30 and 40µg/mL and FSH (10IU/mL. The harvested COCs were observed under an inverted microscope and maturation rates of COCs were determined. Real time RT-PCR and Western blotting were utilized to detect mRNA and protein levels of FSHR and LHR. The concentrations of FSH, LH and caspase-3 were determined using especial ELISA kits for sheep, respectively. Results: Maturation rates of COCs decreased gradually as FRBI concentrations increased from 0 to 40µg/mL, reaching a bottom value of 23.76% of the FRBI-4 group. The maximal apoptosis rate was detected in the FRBI-4 group. IP3 contents of FRBI-3 and FRBI-4 groups were reduced as compared to control group (CG and FSH groups (P<0.05. Levels of FSHR protein of FRBI-3 and FRBI-4 groups as well as LHR protein of FRBI-4 group were significantly less than that of CG and FSH group. FSH contents of four FRBI treatment groups were gradually decreased along with the supplementation doses of FRBI. Caspase-3 contents of FRBI groups were reduced with a maximum reduction of the FRBI-2 group. Conclusion: Our results revealed supplement of FRBI into IVM media could dose-dependently decrease the maturation rate and increase apoptosis rate of sheep COCs. A lower dose of FRBI treatment slightly promoted IP3 production, but a higher dose of FRBI reduced IP3 production. FRBI suppressed the mRNA and protein expression levels of FSHR and LHR in sheep COCs

  19. Muscarinic Receptor Signaling in Colon Cancer

    Energy Technology Data Exchange (ETDEWEB)

    Rosenvinge, Erik C. von, E-mail: evonrose@medicine.umaryland.edu; Raufman, Jean-Pierre [University of Maryland School of Medicine, Division of Gastroenterology & Hepatology, 22 S. Greene Street, N3W62, Baltimore, MD 21201 (United States); Department of Veterans Affairs, VA Maryland Health Care System, 10 North Greene Street, Baltimore, MD 21201 (United States)

    2011-03-02

    According to the adenoma-carcinoma sequence, colon cancer results from accumulating somatic gene mutations; environmental growth factors accelerate and augment this process. For example, diets rich in meat and fat increase fecal bile acids and colon cancer risk. In rodent cancer models, increased fecal bile acids promote colon dysplasia. Conversely, in rodents and in persons with inflammatory bowel disease, low-dose ursodeoxycholic acid treatment alters fecal bile acid composition and attenuates colon neoplasia. In the course of elucidating the mechanism underlying these actions, we discovered that bile acids interact functionally with intestinal muscarinic receptors. The present communication reviews muscarinic receptor expression in normal and neoplastic colon epithelium, the role of autocrine signaling following synthesis and release of acetylcholine from colon cancer cells, post-muscarinic receptor signaling including the role of transactivation of epidermal growth factor receptors and activation of the ERK and PI3K/AKT signaling pathways, the structural biology and metabolism of bile acids and evidence for functional interaction of bile acids with muscarinic receptors on human colon cancer cells. In murine colon cancer models, deficiency of subtype 3 muscarinic receptors attenuates intestinal neoplasia; a proof-of-concept supporting muscarinic receptor signaling as a therapeutic target for colon cancer.

  20. Muscarinic Receptor Signaling in Colon Cancer

    International Nuclear Information System (INIS)

    Rosenvinge, Erik C. von; Raufman, Jean-Pierre

    2011-01-01

    According to the adenoma-carcinoma sequence, colon cancer results from accumulating somatic gene mutations; environmental growth factors accelerate and augment this process. For example, diets rich in meat and fat increase fecal bile acids and colon cancer risk. In rodent cancer models, increased fecal bile acids promote colon dysplasia. Conversely, in rodents and in persons with inflammatory bowel disease, low-dose ursodeoxycholic acid treatment alters fecal bile acid composition and attenuates colon neoplasia. In the course of elucidating the mechanism underlying these actions, we discovered that bile acids interact functionally with intestinal muscarinic receptors. The present communication reviews muscarinic receptor expression in normal and neoplastic colon epithelium, the role of autocrine signaling following synthesis and release of acetylcholine from colon cancer cells, post-muscarinic receptor signaling including the role of transactivation of epidermal growth factor receptors and activation of the ERK and PI3K/AKT signaling pathways, the structural biology and metabolism of bile acids and evidence for functional interaction of bile acids with muscarinic receptors on human colon cancer cells. In murine colon cancer models, deficiency of subtype 3 muscarinic receptors attenuates intestinal neoplasia; a proof-of-concept supporting muscarinic receptor signaling as a therapeutic target for colon cancer

  1. Muscarinic Receptor Signaling in Colon Cancer

    Directory of Open Access Journals (Sweden)

    Jean-Pierre Raufman

    2011-03-01

    Full Text Available According to the adenoma-carcinoma sequence, colon cancer results from accumulating somatic gene mutations; environmental growth factors accelerate and augment this process. For example, diets rich in meat and fat increase fecal bile acids and colon cancer risk. In rodent cancer models, increased fecal bile acids promote colon dysplasia. Conversely, in rodents and in persons with inflammatory bowel disease, low-dose ursodeoxycholic acid treatment alters fecal bile acid composition and attenuates colon neoplasia. In the course of elucidating the mechanism underlying these actions, we discovered that bile acids interact functionally with intestinal muscarinic receptors. The present communication reviews muscarinic receptor expression in normal and neoplastic colon epithelium, the role of autocrine signaling following synthesis and release of acetylcholine from colon cancer cells, post-muscarinic receptor signaling including the role of transactivation of epidermal growth factor receptors and activation of the ERK and PI3K/AKT signaling pathways, the structural biology and metabolism of bile acids and evidence for functional interaction of bile acids with muscarinic receptors on human colon cancer cells. In murine colon cancer models, deficiency of subtype 3 muscarinic receptors attenuates intestinal neoplasia; a proof-of-concept supporting muscarinic receptor signaling as a therapeutic target for colon cancer.

  2. Probing Biased Signaling in Chemokine Receptors

    DEFF Research Database (Denmark)

    Amarandi, Roxana Maria; Hjortø, Gertrud Malene; Rosenkilde, Mette Marie

    2016-01-01

    The chemokine system mediates leukocyte migration during homeostatic and inflammatory processes. Traditionally, it is described as redundant and promiscuous, with a single chemokine ligand binding to different receptors and a single receptor having several ligands. Signaling of chemokine receptors...... of others has been termed signaling bias and can accordingly be grouped into ligand bias, receptor bias, and tissue bias. Bias has so far been broadly overlooked in the process of drug development. The low number of currently approved drugs targeting the chemokine system, as well as the broad range...... of failed clinical trials, reflects the need for a better understanding of the chemokine system. Thus, understanding the character, direction, and consequence of biased signaling in the chemokine system may aid the development of new therapeutics. This review describes experiments to assess G protein...

  3. Erythropoietin Receptor Signaling Is Membrane Raft Dependent

    Science.gov (United States)

    McGraw, Kathy L.; Fuhler, Gwenny M.; Johnson, Joseph O.; Clark, Justine A.; Caceres, Gisela C.; Sokol, Lubomir; List, Alan F.

    2012-01-01

    Upon erythropoietin (Epo) engagement, Epo-receptor (R) homodimerizes to activate JAK2 and Lyn, which phosphorylate STAT5. Although recent investigations have identified key negative regulators of Epo-R signaling, little is known about the role of membrane localization in controlling receptor signal fidelity. Here we show a critical role for membrane raft (MR) microdomains in creation of discrete signaling platforms essential for Epo-R signaling. Treatment of UT7 cells with Epo induced MR assembly and coalescence. Confocal microscopy showed that raft aggregates significantly increased after Epo stimulation (mean, 4.3±1.4(SE) vs. 25.6±3.2 aggregates/cell; p≤0.001), accompanied by a >3-fold increase in cluster size (p≤0.001). Raft fraction immunoblotting showed Epo-R translocation to MR after Epo stimulation and was confirmed by fluorescence microscopy in Epo stimulated UT7 cells and primary erythroid bursts. Receptor recruitment into MR was accompanied by incorporation of JAK2, Lyn, and STAT5 and their activated forms. Raft disruption by cholesterol depletion extinguished Epo induced Jak2, STAT5, Akt and MAPK phosphorylation in UT7 cells and erythroid progenitors. Furthermore, inhibition of the Rho GTPases Rac1 or RhoA blocked receptor recruitment into raft fractions, indicating a role for these GTPases in receptor trafficking. These data establish a critical role for MR in recruitment and assembly of Epo-R and signal intermediates into discrete membrane signaling units. PMID:22509308

  4. Receptor tyrosine kinase signaling: a view from quantitative proteomics

    DEFF Research Database (Denmark)

    Dengjel, Joern; Kratchmarova, Irina; Blagoev, Blagoy

    2009-01-01

    Growth factor receptor signaling via receptor tyrosine kinases (RTKs) is one of the basic cellular communication principals found in all metazoans. Extracellular signals are transferred via membrane spanning receptors into the cytoplasm, reversible tyrosine phosphorylation being the hallmark of all...

  5. Plant cell surface receptor-mediated signaling - a common theme amid diversity.

    Science.gov (United States)

    He, Yunxia; Zhou, Jinggeng; Shan, Libo; Meng, Xiangzong

    2018-01-29

    Sessile plants employ a diverse array of plasma membrane-bound receptors to perceive endogenous and exogenous signals for regulation of plant growth, development and immunity. These cell surface receptors include receptor-like kinases (RLKs) and receptor-like proteins (RLPs) that harbor different extracellular domains for perception of distinct ligands. Several RLK and RLP signaling pathways converge at the somatic embryogenesis receptor kinases (SERKs), which function as shared co-receptors. A repertoire of receptor-like cytoplasmic kinases (RLCKs) associate with the receptor complexes to relay intracellular signaling. Downstream of the receptor complexes, mitogen-activated protein kinase (MAPK) cascades are among the key signaling modules at which the signals converge, and these cascades regulate diverse cellular and physiological responses through phosphorylation of different downstream substrates. In this Review, we summarize the emerging common theme that underlies cell surface receptor-mediated signaling pathways in Arabidopsis thaliana : the dynamic association of RLKs and RLPs with specific co-receptors and RLCKs for signal transduction. We further discuss how signaling specificities are maintained through modules at which signals converge, with a focus on SERK-mediated receptor signaling. © 2018. Published by The Company of Biologists Ltd.

  6. Androgen Receptor Signaling in Bladder Cancer

    Science.gov (United States)

    Li, Peng; Chen, Jinbo; Miyamoto, Hiroshi

    2017-01-01

    Emerging preclinical findings have indicated that steroid hormone receptor signaling plays an important role in bladder cancer outgrowth. In particular, androgen-mediated androgen receptor signals have been shown to correlate with the promotion of tumor development and progression, which may clearly explain some sex-specific differences in bladder cancer. This review summarizes and discusses the available data, suggesting the involvement of androgens and/or the androgen receptor pathways in urothelial carcinogenesis as well as tumor growth. While the precise mechanisms of the functions of the androgen receptor in urothelial cells remain far from being fully understood, current evidence may offer chemopreventive or therapeutic options, using androgen deprivation therapy, in patients with bladder cancer. PMID:28241422

  7. Androgen Receptor Signaling in Bladder Cancer

    Directory of Open Access Journals (Sweden)

    Peng Li

    2017-02-01

    Full Text Available Emerging preclinical findings have indicated that steroid hormone receptor signaling plays an important role in bladder cancer outgrowth. In particular, androgen-mediated androgen receptor signals have been shown to correlate with the promotion of tumor development and progression, which may clearly explain some sex-specific differences in bladder cancer. This review summarizes and discusses the available data, suggesting the involvement of androgens and/or the androgen receptor pathways in urothelial carcinogenesis as well as tumor growth. While the precise mechanisms of the functions of the androgen receptor in urothelial cells remain far from being fully understood, current evidence may offer chemopreventive or therapeutic options, using androgen deprivation therapy, in patients with bladder cancer.

  8. Kinetics in Signal Transduction Pathways Involving Promiscuous Oligomerizing Receptors Can Be Determined by Receptor Specificity : Apoptosis Induction by TRAIL

    NARCIS (Netherlands)

    Szegezdi, Eva; van der Sloot, Almer M.; Mahalingam, Devalingam; O'Leary, Lynda; Cool, Robbert H.; Munoz, Ines G.; Montoya, Guillermo; Quax, Wim J.; de Jong, Steven; Samali, Afshin; Serrano, Luis

    Here we show by computer modeling that kinetics and outcome of signal transduction in case of hetero-oligomerizing receptors of a promiscuous ligand largely depend on the relative amounts of its receptors. Promiscuous ligands can trigger the formation of nonproductive receptor complexes, which slows

  9. Signal transduction by growth factor receptors: signaling in an instant

    DEFF Research Database (Denmark)

    Dengjel, Joern; Akimov, Vyacheslav; Blagoev, Blagoy

    2007-01-01

    Phosphorylation-based signaling events happening within the first minute of receptor stimulation have so far only been analyzed by classical cell biological approaches like live-cell microscopy. The development of a quench flow system with a time resolution of one second coupled to a read...

  10. Erythropoietin receptor signaling is membrane raft dependent

    NARCIS (Netherlands)

    K.L. McGraw (Kathy); G.M. Fuhler (Gwenny); J.O. Johnson (Joseph); J.A. Clark (Justine); G.C. Caceres (Gisela); L. Sokol (Lubomir); A.F. List (Alan)

    2012-01-01

    textabstractUpon erythropoietin (Epo) engagement, Epo-receptor (R) homodimerizes to activate JAK2 and Lyn, which phosphorylate STAT5. Although recent investigations have identified key negative regulators of Epo-R signaling, little is known about the role of membrane localization in controlling

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

    OpenAIRE

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

    2011-01-01

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

  12. Assembly and activation of neurotrophic factor receptor complexes.

    Science.gov (United States)

    Simi, Anastasia; Ibáñez, Carlos F

    2010-04-01

    Neurotrophic factors play important roles in the development and function of both neuronal and glial elements of the central and peripheral nervous systems. Their functional diversity is in part based on their ability to interact with alternative complexes of receptor molecules. This review focuses on our current understanding of the mechanisms that govern the assembly and activation of neurotrophic factor receptor complexes. The realization that many, if not the majority, of these complexes exist in a preassembled form at the plasma membrane has forced the revision of classical ligand-mediated oligomerization models, and led to the discovery of novel mechanisms of receptor activation and generation of signaling diversity which are likely to be shared by many different classes of receptors.

  13. CD54/intercellular adhesion molecule 1 and major histocompatibility complex II signaling induces B cells to express interleukin 2 receptors and complements help provided through CD40 ligation

    DEFF Research Database (Denmark)

    Poudrier, J; Owens, T

    1994-01-01

    We have examined signaling roles for CD54 intercellular adhesion molecule 1 and major histocompatibility complex (MHC) II as contact ligands during T help for B cell activation. We used a T helper 1 (Th1)-dependent helper system that was previously shown to be contact as well as interleukin 2 (IL-2......) dependent to demonstrate the relative roles of CD54, MHC II, and CD40 signaling in the events leading to the induction of B cell proliferation and responsiveness to IL-2. Paraformaldehyde-fixed activated Th1-induced expression of IL-2R alpha, IL-2R beta, and B7, and upregulated MHC II and CD54 on B cells...... resulted in the upregulated expression of MHC II and of CD54 and B7, respectively, analogous to the effect of fixed activated Th1 cells. B7 expression was further enhanced by co-cross-linking CD54 and MHC II. Cross-linking of CD40 achieved comparable effects. Strikingly, cross-linking ligation of CD54...

  14. β-Catenin/POU5F1/SOX2 transcription factor complex mediates IGF-I receptor signaling and predicts poor prognosis in lung adenocarcinoma.

    Science.gov (United States)

    Xu, Chuan; Xie, Dan; Yu, Shi-Cang; Yang, Xiao-Jun; He, Li-Ru; Yang, Jing; Ping, Yi-Fang; Wang, Bin; Yang, Lang; Xu, Sen-Lin; Cui, Wei; Wang, Qing-Liang; Fu, Wen-Juan; Liu, Qing; Qian, Cheng; Cui, You-Hong; Rich, Jeremy N; Kung, Hsiang-Fu; Zhang, Xia; Bian, Xiu-Wu

    2013-05-15

    Cancer stem-like cells (CSLC) are crucial in tumor initiation and progression; however, the underlying mechanism for the self-renewal of cancer cells remains undefined. In the study, immunohistochemical analysis of specimens freshly excised from patients with lung adenocarcinoma showed that high expression of insulin-like growth factor I receptor (IGF-IR) in lung adenocarcinoma cells was positively correlated with the expressions of cancer stem cell markers CD133 and aldehyde dehydrogenase 1 family member A1 (ALDH1A1). IGF-IR activation enhanced POU class 5 homeobox 1 (POU5F1) expression on human lung adenocarcinoma stem-like cells (LACSLC) through PI3K/AKT/GSK3β/β-catenin cascade. POU5F1 could form a novel complex with β-catenin and SOX2 to bind Nanog promoter for transcription to maintain self-renewal of LACSLCs, which was dependent on the functional IGF-IR. Genetic and pharmacologic inhibition of IGF-IR abrogated LACSLC capabilities for self-renewal and tumorigenicity in vitro. In an in vivo xenograft tumor model, knockdown of either IGF-IR or POU5F1 impeded tumorigenic potentials of LACSLCs. By analyzing pathologic specimens excised from 200 patients with lung adenocarcinoma, we found that colocalization of highly expressed IGF-IR with β-catenin and POU5F1 predicted poor prognosis. Taken together, we show that IGF-IR-mediated POU5F1 expression to form a complex with β-catenin and SOX2 is crucial for the self-renewal and oncogenic potentials of LACSLCs, and the integrative clinical detection of the expressions of IGF-IR, β-catenin, and POU5F1 is indicatory for predicting prognosis in the patients of lung adenocarcinoma. ©2013 AACR.

  15. Complex Pharmacology of Free Fatty Acid Receptors

    DEFF Research Database (Denmark)

    Milligan, Graeme; Shimpukade, Bharat; Ulven, Trond

    2017-01-01

    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...

  16. Role of Estrogen Receptor Signaling in Breast Cancer Metastasis

    International Nuclear Information System (INIS)

    Roy, S.S.; Vadlamudi, R.K.

    2012-01-01

    Metastatic breast cancer is a life-threatening stage of cancer and is the leading cause of death in advanced breast cancer patients. Estrogen signaling and the estrogen receptor (ER) are implicated in breast cancer progression, and the majority of the human breast cancers start out as estrogen dependent. Accumulating evidence suggests that ER signaling is complex, involving coregulatory proteins and extranuclear actions. ER-coregualtory proteins are tightly regulated under normal conditions with miss expression primarily reported in cancer. Deregulation of ER coregualtors or ER extranuclear signaling has potential to promote metastasis in ER-positive breast cancer cells. This review summarizes the emerging role of ER signaling in promoting metastasis of breast cancer cells, discusses the molecular mechanisms by which ER signaling contributes to metastasis, and explores possible therapeutic targets to block ER-driven metastasis

  17. Protein Connectivity in Chemotaxis Receptor Complexes.

    Directory of Open Access Journals (Sweden)

    Stephan Eismann

    2015-12-01

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

  18. TAM Receptor Signaling in Immune Homeostasis

    Science.gov (United States)

    Rothlin, Carla V.; Carrera-Silva, Eugenio A.; Bosurgi, Lidia; Ghosh, Sourav

    2015-01-01

    The TAM receptor tyrosine kinases (RTKs)—TYRO3, AXL, and MERTK—together with their cognate agonists GAS6 and PROS1 play an essential role in the resolution of inflammation. Deficiencies in TAM signaling have been associated with chronic inflammatory and autoimmune diseases. Three processes regulated by TAM signaling may contribute, either independently or collectively, to immune homeostasis: the negative regulation of the innate immune response, the phagocytosis of apoptotic cells, and the restoration of vascular integrity. Recent studies have also revealed the function of TAMs in infectious diseases and cancer. Here, we review the important milestones in the discovery of these RTKs and their ligands and the studies that underscore the functional importance of this signaling pathway in physiological immune settings and disease. PMID:25594431

  19. Inositol trisphosphate receptor mediated spatiotemporal calcium signalling.

    Science.gov (United States)

    Miyazaki, S

    1995-04-01

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

  20. Taste Receptor Signaling-- From Tongues to Lungs

    Science.gov (United States)

    Kinnamon, Sue C.

    2013-01-01

    Taste buds are the transducing endorgans of gustation. Each taste bud comprises 50–100 elongated cells, which extend from the basal lamina to the surface of the tongue, where their apical microvilli encounter taste stimuli in the oral cavity. Salts and acids utilize apically located ion channels for transduction, while bitter, sweet and umami (glutamate) stimuli utilize G protein coupled receptors (GPCRs) and second messenger signaling mechanisms. This review will focus on GPCR signaling mechanisms. Two classes of taste GPCRs have been identified, the T1Rs for sweet and umami (glutamate) stimuli, and the T2Rs for bitter stimuli. These low affinity GPCRs all couple to the same downstream signaling effectors that include Gβγ activation of PLCβ2, IP3-mediated release of Ca2+ from intracellular stores, and Ca2+-dependent activation of the monovalent selective cation channel, TrpM5. These events lead to membrane depolarization, action potentials, and release of ATP as a transmitter to activate gustatory afferents. The Gα subunit, α-gustducin, activates a phosphodiesterase to decrease intracellular cAMP levels, although the precise targets of cAMP have not been identified. With the molecular identification of the taste GPCRs, it has become clear that taste signaling is not limited to taste buds, but occurs in many cell types of the airways. These include solitary chemosensory cells, ciliated epithelial cells, and smooth muscle cells. Bitter receptors are most abundantly expressed in the airways, where they respond to irritating chemicals and promote protective airway reflexes, utilizing the same downstream signaling effectors as taste cells. PMID:21481196

  1. Steroid Hormone Receptor Signals as Prognosticators for Urothelial Tumor

    Directory of Open Access Journals (Sweden)

    Hiroki Ide

    2015-01-01

    Full Text Available There is a substantial amount of preclinical or clinical evidence suggesting that steroid hormone receptor-mediated signals play a critical role in urothelial tumorigenesis and tumor progression. These receptors include androgen receptor, estrogen receptors, glucocorticoid receptor, progesterone receptor, vitamin D receptor, retinoid receptors, peroxisome proliferator-activated receptors, and others including orphan receptors. In particular, studies using urothelial cancer tissue specimens have demonstrated that elevated or reduced expression of these receptors as well as alterations of their upstream or downstream pathways correlates with patient outcomes. This review summarizes and discusses available data suggesting that steroid hormone receptors and related signals serve as biomarkers for urothelial carcinoma and are able to predict tumor recurrence or progression.

  2. Synergistic effect of signaling from receptors of soluble platelet agonists and outside-in signaling in formation of a stable fibrinogen-integrin αIIbβ3-actin cytoskeleton complex.

    Science.gov (United States)

    Budnik, Ivan; Shenkman, Boris; Savion, Naphtali

    2015-01-01

    Thrombus formation in the injured vessel wall is a highly complex process involving various blood-born components that go through specific temporal and spatial changes as observed by intravital videomicroscopy. Platelets bind transiently to the developing thrombus and may either become stably incorporated into or disengage from the thrombus. The aim of the present study was to reveal the processes involved in the formation of a stable thrombus. Platelet-rich plasma and washed platelets were studied by the aggregometer. The aggregate stability was challenged by eptifibatide. Platelet Triton-insoluble fraction was prepared and the actin and αIIb content in the cytoskeleton was analyzed by western blot. Maximal actin polymerization is achieved 1min after platelet activation while maximal αIIbβ3-actin cytoskeleton association requires 5 to 10min of activation and fibrinogen-mediated platelet-to-platelet bridging. Thus, actin polymerization is dependent on platelet activation and requires neither αIIbβ3 integrin occupation nor platelet aggregation. Formation of a stable aggregate requires platelet activation for more than 1min, complete increase in actin cytoskeleton fraction and partial association of αIIbβ3 with the actin cytoskeleton. However, direct αIIbβ3 activation is not sufficient for cytoskeleton complex formation. Thus, stable αIIbβ3-fibrinogen interaction, representing stable aggregate, is achieved after more than 1min agonist activation, involving inside-out and outside-in signaling but not after direct integrin activation, involving only outside-in signaling. Formation of a stable fibrinogen-αIIbβ3-actin cytoskeleton complex is the result of the combined effect of platelet stimulation by soluble agonists, activation of αIIbβ3, fibrinogen binding and platelet-to-platelet bridging. Copyright © 2014 Elsevier Ltd. All rights reserved.

  3. Receptor activity-independent recruitment of βarrestin2 reveals specific signalling modes

    Science.gov (United States)

    Terrillon, Sonia; Bouvier, Michel

    2004-01-01

    The roles of βarrestins in regulating G protein coupling and receptor endocytosis following agonist stimulation of G protein-coupled receptors are well characterised. However, their ability to act on their own as direct modulators or activators of signalling remains poorly characterised. Here, βarrestin2 intrinsic signalling properties were assessed by forcing the recruitment of this accessory protein to vasopressin V1a or V2 receptors independently of agonist-promoted activation of the receptors. Such induction of a stable interaction with βarrestin2 initiated receptor endocytosis leading to intracellular accumulation of the βarrestin/receptor complexes. Interestingly, βarrestin2 association to a single receptor protomer was sufficient to elicit receptor dimer internalisation. In addition to recapitulating βarrestin2 classical actions on receptor trafficking, the receptor activity-independent recruitment of βarrestin2 activated the extracellular signal-regulated kinases. In the latter case, recruitment to the receptor itself was not required since kinase activation could be mediated by βarrestin2 translocation to the plasma membrane in the absence of any interacting receptor. These data demonstrate that βarrestin2 can act as a ‘bonafide' signalling molecule even in the absence of activated receptor. PMID:15385966

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

    DEFF Research Database (Denmark)

    Steen, Anne; Larsen, Olav; Thiele, Stefanie

    2014-01-01

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

  5. Acetylcholine Receptor: Complex of Homologous Subunits

    Science.gov (United States)

    Raftery, Michael A.; Hunkapiller, Michael W.; Strader, Catherine D.; Hood, Leroy E.

    1980-06-01

    The acetylcholine receptor from the electric ray Torpedo californica is composed of five subunits; two are identical and the other three are structurally related to them. Microsequence analysis of the four polypeptides demonstrates amino acid homology among the subunits. Further sequence analysis of both membrane-bound and Triton-solubilized, chromatographically purified receptor gave the stoichiometry of the four subunits (40,000:50,000:60,000:65,000 daltons) as 2:1:1:1, indicating that this protein is a pentameric complex with a molecular weight of 255,000 daltons. Genealogical analysis suggests that divergence from a common ancestral gene occurred early in the evolution of the receptor. This shared ancestry argues that each of the four subunits plays a functional role in the receptor's physiological action.

  6. The Pseudo signal peptide of the corticotropin-releasing factor receptor type 2A prevents receptor oligomerization.

    Science.gov (United States)

    Teichmann, Anke; Rutz, Claudia; Kreuchwig, Annika; Krause, Gerd; Wiesner, Burkhard; Schülein, Ralf

    2012-08-03

    N-terminal signal peptides mediate the interaction of native proteins with the translocon complex of the endoplasmic reticulum membrane and are cleaved off during early protein biogenesis. The corticotropin-releasing factor receptor type 2a (CRF(2(a))R) possesses an N-terminal pseudo signal peptide, which represents a so far unique domain within the large protein family of G protein-coupled receptors (GPCRs). In contrast to a conventional signal peptide, the pseudo signal peptide remains uncleaved and consequently forms a hydrophobic extension at the N terminus of the receptor. The functional consequence of the presence of the pseudo signal peptide is not understood. Here, we have analyzed the significance of this domain for receptor dimerization/oligomerization in detail. To this end, we took the CRF(2(a))R and the homologous corticotropin-releasing factor receptor type 1 (CRF(1)R) possessing a conventional cleaved signal peptide and conducted signal peptide exchange experiments. Using single cell and single molecule imaging methods (fluorescence resonance energy transfer and fluorescence cross-correlation spectroscopy, respectively) as well as biochemical experiments, we obtained two novel findings; we could show that (i) the CRF(2(a))R is expressed exclusively as a monomer, and (ii) the presence of the pseudo signal peptide prevents its oligomerization. Thus, we have identified a novel functional domain within the GPCR protein family, which plays a role in receptor oligomerization and which may be useful to study the functional significance of this process in general.

  7. Molecular mechanisms of glucocorticoid receptor signaling

    Directory of Open Access Journals (Sweden)

    Marta Labeur

    2010-10-01

    Full Text Available This review highlights the most recent findings on the molecular mechanisms of the glucocorticoid receptor (GR. Most effects of glucocorticoids are mediated by the intracellular GR which is present in almost every tissue and controls transcriptional activation via direct and indirect mechanisms. Nevertheless the glucocorticoid responses are tissue -and gene- specific. GR associates selectively with corticosteroid ligands produced in the adrenal gland in response to changes of humoral homeostasis. Ligand interaction with GR promotes either GR binding to genomic glucocorticoid response elements, in turn modulating gene transcription, or interaction of GR monomers with other transcription factors activated by other signalling pathways leading to transrepression. The GR regulates a broad spectrum of physiological functions, including cell differentiation, metabolism and inflammatory responses. Thus, disruption or dysregulation of GR function will result in severe impairments in the maintenance of homeostasis and the control of adaptation to stress.

  8. 3D model of amphioxus steroid receptor complexed with estradiol

    Energy Technology Data Exchange (ETDEWEB)

    Baker, Michael E., E-mail: mbaker@ucsd.edu [Department of Medicine, University of California, San Diego, 9500 Gilman Drive, La Jolla, CA 92093-0693 (United States); Chang, David J. [Department of Biology, University of California, San Diego, 9500 Gilman Drive, La Jolla, CA 92093-0693 (United States)

    2009-08-28

    The origins of signaling by vertebrate steroids are not fully understood. An important advance was the report that an estrogen-binding steroid receptor [SR] is present in amphioxus, a basal chordate with a similar body plan as vertebrates. To investigate the evolution of estrogen-binding to steroid receptors, we constructed a 3D model of amphioxus SR complexed with estradiol. This 3D model indicates that although the SR is activated by estradiol, some interactions between estradiol and human ER{alpha} are not conserved in the SR, which can explain the low affinity of estradiol for the SR. These differences between the SR and ER{alpha} in the steroid-binding domain are sufficient to suggest that another steroid is the physiological regulator of the SR. The 3D model predicts that mutation of Glu-346 to Gln will increase the affinity of testosterone for amphioxus SR and elucidate the evolution of steroid-binding to nuclear receptors.

  9. Plant cell wall signalling and receptor-like kinases.

    Science.gov (United States)

    Wolf, Sebastian

    2017-02-15

    Communication between the extracellular matrix and the cell interior is essential for all organisms as intrinsic and extrinsic cues have to be integrated to co-ordinate development, growth, and behaviour. This applies in particular to plants, the growth and shape of which is governed by deposition and remodelling of the cell wall, a rigid, yet dynamic, extracellular network. It is thus generally assumed that cell wall surveillance pathways exist to monitor the state of the wall and, if needed, elicit compensatory responses such as altered expression of cell wall remodelling and biosynthesis genes. Here, I highlight recent advances in the field of cell wall signalling in plants, with emphasis on the role of plasma membrane receptor-like kinase complexes. In addition, possible roles for cell wall-mediated signalling beyond the maintenance of cell wall integrity are discussed. © 2017 The Author(s); published by Portland Press Limited on behalf of the Biochemical Society.

  10. Human GH Receptor-IGF-1 Receptor Interaction: Implications for GH Signaling

    Science.gov (United States)

    Gan, Yujun; Buckels, Ashiya; Liu, Ying; Zhang, Yue; Paterson, Andrew J.; Jiang, Jing; Zinn, Kurt R.

    2014-01-01

    GH signaling yields multiple anabolic and metabolic effects. GH binds the transmembrane GH receptor (GHR) to activate the intracellular GHR-associated tyrosine kinase, Janus kinase 2 (JAK2), and downstream signals, including signal transducer and activator of transcription 5 (STAT5) activation and IGF-1 gene expression. Some GH effects are partly mediated by GH-induced IGF-1 via IGF-1 receptor (IGF-1R), a tyrosine kinase receptor. We previously demonstrated in non-human cells that GH causes formation of a GHR-JAK2-IGF-1R complex and that presence of IGF-1R (even without IGF-1 binding) augments proximal GH signaling. In this study, we use human LNCaP prostate cancer cells as a model system to further study the IGF-1R's role in GH signaling. GH promoted JAK2 and GHR tyrosine phosphorylation and STAT5 activation in LNCaP cells. By coimmunoprecipitation and a new split luciferase complementation assay, we find that GH augments GHR/IGF-1R complex formation, which is inhibited by a Fab of an antagonistic anti-GHR monoclonal antibody. Short hairpin RNA-mediated IGF-1R silencing in LNCaP cells reduced GH-induced GHR, JAK2, and STAT5 phosphorylation. Similarly, a soluble IGF-1R extracellular domain fragment (sol IGF-1R) interacts with GHR in response to GH and blunts GH signaling. Sol IGF-1R also markedly inhibits GH-induced IGF-1 gene expression in both LNCaP cells and mouse primary osteoblast cells. On the basis of these and other findings, we propose a model in which IGF-1R augments GH signaling by allowing a putative IGF-1R-associated molecule that regulates GH signaling to access the activated GHR/JAK2 complex and envision sol IGF-1R as a dominant-negative inhibitor of this IGF-1R-mediated augmentation. Physiological implications of this new model are discussed. PMID:25211187

  11. Sweet Taste Receptor Signaling Network: Possible Implication for Cognitive Functioning

    Directory of Open Access Journals (Sweden)

    Menizibeya O. Welcome

    2015-01-01

    Full Text Available Sweet taste receptors are transmembrane protein network specialized in the transmission of information from special “sweet” molecules into the intracellular domain. These receptors can sense the taste of a range of molecules and transmit the information downstream to several acceptors, modulate cell specific functions and metabolism, and mediate cell-to-cell coupling through paracrine mechanism. Recent reports indicate that sweet taste receptors are widely distributed in the body and serves specific function relative to their localization. Due to their pleiotropic signaling properties and multisubstrate ligand affinity, sweet taste receptors are able to cooperatively bind multiple substances and mediate signaling by other receptors. Based on increasing evidence about the role of these receptors in the initiation and control of absorption and metabolism, and the pivotal role of metabolic (glucose regulation in the central nervous system functioning, we propose a possible implication of sweet taste receptor signaling in modulating cognitive functioning.

  12. ß-Adrenergic Receptor Signaling and Modulation of Long-Term Potentiation in the Mammalian Hippocampus

    Science.gov (United States)

    O'Dell, Thomas J.; Connor, Steven A.; Guglietta, Ryan; Nguyen, Peter V.

    2015-01-01

    Encoding new information in the brain requires changes in synaptic strength. Neuromodulatory transmitters can facilitate synaptic plasticity by modifying the actions and expression of specific signaling cascades, transmitter receptors and their associated signaling complexes, genes, and effector proteins. One critical neuromodulator in the…

  13. β-Adrenergic receptor signaling and modulation of long-term potentiation in the mammalian hippocampus

    OpenAIRE

    O'Dell, Thomas J.; Connor, Steven A.; Guglietta, Ryan; Nguyen, Peter V.

    2015-01-01

    Encoding new information in the brain requires changes in synaptic strength. Neuromodulatory transmitters can facilitate synaptic plasticity by modifying the actions and expression of specific signaling cascades, transmitter receptors and their associated signaling complexes, genes, and effector proteins. One critical neuromodulator in the mammalian brain is norepinephrine (NE), which regulates multiple brain functions such as attention, perception, arousal, sleep, learning, and memory. The m...

  14. Signaling properties and pharmacological analysis of two sulfakinin receptors from the red flour beetle, Tribolium castaneum.

    Directory of Open Access Journals (Sweden)

    Sven Zels

    Full Text Available Sulfakinin is an insect neuropeptide that constitutes an important component of the complex network of hormonal and neural factors that regulate feeding and digestion. The key modulating functions of sulfakinin are mediated by binding and signaling via G-protein coupled receptors. Although a substantial amount of functional data have already been reported on sulfakinins in different insect species, only little information is known regarding the properties of their respective receptors. In this study, we report on the molecular cloning, functional expression and characterization of two sulfakinin receptors in the red flour beetle, Tribolium castaneum. Both receptor open reading frames show extensive sequence similarity with annotated sulfakinin receptors from other insects. Comparison of the sulfakinin receptor sequences with homologous vertebrate cholecystokinin receptors reveals crucial conserved regions for ligand binding and receptor activation. Quantitative reverse transcriptase PCR shows that transcripts of both receptors are primarily expressed in the central nervous system of the beetle. Pharmacological characterization using 29 different peptide ligands clarified the essential requirements for efficient activation of these sulfakinin receptors. Analysis of the signaling pathway in multiple cell lines disclosed that the sulfakinin receptors of T. castaneum can stimulate both the Ca²⁺ and cyclic AMP second messenger pathways. This in depth characterization of two insect sulfakinin receptors may provide useful leads for the further development of receptor ligands with a potential applicability in pest control and crop protection.

  15. Signaling properties and pharmacological analysis of two sulfakinin receptors from the red flour beetle, Tribolium castaneum.

    Science.gov (United States)

    Zels, Sven; Verlinden, Heleen; Dillen, Senne; Vleugels, Rut; Nachman, Ronald J; Vanden Broeck, Jozef

    2014-01-01

    Sulfakinin is an insect neuropeptide that constitutes an important component of the complex network of hormonal and neural factors that regulate feeding and digestion. The key modulating functions of sulfakinin are mediated by binding and signaling via G-protein coupled receptors. Although a substantial amount of functional data have already been reported on sulfakinins in different insect species, only little information is known regarding the properties of their respective receptors. In this study, we report on the molecular cloning, functional expression and characterization of two sulfakinin receptors in the red flour beetle, Tribolium castaneum. Both receptor open reading frames show extensive sequence similarity with annotated sulfakinin receptors from other insects. Comparison of the sulfakinin receptor sequences with homologous vertebrate cholecystokinin receptors reveals crucial conserved regions for ligand binding and receptor activation. Quantitative reverse transcriptase PCR shows that transcripts of both receptors are primarily expressed in the central nervous system of the beetle. Pharmacological characterization using 29 different peptide ligands clarified the essential requirements for efficient activation of these sulfakinin receptors. Analysis of the signaling pathway in multiple cell lines disclosed that the sulfakinin receptors of T. castaneum can stimulate both the Ca²⁺ and cyclic AMP second messenger pathways. This in depth characterization of two insect sulfakinin receptors may provide useful leads for the further development of receptor ligands with a potential applicability in pest control and crop protection.

  16. SOCS proteins in regulation of receptor tyrosine kinase signaling

    DEFF Research Database (Denmark)

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

    2014-01-01

    Receptor tyrosine kinases (RTKs) are a family of cell surface receptors that play critical roles in signal transduction from extracellular stimuli. Many in this family of kinases are overexpressed or mutated in human malignancies and thus became an attractive drug target for cancer treatment....... The signaling mediated by RTKs must be tightly regulated by interacting proteins including protein-tyrosine phosphatases and ubiquitin ligases. The suppressors of cytokine signaling (SOCS) family proteins are well-known negative regulators of cytokine receptors signaling consisting of eight structurally similar...

  17. Nuclear Receptor Signaling Atlas: Opening Access to the Biology of Nuclear Receptor Signaling Pathways.

    Science.gov (United States)

    Becnel, Lauren B; Darlington, Yolanda F; Ochsner, Scott A; Easton-Marks, Jeremy R; Watkins, Christopher M; McOwiti, Apollo; Kankanamge, Wasula H; Wise, Michael W; DeHart, Michael; Margolis, Ronald N; McKenna, Neil J

    2015-01-01

    Signaling pathways involving nuclear receptors (NRs), their ligands and coregulators, regulate tissue-specific transcriptomes in diverse processes, including development, metabolism, reproduction, the immune response and neuronal function, as well as in their associated pathologies. The Nuclear Receptor Signaling Atlas (NURSA) is a Consortium focused around a Hub website (www.nursa.org) that annotates and integrates diverse 'omics datasets originating from the published literature and NURSA-funded Data Source Projects (NDSPs). These datasets are then exposed to the scientific community on an Open Access basis through user-friendly data browsing and search interfaces. Here, we describe the redesign of the Hub, version 3.0, to deploy "Web 2.0" technologies and add richer, more diverse content. The Molecule Pages, which aggregate information relevant to NR signaling pathways from myriad external databases, have been enhanced to include resources for basic scientists, such as post-translational modification sites and targeting miRNAs, and for clinicians, such as clinical trials. A portal to NURSA's Open Access, PubMed-indexed journal Nuclear Receptor Signaling has been added to facilitate manuscript submissions. Datasets and information on reagents generated by NDSPs are available, as is information concerning periodic new NDSP funding solicitations. Finally, the new website integrates the Transcriptomine analysis tool, which allows for mining of millions of richly annotated public transcriptomic data points in the field, providing an environment for dataset re-use and citation, bench data validation and hypothesis generation. We anticipate that this new release of the NURSA database will have tangible, long term benefits for both basic and clinical research in this field.

  18. Nuclear Receptor Signaling Atlas: Opening Access to the Biology of Nuclear Receptor Signaling Pathways.

    Directory of Open Access Journals (Sweden)

    Lauren B Becnel

    Full Text Available Signaling pathways involving nuclear receptors (NRs, their ligands and coregulators, regulate tissue-specific transcriptomes in diverse processes, including development, metabolism, reproduction, the immune response and neuronal function, as well as in their associated pathologies. The Nuclear Receptor Signaling Atlas (NURSA is a Consortium focused around a Hub website (www.nursa.org that annotates and integrates diverse 'omics datasets originating from the published literature and NURSA-funded Data Source Projects (NDSPs. These datasets are then exposed to the scientific community on an Open Access basis through user-friendly data browsing and search interfaces. Here, we describe the redesign of the Hub, version 3.0, to deploy "Web 2.0" technologies and add richer, more diverse content. The Molecule Pages, which aggregate information relevant to NR signaling pathways from myriad external databases, have been enhanced to include resources for basic scientists, such as post-translational modification sites and targeting miRNAs, and for clinicians, such as clinical trials. A portal to NURSA's Open Access, PubMed-indexed journal Nuclear Receptor Signaling has been added to facilitate manuscript submissions. Datasets and information on reagents generated by NDSPs are available, as is information concerning periodic new NDSP funding solicitations. Finally, the new website integrates the Transcriptomine analysis tool, which allows for mining of millions of richly annotated public transcriptomic data points in the field, providing an environment for dataset re-use and citation, bench data validation and hypothesis generation. We anticipate that this new release of the NURSA database will have tangible, long term benefits for both basic and clinical research in this field.

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

    DEFF Research Database (Denmark)

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

    2011-01-01

    -occupied receptor. The β(2) adrenergic receptor (β(2)AR) activation of Gs, the stimulatory G protein for adenylyl cyclase, has long been a model system for GPCR signalling. Here we present the crystal structure of the active state ternary complex composed of agonist-occupied monomeric β(2)AR and nucleotide-free Gs...

  20. Dynamics of the actin cytoskeleton mediates receptor cross talk: An emerging concept in tuning receptor signaling

    Science.gov (United States)

    Mattila, Pieta K.; Batista, Facundo D.

    2016-01-01

    Recent evidence implicates the actin cytoskeleton in the control of receptor signaling. This may be of particular importance in the context of immune receptors, such as the B cell receptor, where dysregulated signaling can result in autoimmunity and malignancy. Here, we discuss the role of the actin cytoskeleton in controlling receptor compartmentalization, dynamics, and clustering as a means to regulate receptor signaling through controlling the interactions with protein partners. We propose that the actin cytoskeleton is a point of integration for receptor cross talk through modulation of protein dynamics and clustering. We discuss the implication of this cross talk via the cytoskeleton for both ligand-induced and low-level constitutive (tonic) signaling necessary for immune cell survival. PMID:26833785

  1. Quantitative phosphoproteomics dissection of seven-transmembrane receptor signaling using full and biased agonists

    DEFF Research Database (Denmark)

    Christensen, Gitte L; Kelstrup, Christian D; Lyngsø, Christina

    2010-01-01

    (q)-dependent and -independent AT(1)R signaling. This study provides substantial novel insight into angiotensin II signal transduction and is the first study dissecting the differences between a full agonist and a biased agonist from a 7TMR on a systems-wide scale. Importantly, it reveals a previously unappreciated diversity......Seven-transmembrane receptors (7TMRs) signal through the well described heterotrimeric G proteins but can also activate G protein-independent signaling pathways of which the impact and complexity are less understood. The angiotensin II type 1 receptor (AT(1)R) is a prototypical 7TMR...... and quantity of Galpha(q) protein-independent signaling and uncovers novel signaling pathways. We foresee that the amount and diversity of G protein-independent signaling may be more pronounced than previously recognized for other 7TMRs as well. Quantitative mass spectrometry is a promising tool for evaluation...

  2. P2X receptor-mediated ATP purinergic signaling in health and disease

    Directory of Open Access Journals (Sweden)

    Jiang LH

    2012-09-01

    Full Text Available Lin-Hua JiangSchool of Biomedical Sciences, Faculty of Biological Sciences, University of Leeds, Leeds, United KingdomAbstract: Purinergic P2X receptors are plasma membrane proteins present in a wide range of mammalian cells where they act as a cellular sensor, enabling cells to detect and respond to extracellular adenosine triphosphate (ATP, an important signaling molecule. P2X receptors function as ligand-gated Ca2+-permeable cationic channels that open upon ATP binding to elevate intracellular Ca2+ concentrations and cause membrane depolarization. In response to sustained activation, P2X receptors induce formation of a pore permeable to large molecules. P2X receptors also interact with distinct functional proteins and membrane lipids to form specialized signaling complexes. Studies have provided compelling evidence to show that such P2X receptor-mediated ATP-signaling mechanisms determine and regulate a growing number and diversity of important physiological processes, including neurotransmission, muscle contraction, and cytokine release. There is accumulating evidence to support strong causative relationships of altered receptor expression and function with chronic pain, inflammatory diseases, cancers, and other pathologies or diseases. Numerous high throughput screening drug discovery programs and preclinical studies have thus far demonstrated the proof of concepts that the P2X receptors are druggable targets and selective receptor antagonism is a promising therapeutics approach. This review will discuss the recent progress in understanding the mammalian P2X receptors with respect to the ATP-signaling mechanisms, physiological and pathophysiological roles, and development and preclinical studies of receptor antagonists.Keywords: extracellular ATP, ion channel, large pore, signaling complex, chronic pain, inflammatory diseases

  3. Role of protein dynamics in transmembrane receptor signalling

    DEFF Research Database (Denmark)

    Wang, Yong; Bugge, Katrine Østergaard; Kragelund, Birthe Brandt

    2018-01-01

    Cells are dependent on transmembrane receptors to communicate and transform chemical and physical signals into intracellular responses. Because receptors transport 'information', conformational changes and protein dynamics play a key mechanistic role. We here review examples where experiment...... to function. Because the receptors function in a heterogeneous environment and need to be able to switch between distinct functional states, they may be particularly sensitive to small perturbations that complicate studies linking dynamics to function....

  4. Pattern-recognition receptors: signaling pathways and dysregulation in canine chronic enteropathies-brief review.

    Science.gov (United States)

    Heilmann, Romy M; Allenspach, Karin

    2017-11-01

    Pattern-recognition receptors (PRRs) are expressed by innate immune cells and recognize pathogen-associated molecular patterns (PAMPs) as well as endogenous damage-associated molecular pattern (DAMP) molecules. With a large potential for synergism or convergence between their signaling pathways, PRRs orchestrate a complex interplay of cellular mediators and transcription factors, and thus play a central role in homeostasis and host defense. Aberrant activation of PRR signaling, mutations of the receptors and/or their downstream signaling molecules, and/or DAMP/PAMP complex-mediated receptor signaling can potentially lead to chronic auto-inflammatory diseases or development of cancer. PRR signaling pathways appear to also present an interesting new avenue for the modulation of inflammatory responses and to serve as potential novel therapeutic targets. Evidence for a dysregulation of the PRR toll-like receptor (TLR)2, TLR4, TLR5, and TLR9, nucleotide-binding oligomerization domain-containing protein (NOD)2, and the receptor of advanced glycation end products (RAGE) exists in dogs with chronic enteropathies. We describe the TLR, NOD2, and RAGE signaling pathways and evaluate the current veterinary literature-in comparison to human medicine-to determine the role of TLRs, NOD2, and RAGE in canine chronic enteropathies.

  5. A Signal-Interleaving Complex Bandpass Sigma-Delta Converter

    DEFF Research Database (Denmark)

    Wad, Paul Emmanuel

    1997-01-01

    Complex or quadrature Sigma-Delta converters operate on complex signals, i.e. signals consisting of a real and an imaginary component, whereas conventional converters operate only on real signals. The advantage of complex signal processing in the discrete-time domain is that the entire sampling...

  6. Lipoprotein receptor LRP1 regulates leptin signaling and energy homeostasis in the adult central nervous system.

    Science.gov (United States)

    Liu, Qiang; Zhang, Juan; Zerbinatti, Celina; Zhan, Yan; Kolber, Benedict J; Herz, Joachim; Muglia, Louis J; Bu, Guojun

    2011-01-11

    Obesity is a growing epidemic characterized by excess fat storage in adipocytes. Although lipoprotein receptors play important roles in lipid uptake, their role in controlling food intake and obesity is not known. Here we show that the lipoprotein receptor LRP1 regulates leptin signaling and energy homeostasis. Conditional deletion of the Lrp1 gene in the brain resulted in an obese phenotype characterized by increased food intake, decreased energy consumption, and decreased leptin signaling. LRP1 directly binds to leptin and the leptin receptor complex and is required for leptin receptor phosphorylation and Stat3 activation. We further showed that deletion of the Lrp1 gene specifically in the hypothalamus by Cre lentivirus injection is sufficient to trigger accelerated weight gain. Together, our results demonstrate that the lipoprotein receptor LRP1, which is critical in lipid metabolism, also regulates food intake and energy homeostasis in the adult central nervous system.

  7. Lipoprotein receptor LRP1 regulates leptin signaling and energy homeostasis in the adult central nervous system.

    Directory of Open Access Journals (Sweden)

    Qiang Liu

    2011-01-01

    Full Text Available Obesity is a growing epidemic characterized by excess fat storage in adipocytes. Although lipoprotein receptors play important roles in lipid uptake, their role in controlling food intake and obesity is not known. Here we show that the lipoprotein receptor LRP1 regulates leptin signaling and energy homeostasis. Conditional deletion of the Lrp1 gene in the brain resulted in an obese phenotype characterized by increased food intake, decreased energy consumption, and decreased leptin signaling. LRP1 directly binds to leptin and the leptin receptor complex and is required for leptin receptor phosphorylation and Stat3 activation. We further showed that deletion of the Lrp1 gene specifically in the hypothalamus by Cre lentivirus injection is sufficient to trigger accelerated weight gain. Together, our results demonstrate that the lipoprotein receptor LRP1, which is critical in lipid metabolism, also regulates food intake and energy homeostasis in the adult central nervous system.

  8. Plant immune and growth receptors share common signalling components but localise to distinct plasma membrane nanodomains.

    Science.gov (United States)

    Bücherl, Christoph A; Jarsch, Iris K; Schudoma, Christian; Segonzac, Cécile; Mbengue, Malick; Robatzek, Silke; MacLean, Daniel; Ott, Thomas; Zipfel, Cyril

    2017-03-06

    Cell surface receptors govern a multitude of signalling pathways in multicellular organisms. In plants, prominent examples are the receptor kinases FLS2 and BRI1, which activate immunity and steroid-mediated growth, respectively. Intriguingly, despite inducing distinct signalling outputs, both receptors employ common downstream signalling components, which exist in plasma membrane (PM)-localised protein complexes. An important question is thus how these receptor complexes maintain signalling specificity. Live-cell imaging revealed that FLS2 and BRI1 form PM nanoclusters. Using single-particle tracking we could discriminate both cluster populations and we observed spatiotemporal separation between immune and growth signalling platforms. This finding was confirmed by visualising FLS2 and BRI1 within distinct PM nanodomains marked by specific remorin proteins and differential co-localisation with the cytoskeleton. Our results thus suggest that signalling specificity between these pathways may be explained by the spatial separation of FLS2 and BRI1 with their associated signalling components within dedicated PM nanodomains.

  9. Modulation of β-catenin signaling by glucagon receptor activation.

    Directory of Open Access Journals (Sweden)

    Jiyuan Ke

    Full Text Available The glucagon receptor (GCGR is a member of the class B G protein-coupled receptor family. Activation of GCGR by glucagon leads to increased glucose production by the liver. Thus, glucagon is a key component of glucose homeostasis by counteracting the effect of insulin. In this report, we found that in addition to activation of the classic cAMP/protein kinase A (PKA pathway, activation of GCGR also induced β-catenin stabilization and activated β-catenin-mediated transcription. Activation of β-catenin signaling was PKA-dependent, consistent with previous reports on the parathyroid hormone receptor type 1 (PTH1R and glucagon-like peptide 1 (GLP-1R receptors. Since low-density-lipoprotein receptor-related protein 5 (Lrp5 is an essential co-receptor required for Wnt protein mediated β-catenin signaling, we examined the role of Lrp5 in glucagon-induced β-catenin signaling. Cotransfection with Lrp5 enhanced the glucagon-induced β-catenin stabilization and TCF promoter-mediated transcription. Inhibiting Lrp5/6 function using Dickkopf-1(DKK1 or by expression of the Lrp5 extracellular domain blocked glucagon-induced β-catenin signaling. Furthermore, we showed that Lrp5 physically interacted with GCGR by immunoprecipitation and bioluminescence resonance energy transfer assays. Together, these results reveal an unexpected crosstalk between glucagon and β-catenin signaling, and may help to explain the metabolic phenotypes of Lrp5/6 mutations.

  10. Quantitative phosphoproteomics dissection of 7TM receptor signaling using full and biased agonists

    DEFF Research Database (Denmark)

    Christensen, Gitte L; Kelstrup, Christian D; Lyngsø, Christina

    2010-01-01

    only activates the Gaq protein-independent signaling.e quantified more than ten thousand phosphorylation sites of which 1183 were regulated by Angiotensin II or its analogue SII Angiotensin II. 36% of the AT1R regulated phosphorylations were regulated by SII Angiotensin II. Analysis of phosphorylation...... into Angiotensin II signal transduction and is the first study dissecting the differences between a full agonist and a biased agonist from a 7TMR on a systems-wide scale. Importantly, it reveals a previously unappreciated diversity and quantity of Gaq protein-independent signaling and uncovers novel signaling......Seven-transmembrane receptors (7TMRs) signal through the well described heterotrimeric G proteins, but can also activate G protein-independent signaling pathways of which the impact and complexity are less understood. The Angiotensin II type 1 receptor (AT1R) is a prototypical 7TMR and an important...

  11. Functionally biased signalling properties of 7TM receptors - opportunities for drug development for the ghrelin receptor

    DEFF Research Database (Denmark)

    Sivertsen, B; Holliday, N; Madsen, A N

    2013-01-01

    UNLABELLED: The ghrelin receptor is a 7 transmembrane (7TM) receptor involved in a variety of physiological functions including growth hormone secretion, increased food intake and fat accumulation as well as modulation of reward and cognitive functions. Because of its important role in metabolism...... and energy expenditure, the ghrelin receptor has become an important therapeutic target for drug design and the development of anti-obesity compounds. However, none of the compounds developed so far have been approved for commercial use. Interestingly, the ghrelin receptor is able to signal through several...... review, we have described how ligands and mutations in the 7TM receptor may bias the receptors to favour either one G-protein over another or to promote G-protein independent signalling pathways rather than G-protein-dependent pathways. For the ghrelin receptor, both agonist and inverse agonists have...

  12. Protein kinase C alpha controls erythropoietin receptor signaling.

    NARCIS (Netherlands)

    M.M. von Lindern (Marieke); M. Parren-Van Amelsvoort (Martine); T.B. van Dijk (Thamar); E. Deiner; B. Löwenberg (Bob); E. van den Akker (Emile); S. van Emst-de Vries (Sjenet); P.J. Willems (Patrick); H. Beug (Hartmut)

    2000-01-01

    textabstractProtein kinase C (PKC) is implied in the activation of multiple targets of erythropoietin (Epo) signaling, but its exact role in Epo receptor (EpoR) signal transduction and in the regulation of erythroid proliferation and differentiation remained elusive. We

  13. Protein kinase C alpha controls erythropoietin receptor signaling

    NARCIS (Netherlands)

    von Lindern, M.; Parren-van Amelsvoort, M.; van Dijk, T.; Deiner, E.; van den Akker, E.; van Emst-de Vries, S.; Willems, P.; Beug, H.; Löwenberg, B.

    2000-01-01

    Protein kinase C (PKC) is implied in the activation of multiple targets of erythropoietin (Epo) signaling, but its exact role in Epo receptor (EpoR) signal transduction and in the regulation of erythroid proliferation and differentiation remained elusive. We analyzed the effect of PKC inhibitors

  14. Human orexin/hypocretin receptors form constitutive homo- and heteromeric complexes with each other and with human CB1 cannabinoid receptors

    International Nuclear Information System (INIS)

    Jäntti, Maria H.; Mandrika, Ilona; Kukkonen, Jyrki P.

    2014-01-01

    Highlights: • OX 1 and OX 2 orexin and CB 1 cannabinoid receptor dimerization was investigated. • Bioluminescence resonance energy transfer method was used. • All receptors readily formed constitutive homo- and heteromeric complexes. - Abstract: Human OX 1 orexin receptors have been shown to homodimerize and they have also been suggested to heterodimerize with CB 1 cannabinoid receptors. The latter has been suggested to be important for orexin receptor responses and trafficking. In this study, we wanted to assess the ability of the other combinations of receptors to also form similar complexes. Vectors for expression of human OX 1 , OX 2 and CB 1 receptors, C-terminally fused with either Renilla luciferase or GFP 2 green fluorescent protein variant, were generated. The constructs were transiently expressed in Chinese hamster ovary cells, and constitutive dimerization between the receptors was assessed by bioluminescence energy transfer (BRET). Orexin receptor subtypes readily formed homo- and hetero(di)mers, as suggested by significant BRET signals. CB 1 receptors formed homodimers, and they also heterodimerized with both orexin receptors. Interestingly, BRET efficiency was higher for homodimers than for almost all heterodimers. This is likely to be due to the geometry of the interaction; the putatively symmetric dimers may place the C-termini in a more suitable orientation in homomers. Fusion of luciferase to an orexin receptor and GFP 2 to CB 1 produced more effective BRET than the opposite fusions, also suggesting differences in geometry. Similar was seen for the OX 1 –OX 2 interaction. In conclusion, orexin receptors have a significant propensity to make homo- and heterodi-/oligomeric complexes. However, it is unclear whether this affects their signaling. As orexin receptors efficiently signal via endocannabinoid production to CB 1 receptors, dimerization could be an effective way of forming signal complexes with optimal cannabinoid concentrations

  15. Exome localization of complex disease association signals

    Directory of Open Access Journals (Sweden)

    Lewis Cathryn M

    2011-02-01

    Full Text Available Abstract Background Genome-wide association studies (GWAS of common diseases have had a tremendous impact on genetic research over the last five years; the field is now moving from microarray-based technology towards next-generation sequencing. To evaluate the potential of association studies for complex diseases based on exome sequencing we analysed the distribution of association signal with respect to protein-coding genes based on GWAS data for seven diseases from the Wellcome Trust Case Control Consortium. Results We find significant concentration of association signal in exons and genes for Crohn's Disease, Type 1 Diabetes and Bipolar Disorder, but also observe enrichment from up to 40 kilobases upstream to 40 kilobases downstream of protein-coding genes for Crohn's Disease and Type 1 Diabetes; the exact extent of the distribution is disease dependent. Conclusions Our work suggests that exome sequencing may be a feasible approach to find genetic variation associated with complex disease. Extending the exome sequencing to include flanking regions therefore promises further improvement of covering disease-relevant variants.

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

    Directory of Open Access Journals (Sweden)

    Sungho Ghil

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

  17. Sortilin Associates with Trk Receptors to Enhance Anterograde Transport and Signaling by Neurotrophins

    DEFF Research Database (Denmark)

    Vægter, Christian Bjerggaard

      Neurotrophins (NT) are neuronal growth factors essential for development and maintenance of the nervous system. They are released in two forms with opposing biological functions. The proforms induce apoptosis by engaging a death signaling complex comprising the p75NTR neurotrophin receptor...

  18. Cysteinyl-Leukotriene Receptors and Cellular Signals

    Directory of Open Access Journals (Sweden)

    G. Enrico Rovati

    2007-01-01

    Full Text Available Cysteinyl-leukotrienes (cysteinyl-LTs exert a range of proinflammatory effects, such as constriction of airways and vascular smooth muscle, increase of endothelial cell permeability leading to plasma exudation and edema, and enhanced mucus secretion. They have proved to be important mediators in asthma, allergic rhinitis, and other inflammatory conditions, including cardiovascular diseases, cancer, atopic dermatitis, and urticaria. The classification into subtypes of the cysteinyl-LT receptors (CysLTRs was based initially on binding and functional data, obtained using the natural agonists and a wide range of antagonists. CysLTRs have proved remarkably resistant to cloning. However, in 1999 and 2000, the CysLT1R and CysLT2R were successfully cloned and both shown to be members of the G-protein coupled receptors (GPCRs superfamily. Molecular cloning has confirmed most of the previous pharmacological characterization and identified distinct expression patterns only partially overlapping. Recombinant CysLTRs couple to the Gq/11 pathway that modulates inositol phospholipids hydrolysis and calcium mobilization, whereas in native systems, they often activate a pertussis toxin-insensitive Gi/o-protein, or are coupled promiscuously to both G-proteins. Interestingly, recent data provide evidence for the existence of an additional receptor subtype that seems to respond to both cysteinyl-LTs and uracil nucleosides, and of an intracellular pool of CysLTRs that may have roles different from those of plasma membrane receptors. Finally, a cross-talk between the cysteinyl-LT and the purine systems is being delineated. This review will summarize recent data derived from studies on the molecular and cellular pharmacology of CysLTRs.

  19. Signal transduction through the IL-4 and insulin receptor families.

    Science.gov (United States)

    Wang, L M; Keegan, A; Frankel, M; Paul, W E; Pierce, J H

    1995-07-01

    Activation of tyrosine kinase-containing receptors and intracellular tyrosine kinases by ligand stimulation is known to be crucial for mediating initial and subsequent events involved in mitogenic signal transduction. Receptors for insulin and insulin-like growth factor 1 (IGF-1) contain cytoplasmic tyrosine kinase domains that undergo autophosphorylation upon ligand stimulation. Activation of these receptors also leads to pronounced and rapid tyrosine phosphorylation of insulin receptor substrate 1 (IRS-1) in cells of connective tissue origin. A related substrate, designated 4PS, is similarly phosphorylated by insulin and IGF-1 stimulation in many hematopoietic cell types. IRS-1 and 4PS possess a number of tyrosine phosphorylation sites that are within motifs that bind specific SH2-containing molecules known to be involved in mitogenic signaling such as PI-3 kinase, SHPTP-2 (Syp) and Grb-2. Thus, they appear to act as docking substrates for a variety of signaling molecules. The majority of hematopoietic cytokines bind to receptors that do not possess intrinsic kinase activity, and these receptors have been collectively termed as members of the hematopoietin receptor superfamily. Despite their lack of tyrosine kinase domains, stimulation of these receptors has been demonstrated to activate intracellular kinases leading to tyrosine phosphorylation of multiple substrates. Recent evidence has demonstrated that activation of different members of the Janus family of tyrosine kinases is involved in mediating tyrosine phosphorylation events by specific cytokines. Stimulation of the interleukin 4 (IL-4) receptor, a member of the hematopoietin receptor superfamily, is thought to result in activation of Jak1, Jak3, and/or Fes tyrosine kinases.(ABSTRACT TRUNCATED AT 250 WORDS)

  20. Targeting CB2-GPR55 Receptor Heteromers Modulates Cancer Cell Signaling*

    Science.gov (United States)

    Moreno, Estefanía; Andradas, Clara; Medrano, Mireia; Caffarel, María M.; Pérez-Gómez, Eduardo; Blasco-Benito, Sandra; Gómez-Cañas, María; Pazos, M. Ruth; Irving, Andrew J.; Lluís, Carme; Canela, Enric I.; Fernández-Ruiz, Javier; Guzmán, Manuel; McCormick, Peter J.; Sánchez, Cristina

    2014-01-01

    The G protein-coupled receptors CB2 (CB2R) and GPR55 are overexpressed in cancer cells and human tumors. Because a modulation of GPR55 activity by cannabinoids has been suggested, we analyzed whether this receptor participates in cannabinoid effects on cancer cells. Here we show that CB2R and GPR55 form heteromers in cancer cells, that these structures possess unique signaling properties, and that modulation of these heteromers can modify the antitumoral activity of cannabinoids in vivo. These findings unveil the existence of previously unknown signaling platforms that help explain the complex behavior of cannabinoids and may constitute new targets for therapeutic intervention in oncology. PMID:24942731

  1. Age-related changes in expression and signaling of TAM receptor inflammatory regulators in monocytes.

    Science.gov (United States)

    Wang, Xiaomei; Malawista, Anna; Qian, Feng; Ramsey, Christine; Allore, Heather G; Montgomery, Ruth R

    2018-02-09

    The multifactorial immune deterioration in aging--termed "inflamm-aging"--is comprised of a state of low-grade, chronic inflammation and complex dysregulation of responses to immune stimulation. The TAM family (Tyro 3, Axl, and Mer) of receptor tyrosine kinases are negative regulators of Toll like receptor-mediated immune responses that broadly inhibit cytokine receptor cascades to inhibit inflammation. Here we demonstrate elevated expression of TAM receptors in monocytes of older adults, and an age-dependent difference in signaling mediator AKT resulting in dysregulated responses to signaling though Mer. Our results may be especially significant in tissue, where levels of Mer are highest, and may present avenues for modulation of chronic tissue inflammation noted in aging.

  2. CSF-1 Receptor Signaling in Myeloid Cells

    Science.gov (United States)

    Stanley, E. Richard; Chitu, Violeta

    2014-01-01

    The CSF-1 receptor (CSF-1R) is activated by the homodimeric growth factors colony-stimulating factor-1 (CSF-1) and interleukin-34 (IL-34). It plays important roles in development and in innate immunity by regulating the development of most tissue macrophages and osteoclasts, of Langerhans cells of the skin, of Paneth cells of the small intestine, and of brain microglia. It also regulates the differentiation of neural progenitor cells and controls functions of oocytes and trophoblastic cells in the female reproductive tract. Owing to this broad tissue expression pattern, it plays a central role in neoplastic, inflammatory, and neurological diseases. In this review we summarize the evolution, structure, and regulation of expression of the CSF-1R gene. We review, the structures of CSF-1, IL-34, and the CSF-1R and the mechanism of ligand binding to and activation of the receptor. We further describe the pathways regulating macrophage survival, proliferation, differentiation, and chemotaxis downstream from the CSF-1R. PMID:24890514

  3. Signal transduction by the platelet-derived growth factor receptor

    International Nuclear Information System (INIS)

    Williams, L.T.; Escobedo, J.A.; Keating, M.T.; Coughlin, S.R.

    1988-01-01

    The mitogenic effects of platelet-derived growth factor (PDGF) are mediated by the PDGF receptor. The mouse PDGF receptor was recently purified on the basis of its ability to become tyrosine phosphorylated in response to the A-B human platelet form of PDGF, and the receptor amino acid sequence was determined from a full-length cDNA clone. Both the human and mouse receptor cDNA sequences have been expressed in Chinese hamster ovary fibroblast (CHO) cells that normally lack PDGF receptors. This paper summarizes recent results using this system to study signal transduction by the PDGF receptor. Some of the findings show that the KI domain of the PDGF receptor plays an important role in the stimulation of DNA synthesis by PDGF. Surprisingly, the kinase insert region is not essential for PDGF stimulation of PtdIns turnover, pH change, increase in cellular calcium, and receptor autophosphorylation. In addition, PDGF stimulates a conformational change in the receptor

  4. Binding of canonical Wnt ligands to their receptor complexes occurs in ordered plasma membrane environments.

    Science.gov (United States)

    Sezgin, Erdinc; Azbazdar, Yagmur; Ng, Xue W; Teh, Cathleen; Simons, Kai; Weidinger, Gilbert; Wohland, Thorsten; Eggeling, Christian; Ozhan, Gunes

    2017-08-01

    While the cytosolic events of Wnt/β-catenin signaling (canonical Wnt signaling) pathway have been widely studied, only little is known about the molecular mechanisms involved in Wnt binding to its receptors at the plasma membrane. Here, we reveal the influence of the immediate plasma membrane environment on the canonical Wnt-receptor interaction. While the receptors are distributed both in ordered and disordered environments, Wnt binding to its receptors selectively occurs in more ordered membrane environments which appear to cointernalize with the Wnt-receptor complex. Moreover, Wnt/β-catenin signaling is significantly reduced when the membrane order is disturbed by specific inhibitors of certain lipids that prefer to localize at the ordered environments. Similarly, a reduction in Wnt signaling activity is observed in Niemann-Pick Type C disease cells where trafficking of ordered membrane lipid components to the plasma membrane is genetically impaired. We thus conclude that ordered plasma membrane environments are essential for binding of canonical Wnts to their receptor complexes and downstream signaling activity. © 2017 The Authors. The FEBS Journal published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.

  5. Signal Diversity of Receptor for Advanced Glycation End Products.

    Science.gov (United States)

    Sakaguchi, Masakiyo; Kinoshita, Rie; Putranto, Endy Widya; Ruma, I Made Winarsa; Sumardika, I Wayan; Youyi, Chen; Tomonobu, Naoko; Yamamoto, Ken-Ichi; Murata, Hitoshi

    2017-12-01

    The receptor for advanced glycation end products (RAGE) is involved in inflammatory pathogenesis. It functions as a receptor to multiple ligands such as AGEs, HMGB1 and S100 proteins, activating multiple intracellular signaling pathways with each ligand binding. The molecular events by which ligand-activated RAGE controls diverse signaling are not well understood, but some progress was made recently. Accumulating evidence revealed that RAGE has multiple binding partners within the cytoplasm and on the plasma membrane. It was first pointed out in 2008 that RAGE's cytoplasmic tail is able to recruit Diaphanous-1 (Dia-1), resulting in the acquisition of increased cellular motility through Rac1/Cdc42 activation. We also observed that within the cytosol, RAGE's cytoplasmic tail behaves similarly to a Toll-like receptor (TLR4)-TIR domain, interacting with TIRAP and MyD88 adaptor molecules that in turn activate multiple downstream signals. Subsequent studies demonstrated the presence of an alternative adaptor molecule, DAP10, on the plasma membrane. The coupling of RAGE with DAP10 is critical for enhancing the RAGE-mediated survival signal. Interestingly, RAGE interaction on the membrane was not restricted to DAP10 alone. The chemotactic G-protein-coupled receptors (GPCRs) formyl peptide receptors1 and 2 (FPR1 and FPR2) also interacted with RAGE on the plasma membrane. Binding interaction between leukotriene B4 receptor 1 (BLT1) and RAGE was also demonstrated. All of the interactions affected the RAGE signal polarity. These findings indicate that functional interactions between RAGE and various molecules within the cytoplasmic area or on the membrane area coordinately regulate multiple ligand-mediated RAGE responses, leading to typical cellular phenotypes in several pathological settings. Here we review RAGE's signaling diversity, to contribute to the understanding of the elaborate functions of RAGE in physiological and pathological contexts.

  6. Hindbrain ghrelin receptor signaling is sufficient to maintain fasting glucose.

    Directory of Open Access Journals (Sweden)

    Michael M Scott

    Full Text Available The neuronal coordination of metabolic homeostasis requires the integration of hormonal signals with multiple interrelated central neuronal circuits to produce appropriate levels of food intake, energy expenditure and fuel availability. Ghrelin, a peripherally produced peptide hormone, circulates at high concentrations during nutrient scarcity. Ghrelin promotes food intake, an action lost in ghrelin receptor null mice and also helps maintain fasting blood glucose levels, ensuring an adequate supply of nutrients to the central nervous system. To better understand mechanisms of ghrelin action, we have examined the roles of ghrelin receptor (GHSR expression in the mouse hindbrain. Notably, selective hindbrain ghrelin receptor expression was not sufficient to restore ghrelin-stimulated food intake. In contrast, the lowered fasting blood glucose levels observed in ghrelin receptor-deficient mice were returned to wild-type levels by selective re-expression of the ghrelin receptor in the hindbrain. Our results demonstrate the distributed nature of the neurons mediating ghrelin action.

  7. Hydrogen Exchange Differences between Chemoreceptor Signaling Complexes Localize to Functionally Important Subdomains

    Science.gov (United States)

    2015-01-01

    The goal of understanding mechanisms of transmembrane signaling, one of many key life processes mediated by membrane proteins, has motivated numerous studies of bacterial chemotaxis receptors. Ligand binding to the receptor causes a piston motion of an α helix in the periplasmic and transmembrane domains, but it is unclear how the signal is then propagated through the cytoplasmic domain to control the activity of the associated kinase CheA. Recent proposals suggest that signaling in the cytoplasmic domain involves opposing changes in dynamics in different subdomains. However, it has been difficult to measure dynamics within the functional system, consisting of extended arrays of receptor complexes with two other proteins, CheA and CheW. We have combined hydrogen exchange mass spectrometry with vesicle template assembly of functional complexes of the receptor cytoplasmic domain to reveal that there are significant signaling-associated changes in exchange, and these changes localize to key regions of the receptor involved in the excitation and adaptation responses. The methylation subdomain exhibits complex changes that include slower hydrogen exchange in complexes in a kinase-activating state, which may be partially consistent with proposals that this subdomain is stabilized in this state. The signaling subdomain exhibits significant protection from hydrogen exchange in complexes in a kinase-activating state, suggesting a tighter and/or larger interaction interface with CheA and CheW in this state. These first measurements of the stability of protein subdomains within functional signaling complexes demonstrate the promise of this approach for measuring functionally important protein dynamics within the various physiologically relevant states of multiprotein complexes. PMID:25420045

  8. Vitamin D Receptor Signaling and Cancer.

    Science.gov (United States)

    Campbell, Moray J; Trump, Donald L

    2017-12-01

    The vitamin D receptor (VDR) binds the secosteroid hormone 1,25(OH) 2 D 3 with high affinity and regulates gene programs that control a serum calcium levels, as well as cell proliferation and differentiation. A significant focus has been to exploit the VDR in cancer settings. Although preclinical studies have been strongly encouraging, to date clinical trials have delivered equivocal findings that have paused the clinical translation of these compounds. However, it is entirely possible that mining of genomic data will help to refine precisely what are the key anticancer actions of vitamin D compounds and where these can be used most effectively. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

  9. Receptor downregulation and desensitization enhance the information processing ability of signalling receptors

    Directory of Open Access Journals (Sweden)

    Resat Haluk

    2007-11-01

    Full Text Available Abstract Background In addition to initiating signaling events, the activation of cell surface receptors also triggers regulatory processes that restrict the duration of signaling. Acute attenuation of signaling can be accomplished either via ligand-induced internalization of receptors (endocytic downregulation or via ligand-induced receptor desensitization. These phenomena have traditionally been viewed in the context of adaptation wherein the receptor system enters a refractory state in the presence of sustained ligand stimuli and thereby prevents the cell from over-responding to the ligand. Here we use the epidermal growth factor receptor (EGFR and G-protein coupled receptors (GPCR as model systems to respectively examine the effects of downregulation and desensitization on the ability of signaling receptors to decode time-varying ligand stimuli. Results Using a mathematical model, we show that downregulation and desensitization mechanisms can lead to tight and efficient input-output coupling thereby ensuring synchronous processing of ligand inputs. Frequency response analysis indicates that upstream elements of the EGFR and GPCR networks behave like low-pass filters with the system being able to faithfully transduce inputs below a critical frequency. Receptor downregulation and desensitization increase the filter bandwidth thereby enabling the receptor systems to decode inputs in a wider frequency range. Further, system-theoretic analysis reveals that the receptor systems are analogous to classical mechanical over-damped systems. This analogy enables us to metaphorically describe downregulation and desensitization as phenomena that make the systems more resilient in responding to ligand perturbations thereby improving the stability of the system resting state. Conclusion Our findings suggest that in addition to serving as mechanisms for adaptation, receptor downregulation and desensitization can play a critical role in temporal information

  10. The Cannabinoid Receptor CB1 Modulates the Signaling Properties of the Lysophosphatidylinositol Receptor GPR55*

    Science.gov (United States)

    Kargl, Julia; Balenga, Nariman; Parzmair, Gerald P.; Brown, Andrew J.; Heinemann, Akos; Waldhoer, Maria

    2012-01-01

    The G protein-coupled receptor (GPCR) 55 (GPR55) and the cannabinoid receptor 1 (CB1R) are co-expressed in many tissues, predominantly in the central nervous system. Seven transmembrane spanning (7TM) receptors/GPCRs can form homo- and heteromers and initiate distinct signaling pathways. Recently, several synthetic CB1 receptor inverse agonists/antagonists, such as SR141716A, AM251, and AM281, were reported to activate GPR55. Of these, SR141716A was marketed as a promising anti-obesity drug, but was withdrawn from the market because of severe side effects. Here, we tested whether GPR55 and CB1 receptors are capable of (i) forming heteromers and (ii) whether such heteromers could exhibit novel signaling patterns. We show that GPR55 and CB1 receptors alter each others signaling properties in human embryonic kidney (HEK293) cells. We demonstrate that the co-expression of FLAG-CB1 receptors in cells stably expressing HA-GPR55 specifically inhibits GPR55-mediated transcription factor activation, such as nuclear factor of activated T-cells and serum response element, as well as extracellular signal-regulated kinases (ERK1/2) activation. GPR55 and CB1 receptors can form heteromers, but the internalization of both receptors is not affected. In addition, we observe that the presence of GPR55 enhances CB1R-mediated ERK1/2 and nuclear factor of activated T-cell activation. Our data provide the first evidence that GPR55 can form heteromers with another 7TM/GPCR and that this interaction with the CB1 receptor has functional consequences in vitro. The GPR55-CB1R heteromer may play an important physiological and/or pathophysiological role in tissues endogenously co-expressing both receptors. PMID:23161546

  11. New insights into how trafficking regulates T cell receptor signaling

    Directory of Open Access Journals (Sweden)

    Jieqiong Lou

    2016-07-01

    Full Text Available AbstractThere is emerging evidence that exocytosis plays an important role in regulating T cell receptor (TCR signaling. The trafficking molecules involved in lytic granule (LG secretion in cytotoxic T lymphocytes (CTL have been well studied due to the immune disorder known as familial hemophagocytic lymphohisiocytosis (FHLH. However, the knowledge of trafficking machineries regulating the exocytosis of receptors and signaling molecules remains quite limited. In this review, we summarize the reported trafficking molecules involved in the transport of the TCR and downstream signaling molecules to the cell surface. By combining this information with the known knowledge of LG exocytosis and general exocytic trafficking machinery, we attempt to draw a more complete picture of how the TCR signaling network and exocytic trafficking matrix are interconnected to facilitate T cell activation. This also highlights how membrane compartmentalization facilitates the spatiotemporal organization of cellular responses that are essential for immune functions.

  12. The Janus face of death receptor signalling during tumour immunoediting.

    Directory of Open Access Journals (Sweden)

    Eimear O' Reilly

    2016-10-01

    Full Text Available Cancer immune-surveillance is essential for the inhibition of carcinogenesis. Malignantly transformed cells can be recognised by both the innate and adaptive immune systems through different mechanisms. Immune effector cells induce extrinsic cell death in the identified tumour cells by expressing death ligand cytokines of the tumour necrosis factor ligand family. However, some tumour cells can escape immune elimination and progress. Acquisition of resistance to the death-ligand induced apoptotic pathway can be obtained through cleavage of effector-cell expressed death-ligands into a poorly active form, mutations or silencing of the death receptors or overexpression of decoy receptors and pro-survival proteins. Although the immune system is highly effective in the elimination of malignantly transformed cells, abnormal/ dysfunctional death-ligand signalling curbs its cytotoxicity. Moreover, death receptors can also transmit pro-survival and pro-migratory signals. Consequently, dysfunctional death receptor-mediated apoptosis/necroptosis signalling does not only give a passive resistance against cell death, but actively drives tumour cell motility, invasion and contributes to consequent metastasis. This dual contribution of the death ligand-death receptor signalling in both the early, elimination phase and then in the late, escape phase of the tumour immunoediting process is discussed in this review. Death receptor agonists still hold potential for cancer therapy since they can execute the tumour-eliminating immune-effector function even in the absence of activation of the immune system against the tumour. The opportunities and challenges of developing death receptor agonists into effective cancer therapeutics are also discussed.

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

    DEFF Research Database (Denmark)

    Hansen, J A; Lindberg, K; Hilton, D J

    1999-01-01

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

  14. Insulin signaling inhibits the 5-HT2C receptor in choroid plexus via MAP kinase

    Directory of Open Access Journals (Sweden)

    Guan Kunliang

    2003-06-01

    Full Text Available Abstract Background G protein-coupled receptors (GPCRs interact with heterotrimeric GTP-binding proteins (G proteins to modulate acute changes in intracellular messenger levels and ion channel activity. In contrast, long-term changes in cellular growth, proliferation and differentiation are often mediated by tyrosine kinase receptors and certain GPCRs by activation of mitogen-activated protein (MAP kinases. Complex interactions occur between these signaling pathways, but the specific mechanisms of such regulatory events are not well-understood. In particular it is not clear whether GPCRs are modulated by tyrosine kinase receptor-MAP kinase pathways. Results Here we describe tyrosine kinase receptor regulation of a GPCR via MAP kinase. Insulin reduced the activity of the 5-HT2C receptor in choroid plexus cells which was blocked by the MAP kinase kinase (MEK inhibitor, PD 098059. We demonstrate that the inhibitory effect of insulin and insulin-like growth factor type 1 (IGF-1 on the 5-HT2C receptor is dependent on tyrosine kinase, RAS and MAP kinase. The effect may be receptor-specific: insulin had no effect on another GPCR that shares the same G protein signaling pathway as the 5-HT2C receptor. This effect is also direct: activated MAP kinase mimicked the effect of insulin, and removing a putative MAP kinase site from the 5-HT2C receptor abolished the effect of insulin. Conclusion These results show that insulin signaling can inhibit 5-HT2C receptor activity and suggest that MAP kinase may play a direct role in regulating the function of a specific GPCR.

  15. Diverse FGF receptor signaling controls astrocyte specification and proliferation

    Energy Technology Data Exchange (ETDEWEB)

    Kang, Kyungjun [School of Life Sciences, Gwangju Institute of Science and Technology, Oryong-dong, Buk-gu, Gwangju 500-712 (Korea, Republic of); Song, Mi-Ryoung, E-mail: msong@gist.ac.kr [School of Life Sciences, Gwangju Institute of Science and Technology, Oryong-dong, Buk-gu, Gwangju 500-712 (Korea, Republic of); Bioimaging Research Center and Cell Dynamics Research Center, Gwangju Institute of Science and Technology, Oryong-dong, Buk-gu, Gwangju 500-712 (Korea, Republic of)

    2010-05-07

    During CNS development, pluripotency neuronal progenitor cells give rise in succession to neurons and glia. Fibroblast growth factor-2 (FGF-2), a major signal that maintains neural progenitors in the undifferentiated state, is also thought to influence the transition from neurogenesis to gliogenesis. Here we present evidence that FGF receptors and underlying signaling pathways transmit the FGF-2 signals that regulate astrocyte specification aside from its mitogenic activity. Application of FGF-2 to cortical progenitors suppressed neurogenesis whereas treatment with an FGFR antagonist in vitro promoted neurogenesis. Introduction of chimeric FGFRs with mutated tyrosine residues into cortical progenitors and drug treatments to specifically block individual downstream signaling pathways revealed that the overall activity of FGFR rather than individual autophosphorylation sites is important for delivering signals for glial specification. In contrast, a signal for cell proliferation by FGFR was mainly delivered by MAPK pathway. Together our findings indicate that FGFR activity promotes astrocyte specification in the developing CNS.

  16. Diverse FGF receptor signaling controls astrocyte specification and proliferation

    International Nuclear Information System (INIS)

    Kang, Kyungjun; Song, Mi-Ryoung

    2010-01-01

    During CNS development, pluripotency neuronal progenitor cells give rise in succession to neurons and glia. Fibroblast growth factor-2 (FGF-2), a major signal that maintains neural progenitors in the undifferentiated state, is also thought to influence the transition from neurogenesis to gliogenesis. Here we present evidence that FGF receptors and underlying signaling pathways transmit the FGF-2 signals that regulate astrocyte specification aside from its mitogenic activity. Application of FGF-2 to cortical progenitors suppressed neurogenesis whereas treatment with an FGFR antagonist in vitro promoted neurogenesis. Introduction of chimeric FGFRs with mutated tyrosine residues into cortical progenitors and drug treatments to specifically block individual downstream signaling pathways revealed that the overall activity of FGFR rather than individual autophosphorylation sites is important for delivering signals for glial specification. In contrast, a signal for cell proliferation by FGFR was mainly delivered by MAPK pathway. Together our findings indicate that FGFR activity promotes astrocyte specification in the developing CNS.

  17. Signalling through C-type lectin receptors: shaping immune responses

    NARCIS (Netherlands)

    Geijtenbeek, Teunis B. H.; Gringhuis, Sonja I.

    2009-01-01

    C-type lectin receptors (CLRs) expressed by dendritic cells are crucial for tailoring immune responses to pathogens. Following pathogen binding, CLRs trigger distinct signalling pathways that induce the expression of specific cytokines which determine T cell polarization fates. Some CLRs can induce

  18. Evidence for functional pre-coupled complexes of receptor heteromers and adenylyl cyclase.

    Science.gov (United States)

    Navarro, Gemma; Cordomí, Arnau; Casadó-Anguera, Verónica; Moreno, Estefanía; Cai, Ning-Sheng; Cortés, Antoni; Canela, Enric I; Dessauer, Carmen W; Casadó, Vicent; Pardo, Leonardo; Lluís, Carme; Ferré, Sergi

    2018-03-28

    G protein-coupled receptors (GPCRs), G proteins and adenylyl cyclase (AC) comprise one of the most studied transmembrane cell signaling pathways. However, it is unknown whether the ligand-dependent interactions between these signaling molecules are based on random collisions or the rearrangement of pre-coupled elements in a macromolecular complex. Furthermore, it remains controversial whether a GPCR homodimer coupled to a single heterotrimeric G protein constitutes a common functional unit. Using a peptide-based approach, we here report evidence for the existence of functional pre-coupled complexes of heteromers of adenosine A 2A receptor and dopamine D 2 receptor homodimers coupled to their cognate Gs and Gi proteins and to subtype 5 AC. We also demonstrate that this macromolecular complex provides the necessary frame for the canonical Gs-Gi interactions at the AC level, sustaining the ability of a Gi-coupled GPCR to counteract AC activation mediated by a Gs-coupled GPCR.

  19. Interactions among the components of the interleukin-10 receptor complex.

    Science.gov (United States)

    Krause, Christopher D; Mei, Erwen; Mirochnitchenko, Olga; Lavnikova, Natasha; Xie, Junxia; Jia, Yiwei; Hochstrasser, Robin M; Pestka, Sidney

    2006-02-10

    We used fluorescence resonance energy transfer previously to show that the interferon-gamma (IFN-gamma) receptor complex is a preformed entity mediated by constitutive interactions between the IFN-gammaR2 and IFN-gammaR1 chains, and that this preassembled entity changes its structure after the treatment of cells with IFN-gamma. We applied this technique to determine the structure of the interleukin-10 (IL-10) receptor complex and whether it undergoes a similar conformational change after treatment of cells with IL-10. We report that, like the IFN-gamma receptor complex, the IL-10 receptor complex is preassembled: constitutive but weaker interactions occur between the IL-10R1 and IL-10R2 chains, and between two IL-10R2 chains. The IL-10 receptor complex undergoes no major conformational changes when cells are treated with cellular or Epstein-Barr viral IL-10. Receptor complex preassembly may be an inherent feature of Class 2 cytokine receptor complexes.

  20. The Androgen Receptor Bridges Stem Cell-Associated Signaling Nodes in Prostate Stem Cells

    Directory of Open Access Journals (Sweden)

    Alastair H. Davies

    2016-01-01

    Full Text Available The therapeutic potential of stem cells relies on dissecting the complex signaling networks that are thought to regulate their pluripotency and self-renewal. Until recently, attention has focused almost exclusively on a small set of “core” transcription factors for maintaining the stem cell state. It is now clear that stem cell regulatory networks are far more complex. In this review, we examine the role of the androgen receptor (AR in coordinating interactions between signaling nodes that govern the balance of cell fate decisions in prostate stem cells.

  1. Identification of the receptor scavenging hemopexin-heme complexes

    DEFF Research Database (Denmark)

    Hvidberg, Vibeke; Maniecki, Maciej B; Jacobsen, Christian

    2005-01-01

    and is suggested to facilitate cellular heme metabolism. Using a ligand-affinity approach, we purified the human hemopexin-heme receptor and identified it as the low-density lipoprotein receptor-related protein (LRP)/CD91, a receptor expressed in several cell types including macrophages, hepatocytes, neurons......, and syncytiotrophoblasts. Binding experiments, including Biacore analysis, showed that hemopexin-heme complex formation elicits the high receptor affinity. Uptake studies of radio-labeled hemopexin-heme complex in LRP/CD91-expressing COS cells and confocal microscopy of the cellular processing of fluorescent hemopexin......-heme complexes are removed by a receptor-mediated pathway showing striking similarities to the CD163-mediated haptoglobin-hemoglobin clearance in macrophages. Furthermore, the data indicate a hitherto unknown role of LRP/CD91 in inflammation....

  2. Estradiol-induced desensitization of 5-HT1A receptor signaling in the paraventricular nucleus of the hypothalamus is independent of estrogen receptor-beta.

    Science.gov (United States)

    Rossi, Dania V; Dai, Ying; Thomas, Peter; Carrasco, Gonzalo A; DonCarlos, Lydia L; Muma, Nancy A; Li, Qian

    2010-08-01

    Estradiol regulates serotonin 1A (5-HT(1A)) receptor signaling. Since desensitization of 5-HT(1A) receptors may be an underlying mechanism by which selective serotonin reuptake inhibitors (SSRIs) mediate their therapeutic effects and combining estradiol with SSRIs enhances the efficacy of the SSRIs, it is important to determine which estrogen receptors are capable of desensitizating 5-HT(1A) receptor function. We previously demonstrated that selective activation of the estrogen receptor, GPR30, desensitizes 5-HT(1A) receptor signaling in rat hypothalamic paraventricular nucleus (PVN). However, since estrogen receptor-beta (ERbeta), is highly expressed in the PVN, we investigated the role of ERbeta in estradiol-induced desensitization of 5-HT(1A) receptor signaling. We first showed that a selective ERbeta agonist, diarylpropionitrile (DPN) has a 100-fold lower binding affinity than estradiol for GPR30. Administration of DPN did not desensitize 5-HT(1A) receptor signaling in rat PVN as demonstrated by agonist-stimulated hormone release. Second, we used a recombinant adenovirus containing ERbeta siRNAs to decrease ERbeta expression in the PVN. Reductions in ERbeta did not alter the estradiol-induced desensitization of 5-HT(1A) receptor signaling in oxytocin cells. In contrast, in animals with reduced ERbeta, estradiol administration, instead of producing desensitization, augmented the ACTH response to a 5-HT(1A) agonist. Combined with the results from the DPN treatment experiments, desensitization of 5-HT(1A) receptor signaling does not appear to be mediated by ERbeta in oxytocin cells, but that ERbeta, together with GPR30, may play a complex role in central regulation of 5-HT(1A)-mediated ACTH release. Determining the mechanisms by which estrogens induce desensitization may aid in the development of better treatments for mood disorders. Copyright 2010 Elsevier Ltd. All rights reserved.

  3. IL-6/IL-12 Cytokine Receptor Shuffling of Extra- and Intracellular Domains Reveals Canonical STAT Activation via Synthetic IL-35 and IL-39 Signaling.

    Science.gov (United States)

    Floss, D M; Schönberg, M; Franke, M; Horstmeier, F C; Engelowski, E; Schneider, A; Rosenfeldt, E M; Scheller, J

    2017-11-09

    IL-35 and IL-39 are recently discovered shared members of the IL-6- and IL-12-type cytokine family with immune-suppressive capacity. IL-35 has been reported to induce the formation of four different receptor complexes: gp130:IL-12β2, gp130:gp130, IL-12β2:IL-12β2, and IL-12β2:WSX-1. IL-39 was proposed to form a gp130:IL-23R receptor complex. IL-35, but not IL-39, has been reported to activate non-conventional STAT signaling, depending on the receptor complex and target cell. Analyses of IL-35 and IL-39 are, however, hampered by the lack of biologically active recombinant IL-35 and IL-39 proteins. Therefore, we engineered chimeric cytokine receptors to accomplish synthetic IL-35 and IL- 39 signaling by shuffling the extra- and intracellular domains of IL-6/IL-12-type cytokine receptors, resulting in biological activity for all previously described IL-35 receptor complexes. Moreover, we found that the proposed IL-39 receptor complex is biologically active and discovered two additional biologically active synthetic receptor combinations, gp130/IL-12Rβ1 and IL-23R/IL-12Rβ2. Surprisingly, synthetic IL-35 activation led to more canonical STAT signaling of all receptor complexes. In summary, our receptor shuffling approach highlights an interchangeable, modular domain structure among IL-6- and IL-12-type cytokine receptors and enabled synthetic IL-35 and IL-39 signaling.

  4. Neurotransmitter receptors as signaling platforms in anterior pituitary cells

    Czech Academy of Sciences Publication Activity Database

    Zemková, Hana; Stojilkovic, S. S.

    2018-01-01

    Roč. 463, C (2018), s. 49-64 ISSN 0303-7207 R&D Projects: GA ČR(CZ) GA16-12695S; GA ČR(CZ) GBP304/12/G069; GA MŠk(CZ) LQ1604; GA MŠk(CZ) ED1.1.00/02.0109 Institutional support: RVO:67985823 Keywords : pituitary * ligand-gated receptor channels * G protein -coupled receptors * neurotransmitters * action potentials * calcium signaling * hormone secretion Subject RIV: FH - Neurology OBOR OECD: Neurosciences (including psychophysiology Impact factor: 3.754, year: 2016

  5. Physiological Signaling and Structure of the HGF Receptor MET

    Directory of Open Access Journals (Sweden)

    Gianluca Baldanzi

    2014-12-01

    Full Text Available The “hepatocyte growth factor” also known as “scatter factor”, is a multifunctional cytokine with the peculiar ability of simultaneously triggering epithelial cell proliferation, movement and survival. The combination of those proprieties results in the induction of an epithelial to mesenchymal transition in target cells, fundamental for embryogenesis but also exploited by tumor cells during metastatization. The hepatocyte growth factor receptor, MET, is a proto-oncogene and a prototypical transmembrane tyrosine kinase receptor. Inhere we discuss the MET molecular structure and the hepatocyte growth factor driven physiological signaling which coordinates epithelial proliferation, motility and morphogenesis.

  6. The Elastin Receptor Complex: a unique matricellular receptor with high anti-tumoral potential

    Directory of Open Access Journals (Sweden)

    Amandine eScandolera

    2016-03-01

    Full Text Available Elastin, one of the longest-lived proteins, confers elasticity to tissues with high mechanical constraints. During aging or pathophysiological conditions such as cancer progression, this insoluble polymer of tropoelastin undergoes an important degradation leading to the release of bioactive elastin-derived peptides (EDP, named elastokines. EDP exhibit several biological functions able to drive tumor development by regulating cell proliferation, invasion, survival, angiogenesis, and matrix metalloproteinase expression in various tumor and stromal cells. Although several receptors have been suggested to bind elastokines (αvβ3 and αvβ5 integrins, galectin-3, their main receptor remains the Elastin Receptor Complex (ERC. This heterotrimer comprises a peripheral subunit, named Elastin Binding Protein (EBP, associated to the Protective Protein/Cathepsin A (PPCA. The latter is bound to a membrane-associated protein called Neuraminidase-1 (Neu-1. The pro-tumoral effects of elastokines have been linked to their binding onto EBP. Additionally, Neu-1 sialidase activity is essential for their signal transduction. Consistently, EDP-EBP interaction and Neu-1 activity emerge as original anti-tumoral targets. Interestingly, besides its direct involvement in cancer progression, the ERC also regulates diabetes outcome and thrombosis, an important risk factor for cancer development and a vascular process highly increased in patients suffering from cancer. In this review, we will describe ERC and elastokines involvement in cancer development suggesting that this unique receptor would be a promising therapeutic target. We will also discuss the pharmacological concepts aiming at blocking its pro-tumoral activities. Finally, its emerging role in cancer-associated complications and pathologies such as diabetes and thrombotic events will be also considered.

  7. The α7 nicotinic acetylcholine receptor complex

    DEFF Research Database (Denmark)

    Thomsen, Morten Skøtt; Mikkelsen, Jens D

    2012-01-01

    The α7 nicotinic acetylcholine receptor (nAChR) is a promising drug target for a number of diseases ranging from schizophrenia and Alzheimer's disease to chronic pain and inflammatory diseases. Focusing on the central nervous system, we describe how endogenous and experimental compounds and prote......The α7 nicotinic acetylcholine receptor (nAChR) is a promising drug target for a number of diseases ranging from schizophrenia and Alzheimer's disease to chronic pain and inflammatory diseases. Focusing on the central nervous system, we describe how endogenous and experimental compounds...

  8. ABA receptors: The START of a new paradigm in phytohormone signalling

    KAUST Repository

    Klingler, John

    2010-06-03

    The phytohormone abscisic acid (ABA) plays a central role in plant development and in plant adaptation to both biotic and abiotic stressors. In recent years, knowledge of ABA metabolism and signal transduction has advanced rapidly to provide detailed glimpses of the hormone\\'s activities at the molecular level. Despite this progress, many gaps in understanding have remained, particularly at the early stages of ABA perception by the plant cell. The search for an ABA receptor protein has produced multiple candidates, including GCR2, GTG1, and GTG2, and CHLH. In addition to these candidates, in 2009 several research groups converged on a novel family of Arabidopsis proteins that bind ABA, and thereby interact directly with a class of protein phosphatases that are well known as critical players in ABA signal transduction. The PYR/PYL/RCAR receptor family is homologous to the Bet v 1-fold and START domain proteins. It consists of 14 members, nearly all of which appear capable of participating in an ABA receptor-signal complex that responds to the hormone by activating the transcription of ABA-responsive genes. Evidence is provided here that PYR/PYL/RCAR receptors can also drive the phosphorylation of the slow anion channel SLAC1 to provide a fast and timely response to the ABA signal. Crystallographic studies have vividly shown the mechanics of ABA binding to PYR/PYL/RCAR receptors, presenting a model that bears some resemblance to the binding of gibberellins to GID1 receptors. Since this ABA receptor family is highly conserved in crop species, its discovery is likely to usher a new wave of progress in the elucidation and manipulation of plant stress responses in agricultural settings. © 2010 The Author(s).

  9. Rapid phospho-turnover by receptor tyrosine kinases impacts downstream signaling and drug binding.

    Science.gov (United States)

    Kleiman, Laura B; Maiwald, Thomas; Conzelmann, Holger; Lauffenburger, Douglas A; Sorger, Peter K

    2011-09-02

    Epidermal growth factor receptors (ErbB1-4) are oncogenic receptor tyrosine kinases (RTKs) that regulate diverse cellular processes. In this study, we combine measurement and mathematical modeling to quantify phospho-turnover at ErbB receptors in human cells and to determine the consequences for signaling and drug binding. We find that phosphotyrosine residues on ErbB1 have half-lives of a few seconds and therefore turn over 100-1000 times in the course of a typical immediate-early response to ligand. Rapid phospho-turnover is also observed for EGF-activated ErbB2 and ErbB3, unrelated RTKs, and multiple intracellular adaptor proteins and signaling kinases. Thus, the complexes formed on the cytoplasmic tail of active receptors and the downstream signaling kinases they control are highly dynamic and antagonized by potent phosphatases. We develop a kinetic scheme for binding of anti-ErbB1 drugs to receptors and show that rapid phospho-turnover significantly impacts their mechanisms of action. Copyright © 2011 Elsevier Inc. All rights reserved.

  10. CD147 is a signaling receptor for cyclophilin B.

    Science.gov (United States)

    Yurchenko, V; O'Connor, M; Dai, W W; Guo, H; Toole, B; Sherry, B; Bukrinsky, M

    2001-11-09

    Cyclophilins A and B (CyPA and CyPB) are cyclosporin A binding proteins that can be secreted in response to inflammatory stimuli. We recently identified CD147 as a cell-surface receptor for CyPA and demonstrated that CD147 is an essential component in the CyPA-initiated signaling cascade that culminates in ERK activation and chemotaxis. Here we demonstrate that CD147 also serves as a receptor for CyPB. CyPB induced Ca(2+) flux and chemotaxis of CD147-transfected, but not control, CHO cells, and the chemotactic response of primary human neutrophils to CyPB was blocked by antibodies to CD147. These results suggest that CD147 serves as a receptor for extracellular cyclophilins. Copyright 2001 Academic Press.

  11. Nuclear receptor TLX inhibits TGF-β signaling in glioblastoma

    Energy Technology Data Exchange (ETDEWEB)

    Johansson, Erik; Zhai, Qiwei [Sahlgrenska Cancer Center at the Sahlgrenska Academy, University of Gothenburg, Box 425, SE 405 30 Gothenburg (Sweden); Zeng, Zhao-jun [Sahlgrenska Cancer Center at the Sahlgrenska Academy, University of Gothenburg, Box 425, SE 405 30 Gothenburg (Sweden); Molecular Biology Research Center, School of Life Sciences, Central South University, 110, Xiangya Road, Changsha, Hunan 410078 (China); Yoshida, Takeshi [Sahlgrenska Cancer Center at the Sahlgrenska Academy, University of Gothenburg, Box 425, SE 405 30 Gothenburg (Sweden); Funa, Keiko, E-mail: keiko.funa@gu.se [Sahlgrenska Cancer Center at the Sahlgrenska Academy, University of Gothenburg, Box 425, SE 405 30 Gothenburg (Sweden)

    2016-05-01

    TLX (also called NR2E1) is an orphan nuclear receptor that maintains stemness of neuronal stem cells. TLX is highly expressed in the most malignant form of glioma, glioblastoma multiforme (GBM), and is important for the proliferation and maintenance of the stem/progenitor cells of the tumor. Transforming Growth Factor-β (TGF-β) is a cytokine regulating many different cellular processes such as differentiation, migration, adhesion, cell death and proliferation. TGF-β has an important function in cancer where it can work as either a tumor suppressor or oncogene, depending on the cancer type and stage of tumor development. Since glioblastoma often have dysfunctional TGF-β signaling we wanted to find out if there is any interaction between TLX and TGF-β in glioblastoma cells. We demonstrate that knockdown of TLX enhances the canonical TGF-β signaling response in glioblastoma cell lines. TLX physically interacts with and stabilizes Smurf1, which can ubiquitinate and target TGF-β receptor II for degradation, whereas knockdown of TLX leads to stabilization of TGF-β receptor II, increased nuclear translocation of Smad2/3 and enhanced expression of TGF-β target genes. The interaction between TLX and TGF-β may play an important role in the regulation of proliferation and tumor-initiating properties of glioblastoma cells. - Highlights: • TLX knockdown enhances TGF-β dependent Smad signaling in glioblastoma cells • TLX knockdown increases the protein level of TGF-β receptor II. • TLX stabilizes and retains Smurf1 in the cytoplasm. • TLX enhances Smurf1-dependent ubiquitination and degradation of TGF-β receptor II.

  12. Nuclear receptor TLX inhibits TGF-β signaling in glioblastoma

    International Nuclear Information System (INIS)

    Johansson, Erik; Zhai, Qiwei; Zeng, Zhao-jun; Yoshida, Takeshi; Funa, Keiko

    2016-01-01

    TLX (also called NR2E1) is an orphan nuclear receptor that maintains stemness of neuronal stem cells. TLX is highly expressed in the most malignant form of glioma, glioblastoma multiforme (GBM), and is important for the proliferation and maintenance of the stem/progenitor cells of the tumor. Transforming Growth Factor-β (TGF-β) is a cytokine regulating many different cellular processes such as differentiation, migration, adhesion, cell death and proliferation. TGF-β has an important function in cancer where it can work as either a tumor suppressor or oncogene, depending on the cancer type and stage of tumor development. Since glioblastoma often have dysfunctional TGF-β signaling we wanted to find out if there is any interaction between TLX and TGF-β in glioblastoma cells. We demonstrate that knockdown of TLX enhances the canonical TGF-β signaling response in glioblastoma cell lines. TLX physically interacts with and stabilizes Smurf1, which can ubiquitinate and target TGF-β receptor II for degradation, whereas knockdown of TLX leads to stabilization of TGF-β receptor II, increased nuclear translocation of Smad2/3 and enhanced expression of TGF-β target genes. The interaction between TLX and TGF-β may play an important role in the regulation of proliferation and tumor-initiating properties of glioblastoma cells. - Highlights: • TLX knockdown enhances TGF-β dependent Smad signaling in glioblastoma cells • TLX knockdown increases the protein level of TGF-β receptor II. • TLX stabilizes and retains Smurf1 in the cytoplasm. • TLX enhances Smurf1-dependent ubiquitination and degradation of TGF-β receptor II.

  13. Internalisation of gonadotrophin-receptor complex in ovarian luteal cells

    International Nuclear Information System (INIS)

    Conn, P.M.; Conti, M.; Harwood, J.P.; Dufau, M.L.; Catt, K.J.

    1978-01-01

    Following evidence that certain protein hormones can enter target cells the present investigation was undertaken which shows that gonadotrophin-induced receptor loss may occur by a process of internalisation of the hormone-receptor complex following the initial interaction of gonadotrophin with the cell surface. Localisation studies were carried out in 33-d old female rats previously treated with pregnant mare serum gonadotrophin and human chorionic gonadotrophin (hCG) to induce ovarian luteinisation. Animals were injected with 125 I-hCG to label the ovarian receptors for luteinising hormone in vivo. Microscope autoradiographs demonstrating distribution of 125 I-hCG in ovaries at various times following injection are shown. The combined results from the autoradiographs and from solubilisation experiments were used to determine the location and nature of the hCG-receptor complex following occupancy and loss of receptors from the plasma membrane of luteinised ovarian cells. (U.K.)

  14. The role of insulin receptor signaling in the brain.

    Science.gov (United States)

    Plum, Leona; Schubert, Markus; Brüning, Jens C

    2005-03-01

    The insulin receptor (IR) is expressed in various regions of the developing and adult brain, and its functions have become the focus of recent research. Insulin enters the central nervous system (CNS) through the blood-brain barrier by receptor-mediated transport to regulate food intake, sympathetic activity and peripheral insulin action through the inhibition of hepatic gluconeogenesis and reproductive endocrinology. On a molecular level, some of the effects of insulin converge with those of the leptin signaling machinery at the point of activation of phosphatidylinositol 3-kinase (PI3K), resulting in the regulation of ATP-dependent potassium channels. Furthermore, insulin inhibits neuronal apoptosis via activation of protein kinase B in vitro, and it regulates phosphorylation of tau, metabolism of the amyloid precursor protein and clearance of beta-amyloid from the brain in vivo. These findings indicate that neuronal IR signaling has a direct role in the link between energy homeostasis, reproduction and the development of neurodegenerative diseases.

  15. Cross-regulation of cytokine signalling: pro-inflammatory cytokines restrict IL-6 signalling through receptor internalisation and degradation.

    Science.gov (United States)

    Radtke, Simone; Wüller, Stefan; Yang, Xiang-ping; Lippok, Barbara E; Mütze, Barbara; Mais, Christine; de Leur, Hildegard Schmitz-Van; Bode, Johannes G; Gaestel, Matthias; Heinrich, Peter C; Behrmann, Iris; Schaper, Fred; Hermanns, Heike M

    2010-03-15

    The inflammatory response involves a complex interplay of different cytokines which act in an auto- or paracrine manner to induce the so-called acute phase response. Cytokines are known to crosstalk on multiple levels, for instance by regulating the mRNA stability of targeted cytokines through activation of the p38-MAPK pathway. In our study we discovered a new mechanism that answers the long-standing question how pro-inflammatory cytokines and environmental stress restrict immediate signalling of interleukin (IL)-6-type cytokines. We show that p38, activated by IL-1beta, TNFalpha or environmental stress, impairs IL-6-induced JAK/STAT signalling through phosphorylation of the common cytokine receptor subunit gp130 and its subsequent internalisation and degradation. We identify MK2 as the kinase that phosphorylates serine 782 in the cytoplasmic part of gp130. Consequently, inhibition of p38 or MK2, deletion of MK2 or mutation of crucial amino acids within the MK2 target site or the di-leucine internalisation motif blocks receptor depletion and restores IL-6-dependent STAT activation as well as gene induction. Hence, a novel negative crosstalk mechanism for cytokine signalling is described, where cytokine receptor turnover is regulated in trans by pro-inflammatory cytokines and stress stimuli to coordinate the inflammatory response.

  16. δ-opioid receptor and somatostatin receptor-4 heterodimerization: possible implications in modulation of pain associated signaling.

    Directory of Open Access Journals (Sweden)

    Rishi K Somvanshi

    Full Text Available Pain relief is the principal action of opioids. Somatostatin (SST, a growth hormone inhibitory peptide is also known to alleviate pain even in cases when opioids fail. Recent studies have shown that mice are prone to sustained pain and devoid of analgesic effect in the absence of somatostatin receptor 4 (SSTR4. In the present study, using brain slices, cultured neurons and HEK-293 cells, we showed that SSTR4 and δ-Opioid receptor (δOR exist in a heteromeric complex and function in synergistic manner. SSTR4 and δOR co-expressed in cortical/striatal brain regions and spinal cord. Using cultured neuronal cells, we describe the heterogeneous complex formation of SSTR4 and δOR at neuronal cell body and processes. Cotransfected cells display inhibition of cAMP/PKA and co-activation of SSTR4 and δOR oppose receptor trafficking induced by individual receptor activation. Furthermore, downstream signaling pathways either associated with withdrawal or pain relief are modulated synergistically with a predominant role of SSTR4. Inhibition of cAMP/PKA and activation of ERK1/2 are the possible cellular adaptations to prevent withdrawal induced by chronic morphine use. Our results reveal direct intra-membrane interaction between SSTR4 and δOR and provide insights for the molecular mechanism for the anti-nociceptive property of SST in combination with opioids as a potential therapeutic approach to avoid undesirable withdrawal symptoms.

  17. Peroxisome Proliferator-Activated Receptor and Vitamin D Receptor Signaling Pathways in Cancer Cells

    Directory of Open Access Journals (Sweden)

    Yasuko Kitagishi

    2013-10-01

    Full Text Available Peroxisome proliferator-activated receptors (PPARs are members of the superfamily of nuclear hormone receptors, which respond to specific ligands such as polyunsaturated fatty acids by altering gene expression. Three subtypes of this receptor have been discovered, each evolving to achieve different biological functions. Like other nuclear receptors, the transcriptional activity of PPARs is affected not only by ligand-stimulation, but also by cross-talk with other molecules. For example, both PPARs and the RXRs are ligand-activated transcription factors that coordinately regulate gene expression. In addition, PPARs and vitamin D receptor (VDR signaling pathways regulate a multitude of genes that are of importance for cellular functions including cell proliferation and cell differentiation. Interaction of the PPARs and VDR signaling pathways has been shown at the level of molecular cross-regulation of their transcription factor. A variety of ligands influencing the PPARs and VDR signaling pathways have been shown to reveal chemopreventive potential by mediating tumor suppressive activities in human cancers. Use of these compounds may represent a potential novel strategy to prevent cancers. This review summarizes the roles of the PPARs and the VDR in pathogenesis and progression of cancer.

  18. Peroxisome Proliferator-Activated Receptor and Vitamin D Receptor Signaling Pathways in Cancer Cells

    Energy Technology Data Exchange (ETDEWEB)

    Matsuda, Satoru, E-mail: smatsuda@cc.nara-wu.ac.jp; Kitagishi, Yasuko [Department of Food Science and Nutrition, Nara Women’s University, Kita-Uoya Nishimachi, Nara 630-8506 (Japan)

    2013-10-21

    Peroxisome proliferator-activated receptors (PPARs) are members of the superfamily of nuclear hormone receptors, which respond to specific ligands such as polyunsaturated fatty acids by altering gene expression. Three subtypes of this receptor have been discovered, each evolving to achieve different biological functions. Like other nuclear receptors, the transcriptional activity of PPARs is affected not only by ligand-stimulation, but also by cross-talk with other molecules. For example, both PPARs and the RXRs are ligand-activated transcription factors that coordinately regulate gene expression. In addition, PPARs and vitamin D receptor (VDR) signaling pathways regulate a multitude of genes that are of importance for cellular functions including cell proliferation and cell differentiation. Interaction of the PPARs and VDR signaling pathways has been shown at the level of molecular cross-regulation of their transcription factor. A variety of ligands influencing the PPARs and VDR signaling pathways have been shown to reveal chemopreventive potential by mediating tumor suppressive activities in human cancers. Use of these compounds may represent a potential novel strategy to prevent cancers. This review summarizes the roles of the PPARs and the VDR in pathogenesis and progression of cancer.

  19. Peroxisome Proliferator-Activated Receptor and Vitamin D Receptor Signaling Pathways in Cancer Cells

    International Nuclear Information System (INIS)

    Matsuda, Satoru; Kitagishi, Yasuko

    2013-01-01

    Peroxisome proliferator-activated receptors (PPARs) are members of the superfamily of nuclear hormone receptors, which respond to specific ligands such as polyunsaturated fatty acids by altering gene expression. Three subtypes of this receptor have been discovered, each evolving to achieve different biological functions. Like other nuclear receptors, the transcriptional activity of PPARs is affected not only by ligand-stimulation, but also by cross-talk with other molecules. For example, both PPARs and the RXRs are ligand-activated transcription factors that coordinately regulate gene expression. In addition, PPARs and vitamin D receptor (VDR) signaling pathways regulate a multitude of genes that are of importance for cellular functions including cell proliferation and cell differentiation. Interaction of the PPARs and VDR signaling pathways has been shown at the level of molecular cross-regulation of their transcription factor. A variety of ligands influencing the PPARs and VDR signaling pathways have been shown to reveal chemopreventive potential by mediating tumor suppressive activities in human cancers. Use of these compounds may represent a potential novel strategy to prevent cancers. This review summarizes the roles of the PPARs and the VDR in pathogenesis and progression of cancer

  20. DMPD: Is HIV infection a TNF receptor signalling-driven disease? [Dynamic Macrophage Pathway CSML Database

    Lifescience Database Archive (English)

    Full Text Available 18178131 Is HIV infection a TNF receptor signalling-driven disease? Herbein G, Khan... KA. Trends Immunol. 2008 Feb;29(2):61-7. (.png) (.svg) (.html) (.csml) Show Is HIV infection a TNF receptor signalling-driven dise...ase? PubmedID 18178131 Title Is HIV infection a TNF receptor signalling-driven diseas

  1. DMPD: TGF-beta signaling from receptors to the nucleus. [Dynamic Macrophage Pathway CSML Database

    Lifescience Database Archive (English)

    Full Text Available 10611754 TGF-beta signaling from receptors to the nucleus. Roberts AB. Microbes Inf...ect. 1999 Dec;1(15):1265-73. (.png) (.svg) (.html) (.csml) Show TGF-beta signaling from receptors to the nucleus.... PubmedID 10611754 Title TGF-beta signaling from receptors to the nucleus. Authors Roberts AB. Publicat

  2. DMPD: Signals and receptors involved in recruitment of inflammatory cells. [Dynamic Macrophage Pathway CSML Database

    Lifescience Database Archive (English)

    Full Text Available 7744810 Signals and receptors involved in recruitment of inflammatory cells. Ben-Ba...ow Signals and receptors involved in recruitment of inflammatory cells. PubmedID 7744810 Title Signals and receptors involved in recr...uitment of inflammatory cells. Authors Ben-Baruch A, Mic

  3. DMPD: Lysophospholipid receptors: signaling and biology. [Dynamic Macrophage Pathway CSML Database

    Lifescience Database Archive (English)

    Full Text Available 15189145 Lysophospholipid receptors: signaling and biology. Ishii I, Fukushima N, Y...e X, Chun J. Annu Rev Biochem. 2004;73:321-54. (.png) (.svg) (.html) (.csml) Show Lysophospholipid receptors...: signaling and biology. PubmedID 15189145 Title Lysophospholipid receptors: signaling and biology. Authors

  4. The Growth Hormone Receptor: Mechanism of Receptor Activation, Cell Signaling, and Physiological Aspects

    Directory of Open Access Journals (Sweden)

    Farhad Dehkhoda

    2018-02-01

    Full Text Available The growth hormone receptor (GHR, although most well known for regulating growth, has many other important biological functions including regulating metabolism and controlling physiological processes related to the hepatobiliary, cardiovascular, renal, gastrointestinal, and reproductive systems. In addition, growth hormone signaling is an important regulator of aging and plays a significant role in cancer development. Growth hormone activates the Janus kinase (JAK–signal transducer and activator of transcription (STAT signaling pathway, and recent studies have provided a new understanding of the mechanism of JAK2 activation by growth hormone binding to its receptor. JAK2 activation is required for growth hormone-mediated activation of STAT1, STAT3, and STAT5, and the negative regulation of JAK–STAT signaling comprises an important step in the control of this signaling pathway. The GHR also activates the Src family kinase signaling pathway independent of JAK2. This review covers the molecular mechanisms of GHR activation and signal transduction as well as the physiological consequences of growth hormone signaling.

  5. Differential modulation of Beta-adrenergic receptor signaling by trace amine-associated receptor 1 agonists.

    Directory of Open Access Journals (Sweden)

    Gunnar Kleinau

    Full Text Available Trace amine-associated receptors (TAAR are rhodopsin-like G-protein-coupled receptors (GPCR. TAAR are involved in modulation of neuronal, cardiac and vascular functions and they are potentially linked with neurological disorders like schizophrenia and Parkinson's disease. Subtype TAAR1, the best characterized TAAR so far, is promiscuous for a wide set of ligands and is activated by trace amines tyramine (TYR, phenylethylamine (PEA, octopamine (OA, but also by thyronamines, dopamine, and psycho-active drugs. Unfortunately, effects of trace amines on signaling of the two homologous β-adrenergic receptors 1 (ADRB1 and 2 (ADRB2 have not been clarified yet in detail. We, therefore, tested TAAR1 agonists TYR, PEA and OA regarding their effects on ADRB1/2 signaling by co-stimulation studies. Surprisingly, trace amines TYR and PEA are partial allosteric antagonists at ADRB1/2, whereas OA is a partial orthosteric ADRB2-antagonist and ADRB1-agonist. To specify molecular reasons for TAAR1 ligand promiscuity and for observed differences in signaling effects on particular aminergic receptors we compared TAAR, tyramine (TAR octopamine (OAR, ADRB1/2 and dopamine receptors at the structural level. We found especially for TAAR1 that the remarkable ligand promiscuity is likely based on high amino acid similarity in the ligand-binding region compared with further aminergic receptors. On the other hand few TAAR specific properties in the ligand-binding site might determine differences in ligand-induced effects compared to ADRB1/2. Taken together, this study points to molecular details of TAAR1-ligand promiscuity and identified specific trace amines as allosteric or orthosteric ligands of particular β-adrenergic receptor subtypes.

  6. Sequence distance via parsing complexity: Heartbeat signals

    International Nuclear Information System (INIS)

    Degli Esposti, M.; Farinelli, C.; Menconi, G.

    2009-01-01

    We compare and discuss the use of different symbolic codings of electrocardiogram (ECG) signals in order to distinguish healthy patients from hospitalized ones. To this aim, we recall a parsing-based similarity distance and compare the performances of several methods of classification of data.

  7. Hydrogen Exchange Mass Spectrometry of Functional Membrane-bound Chemotaxis Receptor Complexes

    Science.gov (United States)

    Koshy, Seena S.; Eyles, Stephen J.; Weis, Robert M.; Thompson, Lynmarie K.

    2014-01-01

    The transmembrane signaling mechanism of bacterial chemotaxis receptors is thought to involve changes in receptor conformation and dynamics. The receptors function in ternary complexes with two other proteins, CheA and CheW, that form extended membrane-bound arrays. Previous studies have shown that attractant binding induces a small (~2 Å) piston displacement of one helix of the periplasmic and transmembrane domains towards the cytoplasm, but it is not clear how this signal propagates through the cytoplasmic domain to control the kinase activity of the CheA bound at the membrane-distal tip, nearly 200 Å away. The cytoplasmic domain has been shown to be highly dynamic, which raises the question of how a small piston motion could propagate through a dynamic domain to control CheA kinase activity. To address this, we have developed a method for measuring dynamics of the receptor cytoplasmic fragment (CF) in functional complexes with CheA and CheW. Hydrogen exchange mass spectrometry (HDX-MS) measurements of global exchange of CF demonstrate that CF exhibits significantly slower exchange in functional complexes than in solution. Since the exchange rates in functional complexes are comparable to that of other proteins of similar structure, the CF appears to be a well-structured protein within these complexes, which is compatible with its role in propagating a signal that appears to be a tiny conformational change in the periplasmic and transmembrane domains of the receptor. We also demonstrate the feasibility of this protocol for local exchange measurements, by incorporating a pepsin digest step to produce peptides with 87% sequence coverage and only 20% back exchange. This method extends HDX-MS to membrane-bound functional complexes without detergents that may perturb the stability or structure of the system. PMID:24274333

  8. Phosphorylation site dynamics of early T-cell receptor signaling

    DEFF Research Database (Denmark)

    Chylek, Lily A; Akimov, Vyacheslav; Dengjel, Jörn

    2014-01-01

    In adaptive immune responses, T-cell receptor (TCR) signaling impacts multiple cellular processes and results in T-cell differentiation, proliferation, and cytokine production. Although individual protein-protein interactions and phosphorylation events have been studied extensively, we lack...... that diverse dynamic patterns emerge within seconds. We detected phosphorylation dynamics as early as 5 s and observed widespread regulation of key TCR signaling proteins by 30 s. Development of a computational model pointed to the presence of novel regulatory mechanisms controlling phosphorylation of sites...... a systems-level understanding of how these components cooperate to control signaling dynamics, especially during the crucial first seconds of stimulation. Here, we used quantitative proteomics to characterize reshaping of the T-cell phosphoproteome in response to TCR/CD28 co-stimulation, and found...

  9. The α7 nicotinic acetylcholine receptor complex

    DEFF Research Database (Denmark)

    Thomsen, Morten S; Mikkelsen, Jens D

    2012-01-01

    The α7 nicotinic acetylcholine receptor (nAChR) is a promising drug target for a number of diseases ranging from schizophrenia and Alzheimer's disease to chronic pain and inflammatory diseases. Focusing on the central nervous system, we describe how endogenous and experimental compounds and prote......The α7 nicotinic acetylcholine receptor (nAChR) is a promising drug target for a number of diseases ranging from schizophrenia and Alzheimer's disease to chronic pain and inflammatory diseases. Focusing on the central nervous system, we describe how endogenous and experimental compounds...... in diseases such as schizophrenia and Alzheimer's disease. Furthermore, α7 nAChR agonists and allosteric modulators differentially alter expression and functionality of the α7 nAChR with repeated administration, which suggests that there may be fundamentally different outcomes of long-term administration...... with these different types of compounds. Finally, we describe the special case of Aβ1-42 binding to the α7 nAChR, which may pose a unique challenge to drug development of α7 nAChR-specific ligands for Alzheimer's disease. Hopefully, a greater knowledge of the many factors influencing α7 nAChR function as well...

  10. Human orexin/hypocretin receptors form constitutive homo- and heteromeric complexes with each other and with human CB{sub 1} cannabinoid receptors

    Energy Technology Data Exchange (ETDEWEB)

    Jäntti, Maria H., E-mail: maria.jantti@helsinki.fi [Department of Veterinary Biosciences, POB 66, FIN-00014 University of Helsinki (Finland); Mandrika, Ilona, E-mail: ilona@biomed.lu.lv [Latvian Biomedical Research and Study Centre, Ratsupites Str. 1, Riga LV 1067 (Latvia); Kukkonen, Jyrki P., E-mail: jyrki.kukkonen@helsinki.fi [Department of Veterinary Biosciences, POB 66, FIN-00014 University of Helsinki (Finland)

    2014-03-07

    Highlights: • OX{sub 1} and OX{sub 2} orexin and CB{sub 1} cannabinoid receptor dimerization was investigated. • Bioluminescence resonance energy transfer method was used. • All receptors readily formed constitutive homo- and heteromeric complexes. - Abstract: Human OX{sub 1} orexin receptors have been shown to homodimerize and they have also been suggested to heterodimerize with CB{sub 1} cannabinoid receptors. The latter has been suggested to be important for orexin receptor responses and trafficking. In this study, we wanted to assess the ability of the other combinations of receptors to also form similar complexes. Vectors for expression of human OX{sub 1}, OX{sub 2} and CB{sub 1} receptors, C-terminally fused with either Renilla luciferase or GFP{sup 2} green fluorescent protein variant, were generated. The constructs were transiently expressed in Chinese hamster ovary cells, and constitutive dimerization between the receptors was assessed by bioluminescence energy transfer (BRET). Orexin receptor subtypes readily formed homo- and hetero(di)mers, as suggested by significant BRET signals. CB{sub 1} receptors formed homodimers, and they also heterodimerized with both orexin receptors. Interestingly, BRET efficiency was higher for homodimers than for almost all heterodimers. This is likely to be due to the geometry of the interaction; the putatively symmetric dimers may place the C-termini in a more suitable orientation in homomers. Fusion of luciferase to an orexin receptor and GFP{sup 2} to CB{sub 1} produced more effective BRET than the opposite fusions, also suggesting differences in geometry. Similar was seen for the OX{sub 1}–OX{sub 2} interaction. In conclusion, orexin receptors have a significant propensity to make homo- and heterodi-/oligomeric complexes. However, it is unclear whether this affects their signaling. As orexin receptors efficiently signal via endocannabinoid production to CB{sub 1} receptors, dimerization could be an effective way

  11. Selective disruption of the AKAP signaling complexes.

    Science.gov (United States)

    Kennedy, Eileen J; Scott, John D

    2015-01-01

    Synthesis of the second messenger cAMP activates a variety of signaling pathways critical for all facets of intracellular regulation. Protein kinase A (PKA) is the major cAMP-responsive effector. Where and when this enzyme is activated has profound implications on the cellular role of PKA. A-Kinase Anchoring Proteins (AKAPs) play a critical role in this process by orchestrating spatial and temporal aspects of PKA action. A popular means of evaluating the impact of these anchored signaling events is to biochemically interfere with the PKA-AKAP interface. Hence, peptide disruptors of PKA anchoring are valuable tools in the investigation of local PKA action. This article outlines the development of PKA isoform-selective disruptor peptides, documents the optimization of cell-soluble peptide derivatives, and introduces alternative cell-based approaches that interrogate other aspects of the PKA-AKAP interface.

  12. Tyrosine Phosphorylation in Toll-Like Receptor Signaling

    Science.gov (United States)

    Chattopadhyay, Saurabh; Sen, Ganes C.

    2014-01-01

    There is a wealth of knowledge about how different Ser/Thr protein kinases participate in Toll-like receptor (TLR) signaling. In many cases, we know the identities of the Ser/Thr residues of various components of the TLR-signaling pathways that are phosphorylated, the functional consequences of the phosphorylation and the responsible protein kinases. In contrast, the analysis of Tyr-phosphorylation of TLRs and their signaling proteins is currently incomplete, because several existing analyses are not systematic or they do not rely on robust experimental data. Nevertheless, it is clear that many TLRs require, for signaling, ligand-dependent phosphorylation of specific Tyr residues in their cytoplasmic domains; the list includes TLR2, TLR3, TLR4, TLR5, TLR8 and TLR9. In this article, we discuss the current status of knowledge on the effect of Tyr-phosphorylation of TLRs and their signaling proteins on their biochemical and biological functions, the possible identities of the relevant protein tyrosine kinases (PTKs) and the nature of regulations of PTK-mediated activation of TLR signaling pathways. PMID:25022196

  13. Astrocyte Ca2+ signalling: an unexpected complexity

    OpenAIRE

    Volterra, Andrea; Liaudet, Nicolas; Savtchouk, Iaroslav

    2014-01-01

    Astrocyte Ca(2+) signalling has been proposed to link neuronal information in different spatial-temporal dimensions to achieve a higher level of brain integration. However, some discrepancies in the results of recent studies challenge this view and highlight key insufficiencies in our current understanding. In parallel, new experimental approaches that enable the study of astrocyte physiology at higher spatial-temporal resolution in intact brain preparations are beginning to reveal an unexpec...

  14. Postoperative ileus involves interleukin-1 receptor signaling in enteric glia.

    Science.gov (United States)

    Stoffels, Burkhard; Hupa, Kristof Johannes; Snoek, Susanne A; van Bree, Sjoerd; Stein, Kathy; Schwandt, Timo; Vilz, Tim O; Lysson, Mariola; Veer, Cornelis Van't; Kummer, Markus P; Hornung, Veit; Kalff, Joerg C; de Jonge, Wouter J; Wehner, Sven

    2014-01-01

    Postoperative ileus (POI) is a common consequence of abdominal surgery that increases the risk of postoperative complications and morbidity. We investigated the cellular mechanisms and immune responses involved in the pathogenesis of POI. We studied a mouse model of POI in which intestinal manipulation leads to inflammation of the muscularis externa and disrupts motility. We used C57BL/6 (control) mice as well as mice deficient in Toll-like receptors (TLRs) and cytokine signaling components (TLR-2(-/-), TLR-4(-/-), TLR-2/4(-/-), MyD88(-/-), MyD88/TLR adaptor molecule 1(-/-), interleukin-1 receptor [IL-1R1](-/-), and interleukin (IL)-18(-/-) mice). Bone marrow transplantation experiments were performed to determine which cytokine receptors and cell types are involved in the pathogenesis of POI. Development of POI did not require TLRs 2, 4, or 9 or MyD88/TLR adaptor molecule 2 but did require MyD88, indicating a role for IL-1R1. IL-1R1(-/-) mice did not develop POI; however, mice deficient in IL-18, which also signals via MyD88, developed POI. Mice given injections of an IL-1 receptor antagonist (anakinra) or antibodies to deplete IL-1α and IL-1β before intestinal manipulation were protected from POI. Induction of POI activated the inflammasome in muscularis externa tissues of C57BL6 mice, and IL-1α and IL-1β were released in ex vivo organ bath cultures. In bone marrow transplantation experiments, the development of POI required activation of IL-1 receptor in nonhematopoietic cells. IL-1R1 was expressed by enteric glial cells in the myenteric plexus layer, and cultured primary enteric glia cells expressed IL-6 and the chemokine monocyte chemotactic protein 1 in response to IL-1β stimulation. Immunohistochemical analysis of human small bowel tissue samples confirmed expression of IL-1R1 in the ganglia of the myenteric plexus. IL-1 signaling, via IL-1R1 and MyD88, is required for development of POI after intestinal manipulation in mice. Agents that interfere with

  15. Nuclear receptor TLX inhibits TGF-β signaling in glioblastoma.

    Science.gov (United States)

    Johansson, Erik; Zhai, Qiwei; Zeng, Zhao-Jun; Yoshida, Takeshi; Funa, Keiko

    2016-05-01

    TLX (also called NR2E1) is an orphan nuclear receptor that maintains stemness of neuronal stem cells. TLX is highly expressed in the most malignant form of glioma, glioblastoma multiforme (GBM), and is important for the proliferation and maintenance of the stem/progenitor cells of the tumor. Transforming Growth Factor-β (TGF-β) is a cytokine regulating many different cellular processes such as differentiation, migration, adhesion, cell death and proliferation. TGF-β has an important function in cancer where it can work as either a tumor suppressor or oncogene, depending on the cancer type and stage of tumor development. Since glioblastoma often have dysfunctional TGF-β signaling we wanted to find out if there is any interaction between TLX and TGF-β in glioblastoma cells. We demonstrate that knockdown of TLX enhances the canonical TGF-β signaling response in glioblastoma cell lines. TLX physically interacts with and stabilizes Smurf1, which can ubiquitinate and target TGF-β receptor II for degradation, whereas knockdown of TLX leads to stabilization of TGF-β receptor II, increased nuclear translocation of Smad2/3 and enhanced expression of TGF-β target genes. The interaction between TLX and TGF-β may play an important role in the regulation of proliferation and tumor-initiating properties of glioblastoma cells. Copyright © 2016. Published by Elsevier Inc.

  16. Optodynamic simulation of β-adrenergic receptor signalling.

    Science.gov (United States)

    Siuda, Edward R; McCall, Jordan G; Al-Hasani, Ream; Shin, Gunchul; Il Park, Sung; Schmidt, Martin J; Anderson, Sonya L; Planer, William J; Rogers, John A; Bruchas, Michael R

    2015-09-28

    Optogenetics has provided a revolutionary approach to dissecting biological phenomena. However, the generation and use of optically active GPCRs in these contexts is limited and it is unclear how well an opsin-chimera GPCR might mimic endogenous receptor activity. Here we show that a chimeric rhodopsin/β2 adrenergic receptor (opto-β2AR) is similar in dynamics to endogenous β2AR in terms of: cAMP generation, MAP kinase activation and receptor internalization. In addition, we develop and characterize a novel toolset of optically active, functionally selective GPCRs that can bias intracellular signalling cascades towards either G-protein or arrestin-mediated cAMP and MAP kinase pathways. Finally, we show how photoactivation of opto-β2AR in vivo modulates neuronal activity and induces anxiety-like behavioural states in both fiber-tethered and wireless, freely moving animals when expressed in brain regions known to contain β2ARs. These new GPCR approaches enhance the utility of optogenetics and allow for discrete spatiotemporal control of GPCR signalling in vitro and in vivo.

  17. Negative regulation of Toll-like receptor signalling 

    Directory of Open Access Journals (Sweden)

    Halina Antosz

    2013-04-01

    Full Text Available The mechanism of innate immunity is based on the pattern recognition receptors (PRR that recognize molecular patterns associated with pathogens (PAMPs. Among PRR receptors Toll-like receptors (TLR are distinguished. As a result of contact with pathogens, TLRs activate specific intracellular signaling pathways. It happens through proteins such as adaptor molecules, e.g. MyD88, TIRAP, TRIF, TRAM, and IPS-1, which participate in the cascade activation of kinases (IKK, MAP, RIP-1, TBK-1 as well as transcription factors (NF-κB, AP-1 and regulatory factor (IRF3. The result of this activation is the production of active proinflammatory cytokines, chemokines, interferons and enzymes. The PRR pathways are controlled by extra – and intracellular molecules to prevent overexpression of PRR. They include soluble receptors (sTLR, transmembrane proteins (ST2, SIGIRR, RP105, TRAIL-R and intracellular inhibitors (SOCS-1, SOCS-3, sMyD88, TOLLIP, IRAK-M, SARM, A20, β-arrestin, CYLD, SHP. These molecules maintain the balance between activation and inhibition and ensure balancing of the beneficial and adverse effects of antigen recognition.

  18. Metazoan-like signaling in a unicellular receptor tyrosine kinase

    Directory of Open Access Journals (Sweden)

    Schultheiss Kira P

    2013-02-01

    Full Text Available Abstract Background Receptor tyrosine kinases (RTKs are crucial components of signal transduction systems in multicellular animals. Surprisingly, numerous RTKs have been identified in the genomes of unicellular choanoflagellates and other protists. Here, we report the first biochemical study of a unicellular RTK, namely RTKB2 from Monosiga brevicollis. Results We cloned, expressed, and purified the RTKB2 kinase, and showed that it is enzymatically active. The activity of RTKB2 is controlled by autophosphorylation, as in metazoan RTKs. RTKB2 possesses six copies of a unique domain (designated RM2 in its C-terminal tail. An isolated RM2 domain (or a synthetic peptide derived from the RM2 sequence served as a substrate for RTKB2 kinase. When phosphorylated, the RM2 domain bound to the Src homology 2 domain of MbSrc1 from M. brevicollis. NMR structural studies of the RM2 domain indicated that it is disordered in solution. Conclusions Our results are consistent with a model in which RTKB2 activation stimulates receptor autophosphorylation within the RM2 domains. This leads to recruitment of Src-like kinases (and potentially other M. brevicollis proteins and further phosphorylation, which may serve to increase or dampen downstream signals. Thus, crucial features of signal transduction circuitry were established prior to the evolution of metazoans from their unicellular ancestors.

  19. Signaling flux redistribution at toll-like receptor pathway junctions.

    Directory of Open Access Journals (Sweden)

    Kumar Selvarajoo

    Full Text Available Various receptors on cell surface recognize specific extracellular molecules and trigger signal transduction altering gene expression in the nucleus. Gain or loss-of-function mutations of one molecule have shown to affect alternative signaling pathways with a poorly understood mechanism. In Toll-like receptor (TLR 4 signaling, which branches into MyD88- and TRAM-dependent pathways upon lipopolysaccharide (LPS stimulation, we investigated the gain or loss-of-function mutations of MyD88. We predict, using a computational model built on the perturbation-response approach and the law of mass conservation, that removal and addition of MyD88 in TLR4 activation, enhances and impairs, respectively, the alternative TRAM-dependent pathway through signaling flux redistribution (SFR at pathway branches. To verify SFR, we treated MyD88-deficient macrophages with LPS and observed enhancement of TRAM-dependent pathway based on increased IRF3 phosphorylation and induction of Cxcl10 and Ifit2. Furthermore, increasing the amount of MyD88 in cultured cells showed decreased TRAM binding to TLR4. Investigating another TLR4 pathway junction, from TRIF to TRAF6, RIP1 and TBK1, the removal of MyD88-dependent TRAF6 increased expression of TRAM-dependent Cxcl10 and Ifit2. Thus, we demonstrate that SFR is a novel mechanism for enhanced activation of alternative pathways when molecules at pathway junctions are removed. Our data suggest that SFR may enlighten hitherto unexplainable intracellular signaling alterations in genetic diseases where gain or loss-of-function mutations are observed.

  20. Crammed signaling motifs in the T-cell receptor.

    Science.gov (United States)

    Borroto, Aldo; Abia, David; Alarcón, Balbino

    2014-09-01

    Although the T cell antigen receptor (TCR) is long known to contain multiple signaling subunits (CD3γ, CD3δ, CD3ɛ and CD3ζ), their role in signal transduction is still not well understood. The presence of at least one immunoreceptor tyrosine-based activation motif (ITAM) in each CD3 subunit has led to the idea that the multiplication of such elements essentially serves to amplify signals. However, the evolutionary conservation of non-ITAM sequences suggests that each CD3 subunit is likely to have specific non-redundant roles at some stage of development or in mature T cell function. The CD3ɛ subunit is paradigmatic because in a relatively short cytoplasmic sequence (∼55 amino acids) it contains several docking sites for proteins involved in intracellular trafficking and signaling, proteins whose relevance in T cell activation is slowly starting to be revealed. In this review we will summarize our current knowledge on the signaling effectors that bind directly to the TCR and we will propose a hierarchy in their response to TCR triggering. Copyright © 2014 Elsevier B.V. All rights reserved.

  1. Collagen Type I as a Ligand for Receptor-Mediated Signaling

    Directory of Open Access Journals (Sweden)

    Iris Boraschi-Diaz

    2017-05-01

    Full Text Available Collagens form the fibrous component of the extracellular matrix in all multi-cellular animals. Collagen type I is the most abundant collagen present in skin, tendons, vasculature, as well as the organic portion of the calcified tissue of bone and teeth. This review focuses on numerous receptors for which collagen acts as a ligand, including integrins, discoidin domain receptors DDR1 and 2, OSCAR, GPVI, G6b-B, and LAIR-1 of the leukocyte receptor complex (LRC and mannose family receptor uPARAP/Endo180. We explore the process of collagen production and self-assembly, as well as its degradation by collagenases and gelatinases in order to predict potential temporal and spatial sites of action of different collagen receptors. While the interactions of the mature collagen matrix with integrins and DDR are well-appreciated, potential signals from immature matrix as well as collagen degradation products are possible but not yet described. The role of multiple collagen receptors in physiological processes and their contribution to pathophysiology of diseases affecting collagen homeostasis require further studies.

  2. Role of Ubiquitination in IGF-1 Receptor Signaling and Degradation

    OpenAIRE

    Sehat, Bita; Andersson, Sandra; Vasilcanu, Radu; Girnita, Leonard; Larsson, Olle

    2007-01-01

    BACKGROUND: The insulin-like growth factor 1 receptor (IGF-1R) plays numerous crucial roles in cancer biology. The majority of knowledge on IGF-1R signaling is concerned with its role in the activation of the canonical phosphatidyl inositol-3 kinase (PI3K)/Akt and MAPK/ERK pathways. However, the role of IGF-1R ubiquitination in modulating IGF-1R function is an area of current research. In light of this we sought to determine the relationship between IGF-1R phosphorylation, ubiquitination, and...

  3. Modulation of EEG Theta Band Signal Complexity by Music Therapy

    Science.gov (United States)

    Bhattacharya, Joydeep; Lee, Eun-Jeong

    The primary goal of this study was to investigate the impact of monochord (MC) sounds, a type of archaic sounds used in music therapy, on the neural complexity of EEG signals obtained from patients undergoing chemotherapy. The secondary goal was to compare the EEG signal complexity values for monochords with those for progressive muscle relaxation (PMR), an alternative therapy for relaxation. Forty cancer patients were randomly allocated to one of the two relaxation groups, MC and PMR, over a period of six months; continuous EEG signals were recorded during the first and last sessions. EEG signals were analyzed by applying signal mode complexity, a measure of complexity of neuronal oscillations. Across sessions, both groups showed a modulation of complexity of beta-2 band (20-29Hz) at midfrontal regions, but only MC group showed a modulation of complexity of theta band (3.5-7.5Hz) at posterior regions. Therefore, the neuronal complexity patterns showed different changes in EEG frequency band specific complexity resulting in two different types of interventions. Moreover, the different neural responses to listening to monochords and PMR were observed after regular relaxation interventions over a short time span.

  4. Brain signal complexity rises with repetition suppression in visual learning.

    Science.gov (United States)

    Lafontaine, Marc Philippe; Lacourse, Karine; Lina, Jean-Marc; McIntosh, Anthony R; Gosselin, Frédéric; Théoret, Hugo; Lippé, Sarah

    2016-06-21

    Neuronal activity associated with visual processing of an unfamiliar face gradually diminishes when it is viewed repeatedly. This process, known as repetition suppression (RS), is involved in the acquisition of familiarity. Current models suggest that RS results from interactions between visual information processing areas located in the occipito-temporal cortex and higher order areas, such as the dorsolateral prefrontal cortex (DLPFC). Brain signal complexity, which reflects information dynamics of cortical networks, has been shown to increase as unfamiliar faces become familiar. However, the complementarity of RS and increases in brain signal complexity have yet to be demonstrated within the same measurements. We hypothesized that RS and brain signal complexity increase occur simultaneously during learning of unfamiliar faces. Further, we expected alteration of DLPFC function by transcranial direct current stimulation (tDCS) to modulate RS and brain signal complexity over the occipito-temporal cortex. Participants underwent three tDCS conditions in random order: right anodal/left cathodal, right cathodal/left anodal and sham. Following tDCS, participants learned unfamiliar faces, while an electroencephalogram (EEG) was recorded. Results revealed RS over occipito-temporal electrode sites during learning, reflected by a decrease in signal energy, a measure of amplitude. Simultaneously, as signal energy decreased, brain signal complexity, as estimated with multiscale entropy (MSE), increased. In addition, prefrontal tDCS modulated brain signal complexity over the right occipito-temporal cortex during the first presentation of faces. These results suggest that although RS may reflect a brain mechanism essential to learning, complementary processes reflected by increases in brain signal complexity, may be instrumental in the acquisition of novel visual information. Such processes likely involve long-range coordinated activity between prefrontal and lower order visual

  5. Optimal experimental design in an epidermal growth factor receptor signalling and down-regulation model.

    Science.gov (United States)

    Casey, F P; Baird, D; Feng, Q; Gutenkunst, R N; Waterfall, J J; Myers, C R; Brown, K S; Cerione, R A; Sethna, J P

    2007-05-01

    We apply the methods of optimal experimental design to a differential equation model for epidermal growth factor receptor signalling, trafficking and down-regulation. The model incorporates the role of a recently discovered protein complex made up of the E3 ubiquitin ligase, Cbl, the guanine exchange factor (GEF), Cool-1 (beta -Pix) and the Rho family G protein Cdc42. The complex has been suggested to be important in disrupting receptor down-regulation. We demonstrate that the model interactions can accurately reproduce the experimental observations, that they can be used to make predictions with accompanying uncertainties, and that we can apply ideas of optimal experimental design to suggest new experiments that reduce the uncertainty on unmeasurable components of the system.

  6. Novel method for the study of receptor Ca2+ signalling exemplified by the NK1 receptor

    DEFF Research Database (Denmark)

    Heding, A; Elling, C E; Schwartz, T W

    2002-01-01

    We have used a novel technology (NovoStar from BMG Labtechnologies) for the study of the Ca2+ signalling of the human tackykinin NK1 (hNK-I receptor). The NovoStar is a microplate reader based on fluorescence and luminescence. The instrument implements a robotic pipettor arm and two microplate...... carriers, typically one for samples and one for cells. The robotic pipettor arm can transfer sample (agonist or antagonist) from the sample plate or other liquid containers to the cell plate, facilitating the study of Ca2+ signalling to such a degree that the instrument can be used for Medium Throughput...

  7. TSH Receptor Signaling Abrogation by a Novel Small Molecule.

    Science.gov (United States)

    Latif, Rauf; Realubit, Ronald B; Karan, Charles; Mezei, Mihaly; Davies, Terry F

    2016-01-01

    Pathological activation of the thyroid-stimulating hormone receptor (TSHR) is caused by thyroid-stimulating antibodies in patients with Graves' disease (GD) or by somatic and rare genomic mutations that enhance constitutive activation of the receptor influencing both G protein and non-G protein signaling. Potential selective small molecule antagonists represent novel therapeutic compounds for abrogation of such abnormal TSHR signaling. In this study, we describe the identification and in vitro characterization of a novel small molecule antagonist by high-throughput screening (HTS). The identification of the TSHR antagonist was performed using a transcription-based TSH-inhibition bioassay. TSHR-expressing CHO cells, which also expressed a luciferase-tagged CRE response element, were optimized using bovine TSH as the activator, in a 384 well plate format, which had a Z score of 0.3-0.6. Using this HTS assay, we screened a diverse library of ~80,000 compounds at a final concentration of 16.7 μM. The selection criteria for a positive hit were based on a mean signal threshold of ≥50% inhibition of control TSH stimulation. The screening resulted in 450 positive hits giving a hit ratio of 0.56%. A secondary confirmation screen against TSH and forskolin - a post receptor activator of adenylyl cyclase - confirmed one TSHR-specific candidate antagonist molecule (named VA-K-14). This lead molecule had an IC 50 of 12.3 μM and a unique chemical structure. A parallel analysis for cell viability indicated that the lead inhibitor was non-cytotoxic at its effective concentrations. In silico docking studies performed using a TSHR transmembrane model showed the hydrophobic contact locations and the possible mode of inhibition of TSHR signaling. Furthermore, this molecule was capable of inhibiting TSHR stimulation by GD patient sera and monoclonal-stimulating TSHR antibodies. In conclusion, we report the identification of a novel small molecule TSHR inhibitor, which has the

  8. Regulation of Drosophila Brain Wiring by Neuropil Interactions via a Slit-Robo-RPTP Signaling Complex.

    Science.gov (United States)

    Oliva, Carlos; Soldano, Alessia; Mora, Natalia; De Geest, Natalie; Claeys, Annelies; Erfurth, Maria-Luise; Sierralta, Jimena; Ramaekers, Ariane; Dascenco, Dan; Ejsmont, Radoslaw K; Schmucker, Dietmar; Sanchez-Soriano, Natalia; Hassan, Bassem A

    2016-10-24

    The axonal wiring molecule Slit and its Round-About (Robo) receptors are conserved regulators of nerve cord patterning. Robo receptors also contribute to wiring brain circuits. Whether molecular mechanisms regulating these signals are modified to fit more complex brain wiring processes is unclear. We investigated the role of Slit and Robo receptors in wiring Drosophila higher-order brain circuits and identified differences in the cellular and molecular mechanisms of Robo/Slit function. First, we find that signaling by Robo receptors in the brain is regulated by the Receptor Protein Tyrosine Phosphatase RPTP69d. RPTP69d increases membrane availability of Robo3 without affecting its phosphorylation state. Second, we detect no midline localization of Slit during brain development. Instead, Slit is enriched in the mushroom body, a neuronal structure covering large areas of the brain. Thus, a divergent molecular mechanism regulates neuronal circuit wiring in the Drosophila brain, partly in response to signals from the mushroom body. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

  9. DMPD: Modulation of Toll-interleukin 1 receptor mediated signaling. [Dynamic Macrophage Pathway CSML Database

    Lifescience Database Archive (English)

    Full Text Available 15662540 Modulation of Toll-interleukin 1 receptor mediated signaling. Li X, Qin J.... J Mol Med. 2005 Apr;83(4):258-66. Epub 2005 Jan 21. (.png) (.svg) (.html) (.csml) Show Modulation of Toll-i...nterleukin 1 receptor mediated signaling. PubmedID 15662540 Title Modulation of Toll-interleukin 1 receptor

  10. Heteroreceptor Complexes Formed by Dopamine D1, Histamine H3, and N-Methyl-D-Aspartate Glutamate Receptors as Targets to Prevent Neuronal Death in Alzheimer's Disease.

    Science.gov (United States)

    Rodríguez-Ruiz, Mar; Moreno, Estefanía; Moreno-Delgado, David; Navarro, Gemma; Mallol, Josefa; Cortés, Antonio; Lluís, Carme; Canela, Enric I; Casadó, Vicent; McCormick, Peter J; Franco, Rafael

    2017-08-01

    Alzheimer's disease (AD) is a neurodegenerative disorder causing progressive memory loss and cognitive dysfunction. Anti-AD strategies targeting cell receptors consider them as isolated units. However, many cell surface receptors cooperate and physically contact each other forming complexes having different biochemical properties than individual receptors. We here report the discovery of dopamine D 1 , histamine H 3 , and N-methyl-D-aspartate (NMDA) glutamate receptor heteromers in heterologous systems and in rodent brain cortex. Heteromers were detected by co-immunoprecipitation and in situ proximity ligation assays (PLA) in the rat cortex where H 3 receptor agonists, via negative cross-talk, and H 3 receptor antagonists, via cross-antagonism, decreased D 1 receptor agonist signaling determined by ERK1/2 or Akt phosphorylation, and counteracted D 1 receptor-mediated excitotoxic cell death. Both D 1 and H 3 receptor antagonists also counteracted NMDA toxicity suggesting a complex interaction between NMDA receptors and D 1 -H 3 receptor heteromer function. Likely due to heteromerization, H 3 receptors act as allosteric regulator for D 1 and NMDA receptors. By bioluminescence resonance energy transfer (BRET), we demonstrated that D 1 or H 3 receptors form heteromers with NR1A/NR2B NMDA receptor subunits. D 1 -H 3 -NMDA receptor complexes were confirmed by BRET combined with fluorescence complementation. The endogenous expression of complexes in mouse cortex was determined by PLA and similar expression was observed in wild-type and APP/PS1 mice. Consistent with allosteric receptor-receptor interactions within the complex, H 3 receptor antagonists reduced NMDA or D 1 receptor-mediated excitotoxic cell death in cortical organotypic cultures. Moreover, H 3 receptor antagonists reverted the toxicity induced by ß 1-42 -amyloid peptide. Thus, histamine H 3 receptors in D 1 -H 3 -NMDA heteroreceptor complexes arise as promising targets to prevent neurodegeneration.

  11. Retinoid receptor signaling and autophagy in acute promyelocytic leukemia

    Energy Technology Data Exchange (ETDEWEB)

    Orfali, Nina [Cork Cancer Research Center, University College Cork, Cork (Ireland); Department of Pharmacology, Weill Cornell Medical College, New York, NY 10065, USA. (United States); McKenna, Sharon L. [Cork Cancer Research Center, University College Cork, Cork (Ireland); Cahill, Mary R. [Department of Hematology, Cork University Hospital, Cork (Ireland); Gudas, Lorraine J., E-mail: ljgudas@med.cornell.edu [Department of Pharmacology, Weill Cornell Medical College, New York, NY 10065, USA. (United States); Mongan, Nigel P., E-mail: nigel.mongan@nottingham.ac.uk [Faculty of Medicine and Health Science, School of Veterinary Medicine and Science, University of Nottingham, LE12 5RD (United Kingdom); Department of Pharmacology, Weill Cornell Medical College, New York, NY 10065, USA. (United States)

    2014-05-15

    Retinoids are a family of signaling molecules derived from vitamin A with well established roles in cellular differentiation. Physiologically active retinoids mediate transcriptional effects on cells through interactions with retinoic acid (RARs) and retinoid-X (RXR) receptors. Chromosomal translocations involving the RARα gene, which lead to impaired retinoid signaling, are implicated in acute promyelocytic leukemia (APL). All-trans-retinoic acid (ATRA), alone and in combination with arsenic trioxide (ATO), restores differentiation in APL cells and promotes degradation of the abnormal oncogenic fusion protein through several proteolytic mechanisms. RARα fusion-protein elimination is emerging as critical to obtaining sustained remission and long-term cure in APL. Autophagy is a degradative cellular pathway involved in protein turnover. Both ATRA and ATO also induce autophagy in APL cells. Enhancing autophagy may therefore be of therapeutic benefit in resistant APL and could broaden the application of differentiation therapy to other cancers. Here we discuss retinoid signaling in hematopoiesis, leukemogenesis, and APL treatment. We highlight autophagy as a potential important regulator in anti-leukemic strategies. - Highlights: • Normal and aberrant retinoid signaling in hematopoiesis and leukemia is reviewed. • We suggest a novel role for RARα in the development of X-RARα gene fusions in APL. • ATRA therapy in APL activates transcription and promotes onco-protein degradation. • Autophagy may be involved in both onco-protein degradation and differentiation. • Pharmacologic autophagy induction may potentiate ATRA's therapeutic effects.

  12. Orphan nuclear receptor TLX regulates astrogenesis by modulating BMP signaling

    Directory of Open Access Journals (Sweden)

    Song eQin

    2014-04-01

    Full Text Available Neural stem cells (NSCs are self-renewing multipotent progenitors that generate both neurons and glia. The precise control of NSC behavior is fundamental to the architecture and function of the central nervous system. We previously demonstrated that the orphan nuclear receptor TLX is required for postnatal NSC activation and neurogenesis in the neurogenic niche. Here, we show that TLX modulates BMP-SMAD signaling to control the timing of postnatal astrogenesis. Genes involved in the BMP signaling pathway, such as Bmp4, Hes1, and Id3, are upregulated in postnatal brains lacking Tlx. Chromatin immunoprecipitation and electrophoretic mobility shift assays reveal that TLX can directly bind the enhancer region of Bmp4. In accordance with elevated BMP signaling, the downstream effectors SMAD1/5/8 are activated by phosphorylation in Tlx mutant mice. Consequently, Tlx mutant brains exhibit an early appearance and increased number of astrocytes with marker expression of glial fibrillary acidic protein (GFAP and S100B. Taken together, these results suggest that TLX tightly controls postnatal astrogenesis through the modulation of BMP-SMAD signaling pathway activity.

  13. Orphan nuclear receptor TLX regulates astrogenesis by modulating BMP signaling.

    Science.gov (United States)

    Qin, Song; Niu, Wenze; Iqbal, Nida; Smith, Derek K; Zhang, Chun-Li

    2014-01-01

    Neural stem cells (NSCs) are self-renewing multipotent progenitors that generate both neurons and glia. The precise control of NSC behavior is fundamental to the architecture and function of the central nervous system. We previously demonstrated that the orphan nuclear receptor TLX is required for postnatal NSC activation and neurogenesis in the neurogenic niche. Here, we show that TLX modulates bone morphogenetic protein (BMP)-SMAD signaling to control the timing of postnatal astrogenesis. Genes involved in the BMP signaling pathway, such as Bmp4, Hes1, and Id3, are upregulated in postnatal brains lacking Tlx. Chromatin immunoprecipitation and electrophoretic mobility shift assays reveal that TLX can directly bind the enhancer region of Bmp4. In accordance with elevated BMP signaling, the downstream effectors SMAD1/5/8 are activated by phosphorylation in Tlx mutant mice. Consequently, Tlx mutant brains exhibit an early appearance and increased number of astrocytes with marker expression of glial fibrillary acidic protein (GFAP) and S100B. Taken together, these results suggest that TLX tightly controls postnatal astrogenesis through the modulation of BMP-SMAD signaling pathway activity.

  14. Retinoid receptor signaling and autophagy in acute promyelocytic leukemia

    International Nuclear Information System (INIS)

    Orfali, Nina; McKenna, Sharon L.; Cahill, Mary R.; Gudas, Lorraine J.; Mongan, Nigel P.

    2014-01-01

    Retinoids are a family of signaling molecules derived from vitamin A with well established roles in cellular differentiation. Physiologically active retinoids mediate transcriptional effects on cells through interactions with retinoic acid (RARs) and retinoid-X (RXR) receptors. Chromosomal translocations involving the RARα gene, which lead to impaired retinoid signaling, are implicated in acute promyelocytic leukemia (APL). All-trans-retinoic acid (ATRA), alone and in combination with arsenic trioxide (ATO), restores differentiation in APL cells and promotes degradation of the abnormal oncogenic fusion protein through several proteolytic mechanisms. RARα fusion-protein elimination is emerging as critical to obtaining sustained remission and long-term cure in APL. Autophagy is a degradative cellular pathway involved in protein turnover. Both ATRA and ATO also induce autophagy in APL cells. Enhancing autophagy may therefore be of therapeutic benefit in resistant APL and could broaden the application of differentiation therapy to other cancers. Here we discuss retinoid signaling in hematopoiesis, leukemogenesis, and APL treatment. We highlight autophagy as a potential important regulator in anti-leukemic strategies. - Highlights: • Normal and aberrant retinoid signaling in hematopoiesis and leukemia is reviewed. • We suggest a novel role for RARα in the development of X-RARα gene fusions in APL. • ATRA therapy in APL activates transcription and promotes onco-protein degradation. • Autophagy may be involved in both onco-protein degradation and differentiation. • Pharmacologic autophagy induction may potentiate ATRA's therapeutic effects

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

    Science.gov (United States)

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

    2016-04-05

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

  16. Targeting fibroblast growth factor receptor signaling inhibits prostate cancer progression.

    Science.gov (United States)

    Feng, Shu; Shao, Longjiang; Yu, Wendong; Gavine, Paul; Ittmann, Michael

    2012-07-15

    Extensive correlative studies in human prostate cancer as well as studies in vitro and in mouse models indicate that fibroblast growth factor receptor (FGFR) signaling plays an important role in prostate cancer progression. In this study, we used a probe compound for an FGFR inhibitor, which potently inhibits FGFR-1-3 and significantly inhibits FGFR-4. The purpose of this study is to determine whether targeting FGFR signaling from all four FGFRs will have in vitro activities consistent with inhibition of tumor progression and will inhibit tumor progression in vivo. Effects of AZ8010 on FGFR signaling and invasion were analyzed using immortalized normal prostate epithelial (PNT1a) cells and PNT1a overexpressing FGFR-1 or FGFR-4. The effect of AZ8010 on invasion and proliferation in vitro was also evaluated in prostate cancer cell lines. Finally, the impact of AZ8010 on tumor progression in vivo was evaluated using a VCaP xenograft model. AZ8010 completely inhibits FGFR-1 and significantly inhibits FGFR-4 signaling at 100 nmol/L, which is an achievable in vivo concentration. This results in marked inhibition of extracellular signal-regulated kinase (ERK) phosphorylation and invasion in PNT1a cells expressing FGFR-1 and FGFR-4 and all prostate cancer cell lines tested. Treatment in vivo completely inhibited VCaP tumor growth and significantly inhibited angiogenesis and proliferation and increased cell death in treated tumors. This was associated with marked inhibition of ERK phosphorylation in treated tumors. Targeting FGFR signaling is a promising new approach to treating aggressive prostate cancer.

  17. Exponential signaling gain at the receptor level enhances signal-to-noise ratio in bacterial chemotaxis.

    Directory of Open Access Journals (Sweden)

    Silke Neumann

    Full Text Available Cellular signaling systems show astonishing precision in their response to external stimuli despite strong fluctuations in the molecular components that determine pathway activity. To control the effects of noise on signaling most efficiently, living cells employ compensatory mechanisms that reach from simple negative feedback loops to robustly designed signaling architectures. Here, we report on a novel control mechanism that allows living cells to keep precision in their signaling characteristics - stationary pathway output, response amplitude, and relaxation time - in the presence of strong intracellular perturbations. The concept relies on the surprising fact that for systems showing perfect adaptation an exponential signal amplification at the receptor level suffices to eliminate slowly varying multiplicative noise. To show this mechanism at work in living systems, we quantified the response dynamics of the E. coli chemotaxis network after genetically perturbing the information flux between upstream and downstream signaling components. We give strong evidence that this signaling system results in dynamic invariance of the activated response regulator against multiplicative intracellular noise. We further demonstrate that for environmental conditions, for which precision in chemosensing is crucial, the invariant response behavior results in highest chemotactic efficiency. Our results resolve several puzzling features of the chemotaxis pathway that are widely conserved across prokaryotes but so far could not be attributed any functional role.

  18. Structure of a prehandover mammalian ribosomal SRP·SRP receptor targeting complex.

    Science.gov (United States)

    Kobayashi, Kan; Jomaa, Ahmad; Lee, Jae Ho; Chandrasekar, Sowmya; Boehringer, Daniel; Shan, Shu-Ou; Ban, Nenad

    2018-04-20

    Signal recognition particle (SRP) targets proteins to the endoplasmic reticulum (ER). SRP recognizes the ribosome synthesizing a signal sequence and delivers it to the SRP receptor (SR) on the ER membrane followed by the transfer of the signal sequence to the translocon. Here, we present the cryo-electron microscopy structure of the mammalian translating ribosome in complex with SRP and SR in a conformation preceding signal sequence handover. The structure visualizes all eukaryotic-specific SRP and SR proteins and reveals their roles in stabilizing this conformation by forming a large protein assembly at the distal site of SRP RNA. We provide biochemical evidence that the guanosine triphosphate hydrolysis of SRP·SR is delayed at this stage, possibly to provide a time window for signal sequence handover to the translocon. Copyright © 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.

  19. Signal interaction of Hedgehog/GLI and epidermal growth factor receptor signaling in cancer development

    International Nuclear Information System (INIS)

    Eberl, M.

    2012-01-01

    The subject of this PhD thesis is based on the cooperation of Hedgehog (HH)/GLI with epidermal growth factor receptor (EGFR) signaling synergistically promoting oncogenic transformation and cancer growth. In previous studies we have demonstrated that the HH/GLI and EGFR signaling pathways interact synergistically resulting not only in selective induction of HH/GLI-EGFR target genes, but also in the onset of oncogenic transformation and tumor formation (Kasper, Schnidar et al. 2006; Schnidar, Eberl et al. 2009). However, the molecular key mediators acting downstream of HH/GLI and EGFR signal cooperation were largely unknown and the in vivo evidence for the therapeutic relevance of HH/GLI and EGFR signal cooperation in HH-associated cancers was lacking. During my PhD thesis I could demonstrate that the integration of EGFR and HH/GLI signaling involves activation of RAS/MEK/ERK and JUN/AP1 signaling in response to EGFR activation. Furthermore I succeeded in identifying genes, including stem cell- (SOX2, SOX9), tumor growth- (JUN, TGFA, FGF19) and metastasis-associated genes (SPP1/osteopontin, CXCR4) that showed synergistic transcriptional activation by HH/GLI-EGFR signal integration. Importantly, I could demonstrate that these genes arrange themselves within a stable interdependent signaling network, which is required for in vivo growth of basal cell carcinoma (BCC) and tumor-initiating pancreatic cancer cells. These data validate EGFR signaling as additional drug target in HH/GLI driven cancers and provide new therapeutic strategies based on combined targeting of cooperative HH/GLI-EGFR signaling and selected downstream target genes (Eberl, Klingler et al. 2012). (author) [de

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

    Science.gov (United States)

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

    2017-08-25

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

  1. Binding of Signal Recognition Particle Gives Ribosome/Nascent Chain Complexes a Competitive Advantage in Endoplasmic Reticulum Membrane Interaction

    Science.gov (United States)

    Neuhof, Andrea; Rolls, Melissa M.; Jungnickel, Berit; Kalies, Kai-Uwe; Rapoport, Tom A.

    1998-01-01

    Most secretory and membrane proteins are sorted by signal sequences to the endoplasmic reticulum (ER) membrane early during their synthesis. Targeting of the ribosome-nascent chain complex (RNC) involves the binding of the signal sequence to the signal recognition particle (SRP), followed by an interaction of ribosome-bound SRP with the SRP receptor. However, ribosomes can also independently bind to the ER translocation channel formed by the Sec61p complex. To explain the specificity of membrane targeting, it has therefore been proposed that nascent polypeptide-associated complex functions as a cytosolic inhibitor of signal sequence- and SRP-independent ribosome binding to the ER membrane. We report here that SRP-independent binding of RNCs to the ER membrane can occur in the presence of all cytosolic factors, including nascent polypeptide-associated complex. Nontranslating ribosomes competitively inhibit SRP-independent membrane binding of RNCs but have no effect when SRP is bound to the RNCs. The protective effect of SRP against ribosome competition depends on a functional signal sequence in the nascent chain and is also observed with reconstituted proteoliposomes containing only the Sec61p complex and the SRP receptor. We conclude that cytosolic factors do not prevent the membrane binding of ribosomes. Instead, specific ribosome targeting to the Sec61p complex is provided by the binding of SRP to RNCs, followed by an interaction with the SRP receptor, which gives RNC–SRP complexes a selective advantage in membrane targeting over nontranslating ribosomes. PMID:9436994

  2. Signal transduction by the major histocompatibility complex class I molecule

    DEFF Research Database (Denmark)

    Pedersen, A E; Skov, Svend; Bregenholt, S

    1999-01-01

    Ligation of cell surface major histocompatibility class I (MHC-I) proteins by antibodies, or by their native counter receptor, the CD8 molecule, mediates transduction of signals into the cells. MHC-I-mediated signaling can lead to both increased and decreased activity of the MHC-I-expressing cell...... and functioning, MHC-I molecules might be of importance for the maintenance of cellular homeostasis not only within the immune system, but also in the interplay between the immune system and other organ systems....

  3. NSP-CAS Protein Complexes: Emerging Signaling Modules in Cancer.

    Science.gov (United States)

    Wallez, Yann; Mace, Peter D; Pasquale, Elena B; Riedl, Stefan J

    2012-05-01

    The CAS (CRK-associated substrate) family of adaptor proteins comprises 4 members, which share a conserved modular domain structure that enables multiple protein-protein interactions, leading to the assembly of intracellular signaling platforms. Besides their physiological role in signal transduction downstream of a variety of cell surface receptors, CAS proteins are also critical for oncogenic transformation and cancer cell malignancy through associations with a variety of regulatory proteins and downstream effectors. Among the regulatory partners, the 3 recently identified adaptor proteins constituting the NSP (novel SH2-containing protein) family avidly bind to the conserved carboxy-terminal focal adhesion-targeting (FAT) domain of CAS proteins. NSP proteins use an anomalous nucleotide exchange factor domain that lacks catalytic activity to form NSP-CAS signaling modules. Additionally, the NSP SH2 domain can link NSP-CAS signaling assemblies to tyrosine-phosphorylated cell surface receptors. NSP proteins can potentiate CAS function by affecting key CAS attributes such as expression levels, phosphorylation state, and subcellular localization, leading to effects on cell adhesion, migration, and invasion as well as cell growth. The consequences of these activities are well exemplified by the role that members of both families play in promoting breast cancer cell invasiveness and resistance to antiestrogens. In this review, we discuss the intriguing interplay between the NSP and CAS families, with a particular focus on cancer signaling networks.

  4. 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.

  5. A gate-latch-lock mechanism for hormone signalling by abscisic acid receptors

    KAUST Repository

    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

    2009-01-01

    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. Retinoid receptor signaling and autophagy in acute promyelocytic leukemia.

    LENUS (Irish Health Repository)

    Orfali, Nina

    2014-05-15

    Retinoids are a family of signaling molecules derived from vitamin A with well established roles in cellular differentiation. Physiologically active retinoids mediate transcriptional effects on cells through interactions with retinoic acid (RARs) and retinoid-X (RXR) receptors. Chromosomal translocations involving the RARα gene, which lead to impaired retinoid signaling, are implicated in acute promyelocytic leukemia (APL). All-trans-retinoic acid (ATRA), alone and in combination with arsenic trioxide (ATO), restores differentiation in APL cells and promotes degradation of the abnormal oncogenic fusion protein through several proteolytic mechanisms. RARα fusion-protein elimination is emerging as critical to obtaining sustained remission and long-term cure in APL. Autophagy is a degradative cellular pathway involved in protein turnover. Both ATRA and ATO also induce autophagy in APL cells. Enhancing autophagy may therefore be of therapeutic benefit in resistant APL and could broaden the application of differentiation therapy to other cancers. Here we discuss retinoid signaling in hematopoiesis, leukemogenesis, and APL treatment. We highlight autophagy as a potential important regulator in anti-leukemic strategies.

  7. Dissection of Signaling Events Downstream of the c-Mpl Receptor in Murine Hematopoietic Stem Cells Via Motif-Engineered Chimeric Receptors.

    Science.gov (United States)

    Saka, Koichiro; Lai, Chen-Yi; Nojima, Masanori; Kawahara, Masahiro; Otsu, Makoto; Nakauchi, Hiromitsu; Nagamune, Teruyuki

    2018-02-01

    Hematopoietic stem cells (HSCs) are a valuable resource in transplantation medicine. Cytokines are often used to culture HSCs aiming at better clinical outcomes through enhancement of HSC reconstitution capability. Roles for each signal molecule downstream of receptors in HSCs, however, remain puzzling due to complexity of the cytokine-signaling network. Engineered receptors that are non-responsive to endogenous cytokines represent an attractive tool for dissection of signaling events. We here tested a previously developed chimeric receptor (CR) system in primary murine HSCs, target cells that are indispensable for analysis of stem cell activity. Each CR contains tyrosine motifs that enable selective activation of signal molecules located downstream of the c-Mpl receptor upon stimulation by an artificial ligand. Signaling through a control CR with a wild-type c-Mpl cytoplasmic tail sufficed to enhance HSC proliferation and colony formation in cooperation with stem cell factor (SCF). Among a series of CRs, only one compatible with selective Stat5 activation showed similar positive effects. The HSCs maintained ex vivo in these environments retained long-term reconstitution ability following transplantation. This ability was also demonstrated in secondary recipients, indicating effective transmission of stem cell-supportive signals into HSCs via these artificial CRs during culture. Selective activation of Stat5 through CR ex vivo favored preservation of lymphoid potential in long-term reconstituting HSCs, but not of myeloid potential, exemplifying possible dissection of signals downstream of c-Mpl. These CR systems therefore offer a useful tool to scrutinize complex signaling pathways in HSCs.

  8. Tyrosine 769 of the keratinocyte growth factor receptor is required for receptor signaling but not endocytosis

    International Nuclear Information System (INIS)

    Ceridono, Mara; Belleudi, Francesca; Ceccarelli, Simona; Torrisi, Maria Rosaria

    2005-01-01

    Keratinocyte growth factor receptor (KGFR) is a receptor tyrosine kinase expressed on epithelial cells which belongs to the family of fibroblast growth factor receptors (FGFRs). Following ligand binding, KGFR is rapidly autophosphorylated on specific tyrosine residues in the intracellular domain, recruits substrate proteins, and is rapidly internalized by clathrin-mediated endocytosis. The role of different autophosphorylation sites in FGFRs, and in particular the role of the tyrosine 766 in FGFR1, first identified as PLCγ binding site, has been extensively studied. We analyzed here the possible role of the tyrosine 769 in KGFR, corresponding to tyrosine 766 in FGFR1, in the regulation of KGFR signal transduction and MAPK activation as well as in the control of the endocytic process of KGFR. A mutant KGFR in which tyrosine 769 was substituted by phenylalanine was generated and transfected in NIH3T3 and HeLa cells. Our results indicate that tyrosine 769 is required for the binding to KGFR and tyrosine phosphorylation of PLCγ as well as for the full activation of MAPKs and for cell proliferation through the regulation of FRS2 tyrosine phosphorylation, suggesting that this residue represents a key regulator of KGFR signal transduction. Our data also show that tyrosine 769 is not involved in the regulation of the endocytic process of KGFR

  9. PSM/SH2-B distributes selected mitogenic receptor signals to distinct components in the PI3-kinase and MAP kinase signaling pathways.

    Science.gov (United States)

    Deng, Youping; Xu, Hu; Riedel, Heimo

    2007-02-15

    The Pro-rich, PH, and SH2 domain containing mitogenic signaling adapter PSM/SH2-B has been implicated as a cellular partner of various mitogenic receptor tyrosine kinases and related signaling mechanisms. Here, we report in a direct comparison of three peptide hormones, that PSM participates in the assembly of distinct mitogenic signaling complexes in response to insulin or IGF-I when compared to PDGF in cultured normal fibroblasts. The complex formed in response to insulin or IGF-I involves the respective peptide hormone receptor and presumably the established components leading to MAP kinase activation. However, our data suggest an alternative link from the PDGF receptor via PSM directly to MEK1/2 and consequently also to p44/42 activation, possibly through a scaffold protein. At least two PSM domains participate, the SH2 domain anticipated to link PSM to the respective receptor and the Pro-rich region in an association with an unidentified downstream component resulting in direct MEK1/2 and p44/42 regulation. The PDGF receptor signaling complex formed in response to PDGF involves PI 3-kinase in addition to the same components and interactions as described for insulin or IGF-I. PSM associates with PI 3-kinase via p85 and in addition the PSM PH domain participates in the regulation of PI 3-kinase activity, presumably through membrane interaction. In contrast, the PSM Pro-rich region appears to participate only in the MAP kinase signal. Both pathways contribute to the mitogenic response as shown by cell proliferation, survival, and focus formation. PSM regulates p38 MAP kinase activity in a pathway unrelated to the mitogenic response.

  10. Crystallization and preliminary crystallographic analysis of the measles virus hemagglutinin in complex with the CD46 receptor

    International Nuclear Information System (INIS)

    Santiago, César; Gutiérrez-Rodríguez, Angel; Tucker, Paul A.; Stehle, Thilo; Casasnovas, José M.

    2009-01-01

    A complex of the measles virus hemagglutinin and the CD46 receptor representing the initial step of the cell infection has been crystallized. The measles virus (MV) hemagglutinin (MV-H) mediates the attachment of MV particles to cell-surface receptors for entry into host cells. MV uses two receptors for attachment to host cells: the complement-control protein CD46 and the signalling lymphocyte activation molecule (SLAM). The MV-H glycoprotein from an Edmonston MV variant and the MV-binding fragment of the CD46 receptor were overproduced in mammalian cells and used to crystallize an MV-H–CD46 complex. Well diffracting crystals containing two complexes in the asymmetric unit were obtained and the structure of the complex was solved by the molecular-replacement method

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

    Science.gov (United States)

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

    2014-11-28

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

  12. The transcriptomics of glucocorticoid receptor signaling in developing zebrafish.

    Directory of Open Access Journals (Sweden)

    Dinushan Nesan

    Full Text Available Cortisol is the primary corticosteroid in teleosts that is released in response to stressor activation of the hypothalamus-pituitary-interrenal axis. The target tissue action of this hormone is primarily mediated by the intracellular glucocorticoid receptor (GR, a ligand-bound transcription factor. In developing zebrafish (Danio rerio embryos, GR transcripts and cortisol are maternally deposited into the oocyte prior to fertilization and influence early embryogenesis. To better understand of the molecular mechanisms involved, we investigated changes in the developmental transcriptome prior to hatch, in response to morpholino oligonucleotide knockdown of GR using the Agilent zebrafish microarray platform. A total of 1313 and 836 mRNA transcripts were significantly changed at 24 and 36 hours post fertilization (hpf, respectively. Functional analysis revealed numerous developmental processes under GR regulation, including neurogenesis, eye development, skeletal and cardiac muscle formation. Together, this study underscores a critical role for glucocorticoid signaling in programming molecular events essential for zebrafish development.

  13. Current Views of Toll-Like Receptor Signaling Pathways

    Directory of Open Access Journals (Sweden)

    Masahiro Yamamoto

    2010-01-01

    Full Text Available On microbial invasion, the host immediately evokes innate immune responses. Recent studies have demonstrated that Toll-like receptors (TLRs play crucial roles in innate responses that lead not only to the clearance of pathogens but also to the efficient establishment of acquired immunity by directly detecting molecules from microbes. In terms of intracellular TLR-mediated signaling pathways, cytoplasmic adaptor molecules containing Toll/IL-1R (TIR domains play important roles in inflammatory immune responses through the production of proinflammatory cytokines, nitric oxide, and type I interferon, and upregulation of costimulatory molecules. In this paper, we will describe our current understanding of the relationship between TLRs and their ligands derived from pathogens such as viruses, bacteria, fungi, and parasites. Moreover, we will review the historical and current literature to describe the mechanisms behind TLR-mediated activation of innate immune responses.

  14. Progesterone receptors (PR) mediate STAT actions: PR and prolactin receptor signaling crosstalk in breast cancer models.

    Science.gov (United States)

    Leehy, Katherine A; Truong, Thu H; Mauro, Laura J; Lange, Carol A

    2018-02-01

    Estrogen is the major mitogenic stimulus of mammary gland development during puberty wherein ER signaling acts to induce abundant PR expression. PR signaling, in contrast, is the primary driver of mammary epithelial cell proliferation in adulthood. The high circulating levels of progesterone during pregnancy signal through PR, inducing expression of the prolactin receptor (PRLR). Cooperation between PR and prolactin (PRL) signaling, via regulation of downstream components in the PRL signaling pathway including JAKs and STATs, facilitates the alveolar morphogenesis observed during pregnancy. Indeed, these pathways are fully integrated via activation of shared signaling pathways (i.e. JAKs, MAPKs) as well as by the convergence of PRs and STATs at target genes relevant to both mammary gland biology and breast cancer progression (i.e. proliferation, stem cell outgrowth, tissue cell type heterogeneity). Thus, rather than a single mediator such as ER, transcription factor cascades (ER>PR>STATs) are responsible for rapid proliferative and developmental programming in the normal mammary gland. It is not surprising that these same mediators typify uncontrolled proliferation in a majority of breast cancers, where ER and PR are most often co-expressed and may cooperate to drive malignant tumor progression. This review will primarily focus on the integration of PR and PRL signaling in breast cancer models and the importance of this cross-talk in cancer progression in the context of mammographic density. Components of these PR/PRL signaling pathways could offer alternative drug targets and logical complements to anti-ER or anti-estrogen-based endocrine therapies. Copyright © 2017 Elsevier Ltd. All rights reserved.

  15. B cell antigen receptor signaling and internalization are mutually exclusive events.

    Directory of Open Access Journals (Sweden)

    Ping Hou

    2006-07-01

    Full Text Available Engagement of the B cell antigen receptor initiates two concurrent processes, signaling and receptor internalization. While both are required for normal humoral immune responses, the relationship between these two processes is unknown. Herein, we demonstrate that following receptor ligation, a small subpopulation of B cell antigen receptors are inductively phosphorylated and selectively retained at the cell surface where they can serve as scaffolds for the assembly of signaling molecules. In contrast, the larger population of non-phosphorylated receptors is rapidly endocytosed. Each receptor can undergo only one of two mutually exclusive fates because the tyrosine-based motifs that mediate signaling when phosphorylated mediate internalization when not phosphorylated. Mathematical modeling indicates that the observed competition between receptor phosphorylation and internalization enhances signaling responses to low avidity ligands.

  16. Crystal structure of NL63 respiratory coronavirus receptor-binding domain complexed with its human receptor

    Energy Technology Data Exchange (ETDEWEB)

    Wu, Kailang; Li, Weikai; Peng, Guiqing; Li, Fang; (Harvard-Med); (UMM-MED)

    2010-03-04

    NL63 coronavirus (NL63-CoV), a prevalent human respiratory virus, is the only group I coronavirus known to use angiotensin-converting enzyme 2 (ACE2) as its receptor. Incidentally, ACE2 is also used by group II SARS coronavirus (SARS-CoV). We investigated how different groups of coronaviruses recognize the same receptor, whereas homologous group I coronaviruses recognize different receptors. We determined the crystal structure of NL63-CoV spike protein receptor-binding domain (RBD) complexed with human ACE2. NL63-CoV RBD has a novel {beta}-sandwich core structure consisting of 2 layers of {beta}-sheets, presenting 3 discontinuous receptor-binding motifs (RBMs) to bind ACE2. NL63-CoV and SARS-CoV have no structural homology in RBD cores or RBMs; yet the 2 viruses recognize common ACE2 regions, largely because of a 'virus-binding hotspot' on ACE2. Among group I coronaviruses, RBD cores are conserved but RBMs are variable, explaining how these viruses recognize different receptors. These results provide a structural basis for understanding viral evolution and virus-receptor interactions.

  17. Adiponectin receptors form homomers and heteromers exhibiting distinct ligand binding and intracellular signaling properties.

    Science.gov (United States)

    Almabouada, Farid; Diaz-Ruiz, Alberto; Rabanal-Ruiz, Yoana; Peinado, Juan R; Vazquez-Martinez, Rafael; Malagon, Maria M

    2013-02-01

    Adiponectin binds to two widely expressed receptors (AdipoR1 and AdipoR2) that contain seven transmembrane domains but, unlike G-protein coupled receptors, present an extracellular C terminus and a cytosolic N terminus. Recently, AdipoR1 was found to associate in high order complexes. However, it is still unknown whether AdipoR2 may also form homomers or heteromers with AdipoR1 or if such interactions may be functionally relevant. Herein, we have analyzed the oligomerization pattern of AdipoRs by FRET and immunoprecipitation and evaluated both the internalization of AdipoRs in response to various adiponectin isoforms and the effect of adiponectin binding to different AdipoR combinations on AMP-activated protein kinase phosphorylation and peroxisome proliferator-activated receptor α activation. Transfection of HEK293AD cells with AdipoR1 and AdipoR2 showed that both receptors colocalize at both the plasma membrane and the endoplasmic reticulum. Co-transfection with the different AdipoR pairs yielded high FRET efficiencies in non-stimulated cells, which indicates that AdipoR1 and AdipoR2 form homo- and heteromeric complexes under resting conditions. Live FRET imaging suggested that both homo- and heteromeric AdipoR complexes dissociate in response to adiponectin, but heteromers separate faster than homomers. Finally, phosphorylation of AMP-activated protein kinase in response to adiponectin was delayed in cells wherein heteromer formation was favored. In sum, our findings indicate that AdipoR1 and AdipoR2 form homo- and heteromers that present unique interaction behaviors and signaling properties. This raises the possibility that the pleiotropic, tissue-dependent functions of adiponectin depend on the expression levels of AdipoR1 and AdipoR2 and, therefore, on the steady-state proportion of homo- and heteromeric complexes.

  18. Fractal Complexity-Based Feature Extraction Algorithm of Communication Signals

    Science.gov (United States)

    Wang, Hui; Li, Jingchao; Guo, Lili; Dou, Zheng; Lin, Yun; Zhou, Ruolin

    How to analyze and identify the characteristics of radiation sources and estimate the threat level by means of detecting, intercepting and locating has been the central issue of electronic support in the electronic warfare, and communication signal recognition is one of the key points to solve this issue. Aiming at accurately extracting the individual characteristics of the radiation source for the increasingly complex communication electromagnetic environment, a novel feature extraction algorithm for individual characteristics of the communication radiation source based on the fractal complexity of the signal is proposed. According to the complexity of the received signal and the situation of environmental noise, use the fractal dimension characteristics of different complexity to depict the subtle characteristics of the signal to establish the characteristic database, and then identify different broadcasting station by gray relation theory system. The simulation results demonstrate that the algorithm can achieve recognition rate of 94% even in the environment with SNR of -10dB, and this provides an important theoretical basis for the accurate identification of the subtle features of the signal at low SNR in the field of information confrontation.

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

    DEFF Research Database (Denmark)

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

    2018-01-01

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

  20. Importance of tyrosine phosphorylation in receptor kinase complexes.

    Science.gov (United States)

    Macho, Alberto P; Lozano-Durán, Rosa; Zipfel, Cyril

    2015-05-01

    Tyrosine phosphorylation is an important post-translational modification that is known to regulate receptor kinase (RK)-mediated signaling in animals. Plant RKs are annotated as serine/threonine kinases, but recent work has revealed that tyrosine phosphorylation is also crucial for the activation of RK-mediated signaling in plants. These initial observations have paved the way for subsequent detailed studies on the mechanism of activation of plant RKs and the biological relevance of tyrosine phosphorylation for plant growth and immunity. In this Opinion article we review recent reports on the contribution of RK tyrosine phosphorylation in plant growth and immunity; we propose that tyrosine phosphorylation plays a major regulatory role in the initiation and transduction of RK-mediated signaling in plants. Copyright © 2015 Elsevier Ltd. All rights reserved.

  1. Signaling and Adaptation Modulate the Dynamics of the Photosensoric Complex of Natronomonas pharaonis.

    Directory of Open Access Journals (Sweden)

    Philipp S Orekhov

    2015-10-01

    Full Text Available Motile bacteria and archaea respond to chemical and physical stimuli seeking optimal conditions for survival. To this end transmembrane chemo- and photoreceptors organized in large arrays initiate signaling cascades and ultimately regulate the rotation of flagellar motors. To unravel the molecular mechanism of signaling in an archaeal phototaxis complex we performed coarse-grained molecular dynamics simulations of a trimer of receptor/transducer dimers, namely NpSRII/NpHtrII from Natronomonas pharaonis. Signaling is regulated by a reversible methylation mechanism called adaptation, which also influences the level of basal receptor activation. Mimicking two extreme methylation states in our simulations we found conformational changes for the transmembrane region of NpSRII/NpHtrII which resemble experimentally observed light-induced changes. Further downstream in the cytoplasmic domain of the transducer the signal propagates via distinct changes in the dynamics of HAMP1, HAMP2, the adaptation domain and the binding region for the kinase CheA, where conformational rearrangements were found to be subtle. Overall these observations suggest a signaling mechanism based on dynamic allostery resembling models previously proposed for E. coli chemoreceptors, indicating similar properties of signal transduction for archaeal photoreceptors and bacterial chemoreceptors.

  2. The plant natriuretic peptide receptor is a guanylyl cyclase and enables cGMP-dependent signaling

    KAUST Repository

    Turek, Ilona

    2016-03-05

    The functional homologues of vertebrate natriuretic peptides (NPs), the plant natriuretic peptides (PNPs), are a novel class of peptidic hormones that signal via guanosine 3′,5′-cyclic monophosphate (cGMP) and systemically affect plant salt and water balance and responses to biotrophic plant pathogens. Although there is increasing understanding of the complex roles of PNPs in plant responses at the systems level, little is known about the underlying signaling mechanisms. Here we report isolation and identification of a novel Leucine-Rich Repeat (LRR) protein that directly interacts with A. thaliana PNP, AtPNP-A. In vitro binding studies revealed that the Arabidopsis AtPNP-A binds specifically to the LRR protein, termed AtPNP-R1, and the active region of AtPNP-A is sufficient for the interaction to occur. Importantly, the cytosolic part of the AtPNP-R1, much like in some vertebrate NP receptors, harbors a catalytic center diagnostic for guanylyl cyclases and the recombinant AtPNP-R1 is capable of catalyzing the conversion of guanosine triphosphate to cGMP. In addition, we show that AtPNP-A causes rapid increases of cGMP levels in wild type (WT) leaf tissue while this response is significantly reduced in the atpnp-r1 mutants. AtPNP-A also causes cGMP-dependent net water uptake into WT protoplasts, and hence volume increases, whereas responses of the protoplasts from the receptor mutant are impaired. Taken together, our results suggest that the identified LRR protein is an AtPNP-A receptor essential for the PNP-dependent regulation of ion and water homeostasis in plants and that PNP- and vertebrate NP-receptors and their signaling mechanisms share surprising similarities. © 2016 Springer Science+Business Media Dordrecht

  3. β1-adrenergic receptor stimulation by agonist Compound 49b restores insulin receptor signal transduction in vivo

    Science.gov (United States)

    Jiang, Youde; Zhang, Qiuhua; Ye, Eun-Ah

    2014-01-01

    Purpose Determine whether Compound 49b treatment ameliorates retinal changes due to the lack of β2-adrenergic receptor signaling. Methods Using retinas from 3-month-old β2-adrenergic receptor-deficient mice, we treated mice with our novel β1-/β2-adrenergic receptor agonist, Compound 49b, to assess the effects of adrenergic agonists acting only on β1-adrenergic receptors due to the absence of β2-adrenergic receptors. Western blotting or enzyme-linked immunosorbent assay (ELISA) analyses were performed for β1- and β2-adrenergic receptors, as well as key insulin resistance proteins, including TNF-α, SOCS3, IRS-1Ser307, and IRTyr960. Analyses were also performed on key anti- and proapoptotic proteins: Akt, Bcl-xL, Bax, and caspase 3. Electroretinogram analyses were conducted to assess functional changes, while histological assessment was conducted for changes in retinal thickness. Results A 2-month treatment of β2-adrenergic receptor-deficient mice with daily eye drops of 1 mM Compound 49b, a novel β1- and β2-adrenergic receptor agonist, reversed the changes in insulin resistance markers (TNF-α and SOCS3) observed in untreated β2-adrenergic receptor-deficient mice, and concomitantly increased morphological integrity (retinal thickness) and functional responses (electroretinogram amplitude). These results suggest that stimulating β1-adrenergic receptors on retinal endothelial cells or Müller cells can compensate for the loss of β2-adrenergic receptor signaling on Müller cells, restore insulin signal transduction, reduce retinal apoptosis, and enhance retinal function. Conclusions Since our previous studies with β1-adrenergic receptor knockout mice confirmed that the reverse also occurs (β2-adrenergic receptor stimulation can compensate for the loss of β1-adrenergic receptor activity), it appears that increased activity in either of these pathways alone is sufficient to block insulin resistance–based retinal cell apoptosis. PMID:24966659

  4. Nonlinear complexity analysis of brain FMRI signals in schizophrenia.

    Directory of Open Access Journals (Sweden)

    Moses O Sokunbi

    Full Text Available We investigated the differences in brain fMRI signal complexity in patients with schizophrenia while performing the Cyberball social exclusion task, using measures of Sample entropy and Hurst exponent (H. 13 patients meeting diagnostic and Statistical Manual of Mental Disorders, 4th Edition (DSM IV criteria for schizophrenia and 16 healthy controls underwent fMRI scanning at 1.5 T. The fMRI data of both groups of participants were pre-processed, the entropy characterized and the Hurst exponent extracted. Whole brain entropy and H maps of the groups were generated and analysed. The results after adjusting for age and sex differences together show that patients with schizophrenia exhibited higher complexity than healthy controls, at mean whole brain and regional levels. Also, both Sample entropy and Hurst exponent agree that patients with schizophrenia have more complex fMRI signals than healthy controls. These results suggest that schizophrenia is associated with more complex signal patterns when compared to healthy controls, supporting the increase in complexity hypothesis, where system complexity increases with age or disease, and also consistent with the notion that schizophrenia is characterised by a dysregulation of the nonlinear dynamics of underlying neuronal systems.

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

    DEFF Research Database (Denmark)

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

    2016-01-01

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

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

    NARCIS (Netherlands)

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

    2008-01-01

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

  7. ZINC-INDUCED EGF RECEPTOR SIGNALING REQUIRES SRC-MEDIATED PHOSPHORYLATION OF THE EGF RECEPTOR ON TYROSINE 845 (Y845)

    Science.gov (United States)

    ZINC-INDUCED EGF RECEPTOR SIGNALING REQUIRES Src-MEDIATED PHOSPHORYLATION OF THE EGF RECEPTOR ON TYROSINE 845 (Y845)Weidong Wu1, Lee M. Graves2, Gordon N. Gill3 and James M. Samet4 1Center for Environmental Medicine and Lung Biology; 2Department of Pharmacology, University o...

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

    Science.gov (United States)

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

    2016-10-14

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

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

    Science.gov (United States)

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

    2015-01-01

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

  10. NMDA receptors mediate neuron-to-glia signaling in mouse cortical astrocytes.

    Science.gov (United States)

    Lalo, Ulyana; Pankratov, Yuri; Kirchhoff, Frank; North, R Alan; Verkhratsky, Alexei

    2006-03-08

    Chemical transmission between neurons and glial cells is an important element of integration in the CNS. Here, we describe currents activated by NMDA in cortical astrocytes, identified in transgenic mice that express enhanced green fluorescent protein under control of the human glial fibrillary acidic protein promoter. Astrocytes were studied by whole-cell voltage clamp either in slices or after gentle nonenzymatic mechanical dissociation. Acutely isolated astrocytes showed a three-component response to glutamate. The initial rapid component was blocked by 1,2,3,4-tetrahydro-6-nitro-2,3-dioxo-benzo[f]quinoxaline-7-sulfonamide (NBQX), which is an antagonist of AMPA receptors (IC50, 2 microM), and the NMDA receptor antagonist D-AP-5 blocked the later sustained component (IC50, 0.6 microM). The third component of glutamate application response was sensitive to D,L-threo-beta-benzyloxyaspartate, a glutamate transporter blocker. Fast application of NMDA evoked concentration-dependent inward currents (EC50, 0.3 microM); these showed use-dependent block by (+)-5-methyl-10,11-dihydro-5H-dibenzo [a,d] cyclohepten-5,10-imine maleate (MK-801). These NMDA-evoked currents were linearly dependent on membrane potential and were not affected by extracellular magnesium at concentrations up to 10 mM. Electrical stimulation of axons in layer IV-VI induced a complex inward current in astrocytes situated in the cortical layer II, part of which was sensitive to MK-801 at holding potential -80 mV and was not affected by the AMPA glutamate receptor antagonist NBQX. The fast miniature spontaneous currents were observed in cortical astrocytes in slices as well. These currents exhibited both AMPA and NMDA receptor-mediated components. We conclude that cortical astrocytes express functional NMDA receptors that are devoid of Mg2+ block, and these receptors are involved in neuronal-glial signal transmission.

  11. Aliasing in the Complex Cepstrum of Linear-Phase Signals

    DEFF Research Database (Denmark)

    Bysted, Tommy Kristensen

    1997-01-01

    Assuming linear-phase of the associated time signal, this paper presents an approximated analytical description of the unavoidable aliasing in practical use of complex cepstrums. The linear-phase assumption covers two major applications of complex cepstrums which are linear- to minimum-phase FIR......-filter transformation and minimum-phase estimation from amplitude specifications. The description is made in the cepstrum domain, the Fourier transform of the complex cepstrum and in the frequency domain. Two examples are given, one for verification of the derived equations and one using the description to reduce...... aliasing in minimum-phase estimation...

  12. Macroscopic law of conservation revealed in the population dynamics of Toll-like receptor signaling

    Directory of Open Access Journals (Sweden)

    Selvarajoo Kumar

    2011-04-01

    Full Text Available Abstract Stimulating the receptors of a single cell generates stochastic intracellular signaling. The fluctuating response has been attributed to the low abundance of signaling molecules and the spatio-temporal effects of diffusion and crowding. At population level, however, cells are able to execute well-defined deterministic biological processes such as growth, division, differentiation and immune response. These data reflect biology as a system possessing microscopic and macroscopic dynamics. This commentary discusses the average population response of the Toll-like receptor (TLR 3 and 4 signaling. Without requiring detailed experimental data, linear response equations together with the fundamental law of information conservation have been used to decipher novel network features such as unknown intermediates, processes and cross-talk mechanisms. For single cell response, however, such simplicity seems far from reality. Thus, as observed in any other complex systems, biology can be considered to possess order and disorder, inheriting a mixture of predictable population level and unpredictable single cell outcomes.

  13. 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

    The glucagon-like peptide-1 incretin receptor (GLP-1R) of family B G protein-coupled receptors (GPCRs) is a major drug target in type-2-diabetes due to its regulatory effect on post-prandial blood-glucose levels. The mechanism(s) controlling GLP-1R mediated signaling are far from fully understood....... 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...

  14. A functional TOC complex contributes to gravity signal transduction in Arabidopsis.

    Science.gov (United States)

    Strohm, Allison K; Barrett-Wilt, Greg A; Masson, Patrick H

    2014-01-01

    Although plastid sedimentation has long been recognized as important for a plant's perception of gravity, it was recently shown that plastids play an additional function in gravitropism. The Translocon at the Outer envelope membrane of Chloroplasts (TOC) complex transports nuclear-encoded proteins into plastids, and a receptor of this complex, Toc132, was previously hypothesized to contribute to gravitropism either by directly functioning as a gravity signal transducer or by indirectly mediating the plastid localization of a gravity signal transducer. Here we show that mutations in multiple genes encoding TOC complex components affect gravitropism in a genetically sensitized background and that the cytoplasmic acidic domain of Toc132 is not required for its involvement in this process. Furthermore, mutations in TOC132 enhance the gravitropic defect of a mutant whose amyloplasts lack starch. Finally, we show that the levels of several nuclear-encoded root proteins are altered in toc132 mutants. These data suggest that the TOC complex indirectly mediates gravity signal transduction in Arabidopsis and support the idea that plastids are involved in gravitropism not only through their ability to sediment but also as part of the signal transduction mechanism.

  15. Gold nanoparticle–choline complexes can block nicotinic acetylcholine receptors

    Directory of Open Access Journals (Sweden)

    Chur Chin

    2010-04-01

    Full Text Available Chur Chin1, In Kyeom Kim2, Dong Yoon Lim3, Ki Suk Kim4, Hyang Ae Lee4, Eun Joo Kim41Department of Pediatrics, Fatima Hospital, Daegu, Korea; 2Department of Pharmacology, School of Medicine, Kyungpook National University, Daegu, Korea; 3Department of Pharmacology, School of Medicine, Chosun University, Gwangju, Korea; 4Korea Institute of Toxicology, Daejeon, KoreaAbstract: We identified a novel class of direct ion-channel blockers of ligand-gated ion channels called the gold nanoparticle–choline complex. Negatively charged gold nanoparticles (1.4 nm block ion pores by binding to the sulfur group of the cysteine loop of nicotinic acetylcholine receptors (nAChRs, and currents evoked by acetylcholine (Ach can break these bonds. The current evoked by ACh in nAChRs was blocked directly in ion pores by the gold nanoparticle–choline complex. In adrenal-gland perfusion studies, the complex also blocked nAChRs by diminishing catecholamine release by about 75%. An in vivo study showed muscle relaxation in rats after injection of the complex. These results will foster the application of gold nanoparticles as a direct ion-channel blocker. Keywords: negatively charged gold nanoparticle, choline, gold–sulfur bond, nicotinic acetylcholine receptor, direct ion-channel blocker

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

    Directory of Open Access Journals (Sweden)

    M. A. Prado

    2001-01-01

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

  17. Agmatine produces antidepressant-like effects by activating AMPA receptors and mTOR signaling.

    Science.gov (United States)

    Neis, Vivian Binder; Moretti, Morgana; Bettio, Luis Eduardo B; Ribeiro, Camille M; Rosa, Priscila Batista; Gonçalves, Filipe Marques; Lopes, Mark William; Leal, Rodrigo Bainy; Rodrigues, Ana Lúcia S

    2016-06-01

    The activation of AMPA receptors and mTOR signaling has been reported as mechanisms underlying the antidepressant effects of fast-acting agents, specially the NMDA receptor antagonist ketamine. In the present study, oral administration of agmatine (0.1mg/kg), a neuromodulator that has been reported to modulate NMDA receptors, caused a significant reduction in the immobility time of mice submitted to the tail suspension test (TST), an effect prevented by the administration of DNQX (AMPA receptor antagonist, 2.5μg/site, i.c.v.), BDNF antibody (1μg/site, i.c.v.), K-252a (TrkB receptor antagonist, 1μg/site, i.c.v.), LY294002 (PI3K inhibitor, 10nmol/site, i.c.v.) or rapamycin (selective mTOR inhibitor, 0.2nmol/site, i.c.v.). Moreover, the administration of lithium chloride (non-selective GSK-3β inhibitor, 10mg/kg, p.o.) or AR-A014418 (selective GSK-3β inhibitor, 0.01μg/site, i.c.v.) in combination with a sub-effective dose of agmatine (0.0001mg/kg, p.o.) reduced the immobility time in the TST when compared with either drug alone. Furthermore, increased immunocontents of BDNF, PSD-95 and GluA1 were found in the prefrontal cortex of mice just 1h after agmatine administration. These results indicate that the antidepressant-like effect of agmatine in the TST may be dependent on the activation of AMPA and TrkB receptors, PI3K and mTOR signaling as well as inhibition of GSK-3β, and increase in synaptic proteins. The results contribute to elucidate the complex signaling pathways involved in the antidepressant effect of agmatine and reinforce the pivotal role of these molecular targets for antidepressant responses. Copyright © 2016 Elsevier B.V. and ECNP. All rights reserved.

  18. Labor Inhibits Placental Mechanistic Target of Rapamycin Complex 1 Signaling

    Science.gov (United States)

    LAGER, Susanne; AYE, Irving L.M.H.; GACCIOLI, Francesca; RAMIREZ, Vanessa I.; JANSSON, Thomas; POWELL, Theresa L.

    2014-01-01

    Introduction Labor induces a myriad of changes in placental gene expression. These changes may represent a physiological adaptation inhibiting placental cellular processes associated with a high demand for oxygen and energy (e.g., protein synthesis and active transport) thereby promoting oxygen and glucose transfer to the fetus. We hypothesized that mechanistic target of rapamycin complex 1 (mTORC1) signaling, a positive regulator of trophoblast protein synthesis and amino acid transport, is inhibited by labor. Methods Placental tissue was collected from healthy, term pregnancies (n=15 no-labor; n=12 labor). Activation of Caspase-1, IRS1/Akt, STAT, mTOR, and inflammatory signaling pathways was determined by Western blot. NFκB p65 and PPARγ DNA binding activity was measured in isolated nuclei. Results Labor increased Caspase-1 activation and mTOR complex 2 signaling, as measured by phosphorylation of Akt (S473). However, mTORC1 signaling was inhibited in response to labor as evidenced by decreased phosphorylation of mTOR (S2448) and 4EBP1 (T37/46 and T70). Labor also decreased NFκB and PPARγ DNA binding activity, while having no effect on IRS1 or STAT signaling pathway. Discussion and conclusion Several placental signaling pathways are affected by labor, which has implications for experimental design in studies of placental signaling. Inhibition of placental mTORC1 signaling in response to labor may serve to down-regulate protein synthesis and amino acid transport, processes that account for a large share of placental oxygen and glucose consumption. We speculate that this response preserves glucose and oxygen for transfer to the fetus during the stressful events of labor. PMID:25454472

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

    Science.gov (United States)

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

    2017-01-01

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

  20. Comparison of GFL–GFRα complexes: further evidence relating GFL bend angle to RET signalling

    International Nuclear Information System (INIS)

    Parkash, Vimal; Goldman, Adrian

    2009-01-01

    The second crystal structure of the GDNF-GFRα1 complex provides further evidence that GFL signalling through RET is determined by the bend angle in the GFL. Glial cell line-derived neurotrophic factor (GDNF) activates the receptor tyrosine kinase RET by binding to the GDNF-family receptor α1 (GFRα1) and forming the GDNF 2 –GFRα1 2 –RET 2 heterohexamer complex. A previous crystal structure of the GDNF 2 –GFRα1 2 complex suggested that differences in signalling in GDNF-family ligand (GFL) complexes might arise from differences in the bend angle between the two monomers in the GFL homodimer. Here, a 2.35 Å resolution structure of the GDNF 2 –GFRα1 2 complex crystallized with new cell dimensions is reported. The structure was refined to a final R factor of 22.5% (R free = 28%). The structures of both biological tetrameric complexes in the asymmetric unit are very similar to 2v5e and different from the artemin–GFRα3 structure, even though there is a small change in the structure of the GDNF. By comparison of all known GDNF and artemin structures, it is concluded that GDNF is more bent and more flexible than artemin and that this may be related to RET signalling. Comparisons also suggest that the differences between artemin and GDNF arise from the increased curvature of the artemin ‘fingers’, which both increases the buried surface area in the monomer–monomer interface and changes the intermonomer bend angle. From sequence comparison, it is suggested that neuturin (the second GFL) adopts an artemin-like conformation, while persephin has a different conformation to the other three

  1. Rictor positively regulates B cell receptor signaling by modulating actin reorganization via ezrin.

    Directory of Open Access Journals (Sweden)

    Lu Huang

    2017-08-01

    Full Text Available As the central hub of the metabolism machinery, the mammalian target of rapamycin complex 2 (mTORC2 has been well studied in lymphocytes. As an obligatory component of mTORC2, the role of Rictor in T cells is well established. However, the role of Rictor in B cells still remains elusive. Rictor is involved in B cell development, especially the peripheral development. However, the role of Rictor on B cell receptor (BCR signaling as well as the underlying cellular and molecular mechanism is still unknown. This study used B cell-specfic Rictor knockout (KO mice to investigate how Rictor regulates BCR signaling. We found that the key positive and negative BCR signaling molecules, phosphorylated Brutons tyrosine kinase (pBtk and phosphorylated SH2-containing inositol phosphatase (pSHIP, are reduced and enhanced, respectively, in Rictor KO B cells. This suggests that Rictor positively regulates the early events of BCR signaling. We found that the cellular filamentous actin (F-actin is drastically increased in Rictor KO B cells after BCR stimulation through dysregulating the dephosphorylation of ezrin. The high actin-ezrin intensity area restricts the lateral movement of BCRs upon stimulation, consequently reducing BCR clustering and BCR signaling. The reduction in the initiation of BCR signaling caused by actin alteration is associated with a decreased humoral immune response in Rictor KO mice. The inhibition of actin polymerization with latrunculin in Rictor KO B cells rescues the defects of BCR signaling and B cell differentiation. Overall, our study provides a new pathway linking cell metablism to BCR activation, in which Rictor regulates BCR signaling via actin reorganization.

  2. Innate immune receptor Toll-like receptor 4 signalling in neuropsychiatric diseases.

    Science.gov (United States)

    García Bueno, B; Caso, J R; Madrigal, J L M; Leza, J C

    2016-05-01

    The innate immunity is a stereotyped first line of defense against pathogens and unspecified damage signals. One of main actors of innate immunity are the Toll-like receptors (TLRs), and one of the better characterized members of this family is TLR-4, that it is mainly activated by Gram-negative bacteria lipopolysaccharide. In brain, TLR-4 organizes innate immune responses against infections or cellular damage, but also possesses other physiological functions. In the last years, some evidences suggest a role of TLR-4 in stress and stress-related neuropsychiatric diseases. Peripheral and brain TLR-4 activation triggers sickness behavior, and its expression is a risk factor of depression. Some elements of the TLR-4 signaling pathway are up-regulated in peripheral samples and brain post-mortem tissue from depressed and suicidal patients. The "leaky gut" hypothesis of neuropsychiatric diseases is based on the existence of an increase of the intestinal permeability which results in bacterial translocation able to activate TLR-4. Enhanced peripheral TLR-4 expression/activity has been described in subjects diagnosed with schizophrenia, bipolar disorder and in autistic children. A role for TLR-4 in drugs abuse has been also proposed. The therapeutic potential of pharmacological/genetic modulation of TLRs signaling pathways in neuropsychiatry is promising, but a great preclinical/clinical scientific effort is still needed. Copyright © 2016 Elsevier Ltd. All rights reserved.

  3. SGIP1 alters internalization and modulates signaling of activated cannabinoid receptor 1 in a biased manner

    Czech Academy of Sciences Publication Activity Database

    Hájková, Alena; Techlovská, Šárka; Dvořáková, Michaela; Chambers, Jayne Nicole; Kumpošt, Jiří; Hubálková, Pavla; Prezeau, L.; Blahoš, Jaroslav

    2016-01-01

    Roč. 107, léto (2016), s. 201-214 ISSN 0028-3908 R&D Projects: GA ČR GAP303/12/2408 Institutional support: RVO:68378050 Keywords : Seven transmembrane receptors * G-protein coupled receptors * Cannabinoid receptor 1 * Protein-protein interactions * Bias signaling * Receptor endocytosis Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 5.012, year: 2016

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

    Energy Technology Data Exchange (ETDEWEB)

    Rasmussen, Søren G.F.; DeVree, Brian T; Zou, Yaozhong; Kruse, Andrew C; Chung, Ka Young; Kobilka, Tong Sun; Thian, Foon Sun; Chae, Pil Seok; Pardon, Els; Calinski, Diane; Mathiesen, Jesper M; Shah, Syed T.A.; Lyons, Joseph A; Caffrey, Martin; Gellman, Samuel H; Steyaert, Jan; Skiniotis, Georgios; Weis, William I; Sunahara, Roger K; Kobilka, Brian K [Brussels; (Trinity); (Michigan); (Stanford-MED); (Michigan-Med); (UW)

    2011-12-07

    G protein-coupled receptors (GPCRs) are responsible for the majority of cellular responses to hormones and neurotransmitters as well as the senses of sight, olfaction and taste. The paradigm of GPCR signalling is the activation of a heterotrimeric GTP binding protein (G protein) by an agonist-occupied receptor. The β2 adrenergic receptor2AR) activation of Gs, the stimulatory G protein for adenylyl cyclase, has long been a model system for GPCR signalling. Here we present the crystal structure of the active state ternary complex composed of agonist-occupied monomeric β2AR and nucleotide-free Gs heterotrimer. The principal interactions between the β2AR and Gs involve the amino- and carboxy-terminal α-helices of Gs, with conformational changes propagating to the nucleotide-binding pocket. The largest conformational changes in the β2AR include a 14Å outward movement at the cytoplasmic end of transmembrane segment 6 (TM6) and an α-helical extension of the cytoplasmic end of TM5. The most surprising observation is a major displacement of the α-helical domain of Gαs relative to the Ras-like GTPase domain. This crystal structure represents the first high-resolution view of transmembrane signalling by a GPCR.

  5. Quercetin suppresses insulin receptor signaling through inhibition of the insulin ligand–receptor binding and therefore impairs cancer cell proliferation

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Feng [Department of Gastroenterology, The Tenth People’s Hospital of Shanghai, Tongji University, Shanghai 200072 (China); Department of Nanomedicine, Houston Methodist Research Institute, Houston, TX 77030 (United States); Yang, Yong, E-mail: yyang@houstonmethodist.org [Department of Nanomedicine, Houston Methodist Research Institute, Houston, TX 77030 (United States); Department of Medicine, Weill Cornell Medical College, New York, NY 10065 (United States)

    2014-10-03

    Graphical abstract: - Highlights: • Quercetin inhibits insulin ligand–receptor interactions. • Quercetin reduces downstream insulin receptor signaling. • Quercetin blocks insulin induced glucose uptake. • Quercetin suppresses insulin stimulated cancer cell proliferation and tumor growth. - Abstract: Although the flavonoid quercetin is known to inhibit activation of insulin receptor signaling, the inhibitory mechanism is largely unknown. In this study, we demonstrate that quercetin suppresses insulin induced dimerization of the insulin receptor (IR) through interfering with ligand–receptor interactions, which reduces the phosphorylation of IR and Akt. This inhibitory effect further inhibits insulin stimulated glucose uptake due to decreased cell membrane translocation of glucose transporter 4 (GLUT4), resulting in impaired cancer cell proliferation. The effect of quercetin in inhibiting tumor growth was also evident in an in vivo model, indicating a potential future application for quercetin in the treatment of cancers.

  6. Quercetin suppresses insulin receptor signaling through inhibition of the insulin ligand–receptor binding and therefore impairs cancer cell proliferation

    International Nuclear Information System (INIS)

    Wang, Feng; Yang, Yong

    2014-01-01

    Graphical abstract: - Highlights: • Quercetin inhibits insulin ligand–receptor interactions. • Quercetin reduces downstream insulin receptor signaling. • Quercetin blocks insulin induced glucose uptake. • Quercetin suppresses insulin stimulated cancer cell proliferation and tumor growth. - Abstract: Although the flavonoid quercetin is known to inhibit activation of insulin receptor signaling, the inhibitory mechanism is largely unknown. In this study, we demonstrate that quercetin suppresses insulin induced dimerization of the insulin receptor (IR) through interfering with ligand–receptor interactions, which reduces the phosphorylation of IR and Akt. This inhibitory effect further inhibits insulin stimulated glucose uptake due to decreased cell membrane translocation of glucose transporter 4 (GLUT4), resulting in impaired cancer cell proliferation. The effect of quercetin in inhibiting tumor growth was also evident in an in vivo model, indicating a potential future application for quercetin in the treatment of cancers

  7. Molecular and biochemical analysis of symbiotic plant receptor kinase complexes

    Energy Technology Data Exchange (ETDEWEB)

    Cook, Douglas R; Riely, Brendan K

    2010-09-01

    DE-FG02-01ER15200 was a 36-month project, initiated on Sept 1, 2005 and extended with a one-year no cost extension to August 31, 2009. During the project period we published seven manuscripts (2 in review). Including the prior project period (2002-2005) we published 12 manuscripts in journals that include Science, PNAS, The Plant Cell, Plant Journal, Plant Physiology, and MPMI. The primary focus of this work was to further elucidate the function of the Nod factor signaling pathway that is involved in initiation of the legume-rhizobium symbiosis and in particular to explore the relationship between receptor kinase-like proteins and downstream effectors of symbiotic development. During the project period we have map-base cloned two additional players in symbiotic development, including an ERF transcription factor and an ethylene pathway gene (EIN2) that negatively regulates symbiotic signaling; we have also further characterized the subcellular distribution and function of a nuclear-localized symbiosis-specific ion channel, DMI1. The major outcome of the work has been the development of systems for exploring and validating protein-protein interactions that connect symbiotic receptor-like proteins to downstream responses. In this regard, we have developed both homologous (i.e., in planta) and heterologous (i.e., in yeast) systems to test protein interactions. Using yeast 2-hybrid screens we isolated the only known interactor of the nuclear-localized calcium-responsive kinase DMI3. We have also used yeast 2-hybrid methodology to identify interactions between symbiotic signaling proteins and certain RopGTPase/RopGEF proteins that regulate root hair polar growth. More important to the long-term goals of our work, we have established a TAP tagging system that identifies in planta interactions based on co-immuno precipitation and mass spectrometry. The validity of this approach has been shown using known interactors that either co-iummnoprecipate (i.e., remorin) or co

  8. Cell surface receptors for signal transduction and ligand transport: a design principles study.

    Directory of Open Access Journals (Sweden)

    Harish Shankaran

    2007-06-01

    Full Text Available Receptors constitute the interface of cells to their external environment. These molecules bind specific ligands involved in multiple processes, such as signal transduction and nutrient transport. Although a variety of cell surface receptors undergo endocytosis, the systems-level design principles that govern the evolution of receptor trafficking dynamics are far from fully understood. We have constructed a generalized mathematical model of receptor-ligand binding and internalization to understand how receptor internalization dynamics encodes receptor function and regulation. A given signaling or transport receptor system represents a particular implementation of this module with a specific set of kinetic parameters. Parametric analysis of the response of receptor systems to ligand inputs reveals that receptor systems can be characterized as being: i avidity-controlled where the response control depends primarily on the extracellular ligand capture efficiency, ii consumption-controlled where the ability to internalize surface-bound ligand is the primary control parameter, and iii dual-sensitivity where both the avidity and consumption parameters are important. We show that the transferrin and low-density lipoprotein receptors are avidity-controlled, the vitellogenin receptor is consumption-controlled, and the epidermal growth factor receptor is a dual-sensitivity receptor. Significantly, we show that ligand-induced endocytosis is a mechanism to enhance the accuracy of signaling receptors rather than merely serving to attenuate signaling. Our analysis reveals that the location of a receptor system in the avidity-consumption parameter space can be used to understand both its function and its regulation.

  9. The mediator complex in genomic and non-genomic signaling in cancer.

    Science.gov (United States)

    Weber, Hannah; Garabedian, Michael J

    2018-05-01

    Mediator is a conserved, multi-subunit macromolecular machine divided structurally into head, middle, and tail modules, along with a transiently associating kinase module. Mediator functions as an integrator of transcriptional regulatory activity by interacting with DNA-bound transcription factors and with RNA polymerase II (Pol II) to both activate and repress gene expression. Mediator has been shown to affect multiple steps in transcription, including chromatin looping between enhancers and promoters, pre-initiation complex formation, transcriptional elongation, and mRNA splicing. Individual Mediator subunits participate in regulation of gene expression by the estrogen and androgen receptors and are altered in a number of endocrine cancers, including breast and prostate cancer. In addition to its role in genomic signaling, MED12 has been implicated in non-genomic signaling by interacting with and activating TGF-beta receptor 2 in the cytoplasm. Recent structural studies have revealed extensive inter-domain interactions and complex architecture of the Mediator-Pol II complex, suggesting that Mediator is capable of reorganizing its conformation and composition to fit cellular needs. We propose that alterations in Mediator subunit expression that occur in various cancers could impact the organization and function of Mediator, resulting in changes in gene expression that promote malignancy. A better understanding of the role of Mediator in cancer could reveal new approaches to the diagnosis and treatment of Mediator-dependent endocrine cancers, especially in settings of therapy resistance. Copyright © 2017 Elsevier Inc. All rights reserved.

  10. Jasmonate signalling in Arabidopsis involves SGT1b-HSP70-HSP90 chaperone complexes.

    Science.gov (United States)

    Zhang, Xue-Cheng; Millet, Yves A; Cheng, Zhenyu; Bush, Jenifer; Ausubel, Frederick M

    Plant hormones play pivotal roles in growth, development and stress responses. Although it is essential to our understanding of hormone signalling, how plants maintain a steady state level of hormone receptors is poorly understood. We show that mutation of the Arabidopsis thaliana co-chaperone SGT1b impairs responses to the plant hormones jasmonate, auxin and gibberellic acid, but not brassinolide and abscisic acid, and that SGT1b and its homologue SGT1a are involved in maintaining the steady state levels of the F-box proteins COI1 and TIR1, receptors for jasmonate and auxin, respectively. The association of SGT1b with COI1 is direct and is independent of the Arabidopsis SKP1 protein, ASK1. We further show that COI1 is a client protein of SGT1b-HSP70-HSP90 chaperone complexes and that the complexes function in hormone signalling by stabilizing the COI1 protein. This study extends the SGT1b-HSP90 client protein list and broadens the functional scope of SGT1b-HSP70-HSP90 chaperone complexes.

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

    Directory of Open Access Journals (Sweden)

    Claire J Fenech

    2009-10-01

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

  12. Orexin A/Hypocretin Modulates Leptin Receptor-Mediated Signaling by Allosteric Modulations Mediated by the Ghrelin GHS-R1A Receptor in Hypothalamic Neurons.

    Science.gov (United States)

    Medrano, Mireia; Aguinaga, David; Reyes-Resina, Irene; Canela, Enric I; Mallol, Josefa; Navarro, Gemma; Franco, Rafael

    2018-06-01

    The hypothalamus is a key integrator of nutrient-seeking signals in the form of hormones and metabolites originated in both the central nervous system and the periphery. The main autocrine and paracrine target of orexinergic-related hormones such as leptin, orexin/hypocretin, and ghrelin are neuropeptide Y neurons located in the arcuate nucleus of the hypothalamus. The aim of this study was to investigate the expression and the molecular and functional relationships between leptin, orexin/hypocretin and ghrelin receptors. Biophysical studies in a heterologous system showed physical interactions between them, with potential formation of heterotrimeric complexes. Functional assays showed robust allosteric interactions particularly different when the three receptors are expressed together. Further biochemical and pharmacological assays provided evidence of heterotrimer functional expression in primary cultures of hypothalamic neurons. These findings constitute evidence of close relationships in the action of the three hormones already starting at the receptor level in hypothalamic cells.

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

    Science.gov (United States)

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

    2017-10-01

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

  14. Vitamin D receptor signaling and its therapeutic implications: Genome-wide and structural view.

    Science.gov (United States)

    Carlberg, Carsten; Molnár, Ferdinand

    2015-05-01

    Vitamin D3 is one of the few natural compounds that has, via its metabolite 1α,25-dihydroxyvitamin D3 (1,25(OH)2D3) and the transcription factor vitamin D receptor (VDR), a direct effect on gene regulation. For efficiently applying the therapeutic and disease-preventing potential of 1,25(OH)2D3 and its synthetic analogs, the key steps in vitamin D signaling need to be understood. These are the different types of molecular interactions with the VDR, such as (i) the complex formation of VDR with genomic DNA, (ii) the interaction of VDR with its partner transcription factors, (iii) the binding of 1,25(OH)2D3 or its synthetic analogs within the ligand-binding pocket of the VDR, and (iv) the resulting conformational change on the surface of the VDR leading to a change of the protein-protein interaction profile of the receptor with other proteins. This review will present the latest genome-wide insight into vitamin D signaling, and will discuss its therapeutic implications.

  15. Acute inhibition of selected membrane-proximal mouse T cell receptor signaling by mitochondrial antagonists.

    Directory of Open Access Journals (Sweden)

    Kwangmi Kim

    2009-11-01

    Full Text Available T cells absorb nanometric membrane vesicles, prepared from plasma membrane of antigen presenting cells, via dual receptor/ligand interactions of T cell receptor (TCR with cognate peptide/major histocompatibility complex (MHC plus lymphocyte function-associated antigen 1 (LFA-1 with intercellular adhesion molecule 1. TCR-mediated signaling for LFA-1 activation is also required for the vesicle absorption. Exploiting those findings, we had established a high throughput screening (HTS platform and screened a library for isolation of small molecules inhibiting the vesicle absorption. Follow-up studies confirmed that treatments (1 hour with various mitochondrial antagonists, including a class of anti-diabetic drugs (i.e., Metformin and Phenformin, resulted in ubiquitous inhibition of the vesicle absorption without compromising viability of T cells. Further studies revealed that the mitochondrial drug treatments caused impairment of specific membrane-proximal TCR signaling event(s. Thus, activation of Akt and PLC-gamma1 and entry of extracellular Ca(2+ following TCR stimulation were attenuated while polymerization of monomeric actins upon TCR triggering progressed normally after the treatments. Dynamic F-actin rearrangement concurring with the vesicle absorption was also found to be impaired by the drug treatments, implying that the inhibition by the drug treatments of downstream signaling events (and the vesicle absorption could result from lack of directional relocation of signaling and cell surface molecules. We also assessed the potential application of mitochondrial antagonists as immune modulators by probing effects of the long-term drug treatments (24 hours on viability of resting primary T cells and cell cycle progression of antigen-stimulated T cells. This study unveils a novel regulatory mechanism for T cell immunity in response to environmental factors having effects on mitochondrial function.

  16. Destabilization of strigolactone receptor DWARF14 by binding of ligand and E3-ligase signaling effector DWARF3

    Science.gov (United States)

    Zhao, Li-Hua; Zhou, X Edward; Yi, Wei; Wu, Zhongshan; Liu, Yue; Kang, Yanyong; Hou, Li; de Waal, Parker W; Li, Suling; Jiang, Yi; Scaffidi, Adrian; Flematti, Gavin R; Smith, Steven M; Lam, Vinh Q; Griffin, Patrick R; Wang, Yonghong; Li, Jiayang; Melcher, Karsten; Xu, H Eric

    2015-01-01

    Strigolactones (SLs) are endogenous hormones and exuded signaling molecules in plant responses to low levels of mineral nutrients. Key mediators of the SL signaling pathway in rice include the α/β-fold hydrolase DWARF 14 (D14) and the F-box component DWARF 3 (D3) of the ubiquitin ligase SCFD3 that mediate ligand-dependent degradation of downstream signaling repressors. One perplexing feature is that D14 not only functions as the SL receptor but is also an active enzyme that slowly hydrolyzes diverse natural and synthetic SLs including GR24, preventing the crystallization of a binary complex of D14 with an intact SL as well as the ternary D14/SL/D3 complex. Here we overcome these barriers to derive a structural model of D14 bound to intact GR24 and identify the interface that is required for GR24-mediated D14-D3 interaction. The mode of GR24-mediated signaling, including ligand recognition, hydrolysis by D14, and ligand-mediated D14-D3 interaction, is conserved in structurally diverse SLs. More importantly, D14 is destabilized upon the binding of ligands and D3, thus revealing an unusual mechanism of SL recognition and signaling, in which the hormone, the receptor, and the downstream effectors are systematically destabilized during the signal transduction process. PMID:26470846

  17. Med1 subunit of the mediator complex in nuclear receptor-regulated energy metabolism, liver regeneration, and hepatocarcinogenesis.

    Science.gov (United States)

    Jia, Yuzhi; Viswakarma, Navin; Reddy, Janardan K

    2014-01-01

    Several nuclear receptors regulate diverse metabolic functions that impact on critical biological processes, such as development, differentiation, cellular regeneration, and neoplastic conversion. In the liver, some members of the nuclear receptor family, such as peroxisome proliferator-activated receptors (PPARs), constitutive androstane receptor (CAR), farnesoid X receptor (FXR), liver X receptor (LXR), pregnane X receptor (PXR), glucocorticoid receptor (GR), and others, regulate energy homeostasis, the formation and excretion of bile acids, and detoxification of xenobiotics. Excess energy burning resulting from increases in fatty acid oxidation systems in liver generates reactive oxygen species, and the resulting oxidative damage influences liver regeneration and liver tumor development. These nuclear receptors are important sensors of exogenous activators as well as receptor-specific endogenous ligands. In this regard, gene knockout mouse models revealed that some lipid-metabolizing enzymes generate PPARα-activating ligands, while others such as ACOX1 (fatty acyl-CoA oxidase1) inactivate these endogenous PPARα activators. In the absence of ACOX1, the unmetabolized ACOX1 substrates cause sustained activation of PPARα, and the resulting increase in energy burning leads to hepatocarcinogenesis. Ligand-activated nuclear receptors recruit the multisubunit Mediator complex for RNA polymerase II-dependent gene transcription. Evidence indicates that the Med1 subunit of the Mediator is essential for PPARα, PPARγ, CAR, and GR signaling in liver. Med1 null hepatocytes fail to respond to PPARα activators in that these cells do not show induction of peroxisome proliferation and increases in fatty acid oxidation enzymes. Med1-deficient hepatocytes show no increase in cell proliferation and do not give rise to liver tumors. Identification of nuclear receptor-specific coactivators and Mediator subunits should further our understanding of the complexities of metabolic

  18. The Role of (BETA)-Catenin in Androgen Receptor Signaling

    National Research Council Canada - National Science Library

    Bhowmick, Neil A

    2006-01-01

    .... Our preliminary data seem indicate stromally derived paracrine Wnt family members activate theepithelial frizzled receptor to enable prostate epithelial survival in an androgen deficient environment...

  19. Spatio-temporal dependence of the signaling response in immune-receptor trafficking networks regulated by cell density: a theoretical model.

    Directory of Open Access Journals (Sweden)

    Pilar García-Peñarrubia

    Full Text Available Cell signaling processes involve receptor trafficking through highly connected networks of interacting components. The binding of surface receptors to their specific ligands is a key factor for the control and triggering of signaling pathways. In most experimental systems, ligand concentration and cell density vary within a wide range of values. Dependence of the signal response on cell density is related with the extracellular volume available per cell. This dependence has previously been studied using non-spatial models which assume that signaling components are well mixed and uniformly distributed in a single compartment. In this paper, a mathematical model that shows the influence exerted by cell density on the spatio-temporal evolution of ligands, cell surface receptors, and intracellular signaling molecules is developed. To this end, partial differential equations were used to model ligand and receptor trafficking dynamics through the different domains of the whole system. This enabled us to analyze several interesting features involved with these systems, namely: a how the perturbation caused by the signaling response propagates through the system; b receptor internalization dynamics and how cell density affects the robustness of dose-response curves upon variation of the binding affinity; and c that enhanced correlations between ligand input and system response are obtained under conditions that result in larger perturbations of the equilibrium ligand + surface receptor [Please see text] ligand - receptor complex. Finally, the results are compared with those obtained by considering that the above components are well mixed in a single compartment.

  20. SUMO-, MAPK- and resistance protein-signaling converge at transcription complexes that regulate plant innate immunity

    NARCIS (Netherlands)

    Burg, van den H.A.; Takken, F.L.W.

    2010-01-01

    Upon pathogen perception plant innate immune receptors activate various signaling pathways that trigger host defenses. PAMP-triggered defense signaling requires mitogen-activated protein kinase (MAPK) pathways, which modulate the activity of transcription factors through phosphorylation. Here, we

  1. SUMO-, MAPK-, and resistance protein-signaling converge at transcription complexes that regulate plant innate immunity

    NARCIS (Netherlands)

    van den Burg, H.A.; Takken, F.L.W.

    2010-01-01

    Upon pathogen perception plant innate immune receptors activate various signaling pathways that trigger host defenses. PAMP-triggered defense signaling requires mitogen-activated protein kinase (MAPK) pathways, which modulate the activity of transcription factors through phosphorylation. Here, we

  2. Simulation of Weak Signals of Nanotechnology Innovation in Complex System

    Directory of Open Access Journals (Sweden)

    Sun Hi Yoo

    2018-02-01

    Full Text Available It is especially indispensable for new businesses or industries to predict the innovation of new technologies. This requires an understanding of how the complex process of innovation, which is accomplished through more efficient products, processes, services, technologies, or ideas, is adopted and diffused in the market, government, and society. Furthermore, detecting “weak signals” (signs of change in science and technology (S&T is also important to foretell events associated with innovations in technology. Thus, we explore the dynamic behavior of weak signals of a specific technological innovation using the agent-based simulating tool NetLogo. This study provides a deeper understanding of the early stages of complex technology innovation, and the models are capable of analyzing initial complex interaction structures between components of technologies and between agents engaged in collective invention.

  3. Phenobarbital indirectly activates the constitutive active androstane receptor (CAR) by inhibition of epidermal growth factor receptor signaling.

    Science.gov (United States)

    Mutoh, Shingo; Sobhany, Mack; Moore, Rick; Perera, Lalith; Pedersen, Lee; Sueyoshi, Tatsuya; Negishi, Masahiko

    2013-05-07

    Phenobarbital is a central nervous system depressant that also indirectly activates nuclear receptor constitutive active androstane receptor (CAR), which promotes drug and energy metabolism, as well as cell growth (and death), in the liver. We found that phenobarbital activated CAR by inhibiting epidermal growth factor receptor (EGFR) signaling. Phenobarbital bound to EGFR and potently inhibited the binding of EGF, which prevented the activation of EGFR. This abrogation of EGFR signaling induced the dephosphorylation of receptor for activated C kinase 1 (RACK1) at Tyr(52), which then promoted the dephosphorylation of CAR at Thr(38) by the catalytic core subunit of protein phosphatase 2A. The findings demonstrated that the phenobarbital-induced mechanism of CAR dephosphorylation and activation is mediated through its direct interaction with and inhibition of EGFR.

  4. Testin, a novel binding partner of the calcium-sensing receptor, enhances receptor-mediated Rho-kinase signalling

    International Nuclear Information System (INIS)

    Magno, Aaron L.; Ingley, Evan; Brown, Suzanne J.; Conigrave, Arthur D.; Ratajczak, Thomas; Ward, Bryan K.

    2011-01-01

    Highlights: → A yeast two-hybrid screen revealed testin bound to the calcium-sensing receptor. → The second zinc finger of LIM domain 1 of testin is critical for interaction. → Testin bound to a region of the receptor tail important for cell signalling. → Testin and receptor interaction was confirmed in mammalian (HEK293) cells. → Overexpression of testin enhanced receptor-mediated Rho signalling in HEK293 cells. -- Abstract: The calcium-sensing receptor (CaR) plays an integral role in calcium homeostasis and the regulation of other cellular functions including cell proliferation and cytoskeletal organisation. The multifunctional nature of the CaR is manifested through ligand-dependent stimulation of different signalling pathways that are also regulated by partner binding proteins. Following a yeast two-hybrid library screen using the intracellular tail of the CaR as bait, we identified several novel binding partners including the focal adhesion protein, testin. Testin has not previously been shown to interact with cell surface receptors. The sites of interaction between the CaR and testin were mapped to the membrane proximal region of the receptor tail and the second zinc-finger of LIM domain 1 of testin, the integrity of which was found to be critical for the CaR-testin interaction. The CaR-testin association was confirmed in HEK293 cells by coimmunoprecipitation and confocal microscopy studies. Ectopic expression of testin in HEK293 cells stably expressing the CaR enhanced CaR-stimulated Rho activity but had no effect on CaR-stimulated ERK signalling. These results suggest an interplay between the CaR and testin in the regulation of CaR-mediated Rho signalling with possible effects on the cytoskeleton.

  5. A new inhibitor of the β-arrestin/AP2 endocytic complex reveals interplay between GPCR internalization and signalling

    Science.gov (United States)

    Beautrait, Alexandre; Paradis, Justine S.; Zimmerman, Brandon; Giubilaro, Jenna; Nikolajev, Ljiljana; Armando, Sylvain; Kobayashi, Hiroyuki; Yamani, Lama; Namkung, Yoon; Heydenreich, Franziska M.; Khoury, Etienne; Audet, Martin; Roux, Philippe P.; Veprintsev, Dmitry B.; Laporte, Stéphane A.; Bouvier, Michel

    2017-04-01

    In addition to G protein-coupled receptor (GPCR) desensitization and endocytosis, β-arrestin recruitment to ligand-stimulated GPCRs promotes non-canonical signalling cascades. Distinguishing the respective contributions of β-arrestin recruitment to the receptor and β-arrestin-promoted endocytosis in propagating receptor signalling has been limited by the lack of selective analytical tools. Here, using a combination of virtual screening and cell-based assays, we have identified a small molecule that selectively inhibits the interaction between β-arrestin and the β2-adaptin subunit of the clathrin adaptor protein AP2 without interfering with the formation of receptor/β-arrestin complexes. This selective β-arrestin/β2-adaptin inhibitor (Barbadin) blocks agonist-promoted endocytosis of the prototypical β2-adrenergic (β2AR), V2-vasopressin (V2R) and angiotensin-II type-1 (AT1R) receptors, but does not affect β-arrestin-independent (transferrin) or AP2-independent (endothelin-A) receptor internalization. Interestingly, Barbadin fully blocks V2R-stimulated ERK1/2 activation and blunts cAMP accumulation promoted by both V2R and β2AR, supporting the concept of β-arrestin/AP2-dependent signalling for both G protein-dependent and -independent pathways.

  6. The orphan receptor ALK7 and the Activin receptor ALK4 mediate signaling by Nodal proteins during vertebrate development

    Science.gov (United States)

    Reissmann, Eva; Jörnvall, Henrik; Blokzijl, Andries; Andersson, Olov; Chang, Chenbei; Minchiotti, Gabriella; Persico, M. Graziella; Ibáñez, Carlos F.; Brivanlou, Ali H.

    2001-01-01

    Nodal proteins have crucial roles in mesendoderm formation and left–right patterning during vertebrate development. The molecular mechanisms of signal transduction by Nodal and related ligands, however, are not fully understood. In this paper, we present biochemical and functional evidence that the orphan type I serine/threonine kinase receptor ALK7 acts as a receptor for mouse Nodal and Xenopus Nodal-related 1 (Xnr1). Receptor reconstitution experiments indicate that ALK7 collaborates with ActRIIB to confer responsiveness to Xnr1 and Nodal. Both receptors can independently bind Xnr1. In addition, Cripto, an extracellular protein genetically implicated in Nodal signaling, can independently interact with both Xnr1 and ALK7, and its expression greatly enhances the ability of ALK7 and ActRIIB to respond to Nodal ligands. The Activin receptor ALK4 is also able to mediate Nodal signaling but only in the presence of Cripto, with which it can also interact directly. A constitutively activated form of ALK7 mimics the mesendoderm-inducing activity of Xnr1 in Xenopus embryos, whereas a dominant-negative ALK7 specifically blocks the activities of Nodal and Xnr1 but has little effect on other related ligands. In contrast, a dominant-negative ALK4 blocks all mesoderm-inducing ligands tested, including Nodal, Xnr1, Xnr2, Xnr4, and Activin. In agreement with a role in Nodal signaling, ALK7 mRNA is localized to the ectodermal and organizer regions of Xenopus gastrula embryos and is expressed during early stages of mouse embryonic development. Therefore, our results indicate that both ALK4 and ALK7 can mediate signal transduction by Nodal proteins, although ALK7 appears to be a receptor more specifically dedicated to Nodal signaling. PMID:11485994

  7. Molecular and functional profiling of histamine receptor-mediated calcium ion signals in different cell lines.

    Science.gov (United States)

    Meisenberg, Annika; Kaschuba, Dagmar; Balfanz, Sabine; Jordan, Nadine; Baumann, Arnd

    2015-10-01

    Calcium ions (Ca(2+)) play a pivotal role in cellular physiology. Often Ca(2+)-dependent processes are studied in commonly available cell lines. To induce Ca(2+) signals on demand, cells may need to be equipped with additional proteins. A prominent group of membrane proteins evoking Ca(2+) signals are G-protein coupled receptors (GPCRs). These proteins register external signals such as photons, odorants, and neurotransmitters and convey ligand recognition into cellular responses, one of which is Ca(2+) signaling. To avoid receptor cross-talk or cross-activation with introduced proteins, the repertoire of cell-endogenous receptors must be known. Here we examined the presence of histamine receptors in six cell lines frequently used as hosts to study cellular signaling processes. In a concentration-dependent manner, histamine caused a rise in intracellular Ca(2+) in HeLa, HEK 293, and COS-1 cells. The concentration for half-maximal activation (EC50) was in the low micromolar range. In individual cells, transient Ca(2+) signals and Ca(2+) oscillations were uncovered. The results show that (i) HeLa, HEK 293, and COS-1 cells express sufficient amounts of endogenous receptors to study cellular Ca(2+) signaling processes directly and (ii) these cell lines are suitable for calibrating Ca(2+) biosensors in situ based on histamine receptor evoked responses. Copyright © 2015 Elsevier Inc. All rights reserved.

  8. Differences in signal activation by LH and hCG are mediated by the LH/CG receptor`s extracellular hinge region

    Directory of Open Access Journals (Sweden)

    Paul eGrzesik

    2015-09-01

    Full Text Available The human lutropin/choriogonadotropin receptor (LHCGR can be activated by binding two slightly different gonadotropic glycoprotein hormones, choriogonadotropin (CG - secreted by the placenta, and lutropin (LH - produced by the pituitary. They induce different signaling profiles at the LHCGR. This cannot be explained by binding to the receptor's leucine-rich repeat domain (LRRD, as this binding is similar for the two hormones. We therefore speculate that there are previously unknown differences in the hormone/receptor interaction at the extracellular hinge region, which might help to understand functional differences between the two hormones. We have therefore performed a detailed study of the binding and action of LH and CG at the LHCGR hinge region. We focused on a primate-specific additional exon in the hinge region, which is located between LRRD and the serpentine domain. The segment of the hinge region encoded by exon10 was previously reported to be only relevant to hLH signaling, as the exon10-deletion receptor exhibits decreased hLH signaling, but unchanged hCG signaling. We designed an advanced homology model of the hormone/LHCGR complex, followed by experimental characterization of relevant fragments in the hinge region. In addition, we examined predictions of a helical exon10-encoded conformation by block-wise polyalanine (helix supporting mutations. These helix preserving modifications showed no effect on hormone induced signaling. However, introduction of a structure-disturbing double-proline mutant LHCGR-Q303P/E305P within the exon10-helix has, in contrast to exon10 deletion, no impact on hLH, but only on hCG signaling. This opposite effect on signaling by hLH and hCG can be explained by distinct sites of hormone interaction in the hinge region s. In conclusion, our analysis provides details of the differences between hLH- and hCG-induced signaling that are mainly determined in the L2-beta loop of the hormones and in the hinge region

  9. Entropy for the Complexity of Physiological Signal Dynamics.

    Science.gov (United States)

    Zhang, Xiaohua Douglas

    2017-01-01

    Recently, the rapid development of large data storage technologies, mobile network technology, and portable medical devices makes it possible to measure, record, store, and track analysis of biological dynamics. Portable noninvasive medical devices are crucial to capture individual characteristics of biological dynamics. The wearable noninvasive medical devices and the analysis/management of related digital medical data will revolutionize the management and treatment of diseases, subsequently resulting in the establishment of a new healthcare system. One of the key features that can be extracted from the data obtained by wearable noninvasive medical device is the complexity of physiological signals, which can be represented by entropy of biological dynamics contained in the physiological signals measured by these continuous monitoring medical devices. Thus, in this chapter I present the major concepts of entropy that are commonly used to measure the complexity of biological dynamics. The concepts include Shannon entropy, Kolmogorov entropy, Renyi entropy, approximate entropy, sample entropy, and multiscale entropy. I also demonstrate an example of using entropy for the complexity of glucose dynamics.

  10. Glycosylation as a Main Regulator of Growth and Death Factor Receptors Signaling

    Directory of Open Access Journals (Sweden)

    Inês Gomes Ferreira

    2018-02-01

    Full Text Available Glycosylation is a very frequent and functionally important post-translational protein modification that undergoes profound changes in cancer. Growth and death factor receptors and plasma membrane glycoproteins, which upon activation by extracellular ligands trigger a signal transduction cascade, are targets of several molecular anti-cancer drugs. In this review, we provide a thorough picture of the mechanisms bywhich glycosylation affects the activity of growth and death factor receptors in normal and pathological conditions. Glycosylation affects receptor activity through three non-mutually exclusive basic mechanisms: (1 by directly regulating intracellular transport, ligand binding, oligomerization and signaling of receptors; (2 through the binding of receptor carbohydrate structures to galectins, forming a lattice thatregulates receptor turnover on the plasma membrane; and (3 by receptor interaction with gangliosides inside membrane microdomains. Some carbohydrate chains, for example core fucose and β1,6-branching, exert a stimulatory effect on all receptors, while other structures exert opposite effects on different receptors or in different cellular contexts. In light of the crucial role played by glycosylation in the regulation of receptor activity, the development of next-generation drugs targeting glyco-epitopes of growth factor receptors should be considered a therapeutically interesting goal.

  11. Cross-talk between an activator of nuclear receptors-mediated transcription and the D1 dopamine receptor signaling pathway.

    Science.gov (United States)

    Schmidt, Azriel; Vogel, Robert; Rutledge, Su Jane; Opas, Evan E; Rodan, Gideon A; Friedman, Eitan

    2005-03-01

    Nuclear receptors are transcription factors that usually interact, in a ligand-dependent manner, with specific DNA sequences located within promoters of target genes. The nuclear receptors can also be controlled in a ligand-independent manner via the action of membrane receptors and cellular signaling pathways. 5-Tetradecyloxy-2-furancarboxylic acid (TOFA) was shown to stimulate transcription from the MMTV promoter via chimeric receptors that consist of the DNA binding domain of GR and the ligand binding regions of the PPARbeta or LXRbeta nuclear receptors (GR/PPARbeta and GR/LXRbeta). TOFA and hydroxycholesterols also modulate transcription from NF-kappaB- and AP-1-controlled reporter genes and induce neurite differentiation in PC12 cells. In CV-1 cells that express D(1) dopamine receptors, D(1) dopamine receptor stimulation was found to inhibit TOFA-stimulated transcription from the MMTV promoter that is under the control of chimeric GR/PPARbeta and GR/LXRbeta receptors. Treatment with the D(1) dopamine receptor antagonist, SCH23390, prevented dopamine-mediated suppression of transcription, and by itself increased transcription controlled by GR/LXRbeta. Furthermore, combined treatment of CV-1 cells with TOFA and SCH23390 increased transcription controlled by the GR/LXRbeta chimeric receptor synergistically. The significance of this in vitro synergy was demonstrated in vivo, by the observation that SCH23390 (but not haloperidol)-mediated catalepsy in rats was potentiated by TOFA, thus showing that an agent that mimics the in vitro activities of compounds that activate members of the LXR and PPAR receptor families can influence D1 dopamine receptor elicited responses.

  12. Receptor protein tyrosine phosphatase alpha enhances rheumatoid synovial fibroblast signaling and promotes arthritis in mice

    NARCIS (Netherlands)

    Stanford, Stephanie M; Svensson, Mattias N D; Sacchetti, Cristiano; Pilo, Caila A; Wu, Dennis J; Kiosses, William B; Hellvard, Annelie; Bergum, Brith; Aleman Muench, German R; Elly, Christian; Liu, Yun-Cai; den Hertog, Jeroen; Elson, Ari; Sap, Jan; Mydel, Piotr; Boyle, David L; Corr, Maripat; Firestein, Gary S; Bottini, Nunzio

    2016-01-01

    OBJECTIVE: During rheumatoid arthritis (RA), fibroblast-like synoviocytes (FLS) critically promote disease pathogenesis by aggressively invading the joint extracellular matrix. The focal adhesion kinase (FAK) signaling pathway is emerging as a contributor to RA FLS anomalous behavior. The receptor

  13. The Role of Stat3 Activation in Androgen Receptor Signaling and Prostate Cancer

    National Research Council Canada - National Science Library

    Gao, Allen C

    2006-01-01

    .... The experiments proposed in this application are based upon the hypothesis that Stat3 activation alters androgen receptor signaling pathways which in turn results in the loss of growth control in prostate cancer cells...

  14. The Role of Stat3 Activation in Androgen Receptor Signaling and Prostate Cancer

    National Research Council Canada - National Science Library

    Gao, Allen

    2004-01-01

    .... The experiments proposed in this application are based upon the hypothesis that stat3 activation alters androgen receptor signaling pathways, that in turn results in the loss of growth control in prostate cancer cells...

  15. The Role of Stat3 Activation in Androgen Receptor Signaling and Prostate Cancer

    National Research Council Canada - National Science Library

    Gao, Allen

    2002-01-01

    .... The experiments proposed in this application are based upon the hypothesis that Stat3 activation alters androgen receptor signaling pathways, that in turn results in the loss of growth control in prostate cancer cells...

  16. The Role of Stat3 Activation in Androgen Receptor Signaling and Prostate Cancer

    National Research Council Canada - National Science Library

    Gao, Allen C

    2005-01-01

    .... The experiments proposed in this application are based upon the hypothesis that Stat3 activation alters androgen receptor signaling pathways, that in turn results in the loss of growth control in prostate cancer cells...

  17. The Role of Stat3 Activation in Androgen Receptor Signaling and Prostate Cancer

    National Research Council Canada - National Science Library

    Gao, Allen

    2003-01-01

    .... The experiments proposed in this application are based upon the hypothesis that Stat3 activation alters androgen receptor signaling pathways, that in turn results in the loss of growth control in prostate cancer cells...

  18. Regulation of EGF Receptor Signaling by Histone Deacetylase 6 (HDAC6)-Mediated Reversible Acetylation

    National Research Council Canada - National Science Library

    Kovacs, Jeffrey J

    2005-01-01

    One of the hallmarks of cancer is uncontrolled cell growth and proliferation. In cells, a group of proteins called growth factor receptors are responsible for responding to the signals that trigger proliferation...

  19. The Fyn tyrosine kinase binds Irs-1 and forms a distinct signaling complex during insulin stimulation.

    Science.gov (United States)

    Sun, X J; Pons, S; Asano, T; Myers, M G; Glasheen, E; White, M F

    1996-05-03

    Irs-proteins link the receptors for insulin/IGF-1, growth hormones, and several interleukins and interferons to signaling proteins that contain Src homology-2 (SH2). To identify new Irs-1-binding proteins, we screened a mouse embryo expression library with recombinant [32P]Irs-1, which revealed a specific association between p59fyn and Irs-1. The SH2 domain in p59fyn bound to phosphorylated Tyr895 and Tyr1172, which are located in YXX(L/I) motifs. Mutation of p59fyn at the COOH-terminal tyrosine phosphorylation site (Tyr531) enhanced its binding to Irs-1 during insulin stimulation. Binding experiments with various SH2 protein revealed that Grb-2 was largely excluded from Irs-1 complexes containing p59fyn, whereas Grb-2 and p85 occurred in the same Irs-1 complex. By comparison with the insulin receptor, p59fyn kinase phosphorylated a unique cohort of tyrosine residues in Irs-1. These results outline a role for p59fyn or other related Src-kinases during insulin and cytokine signaling.

  20. β1-adrenergic receptors activate two distinct signaling pathways in striatal neurons

    Science.gov (United States)

    Meitzen, John; Luoma, Jessie I.; Stern, Christopher M.; Mermelstein, Paul G.

    2010-01-01

    Monoamine action in the dorsal striatum and nucleus accumbens plays essential roles in striatal physiology. Although research often focuses on dopamine and its receptors, norepinephrine and adrenergic receptors are also crucial in regulating striatal function. While noradrenergic neurotransmission has been identified in the striatum, little is known regarding the signaling pathways activated by β-adrenergic receptors in this brain region. Using cultured striatal neurons, we characterized a novel signaling pathway by which activation of β1-adrenergic receptors leads to the rapid phosphorylation of cAMP Response Element Binding Protein (CREB), a transcription-factor implicated as a molecular switch underlying long-term changes in brain function. Norepinephrine-mediated CREB phosphorylation requires β1-adrenergic receptor stimulation of a receptor tyrosine kinase, ultimately leading to the activation of a Ras/Raf/MEK/MAPK/MSK signaling pathway. Activation of β1-adrenergic receptors also induces CRE-dependent transcription and increased c-fos expression. In addition, stimulation of β1-adrenergic receptors produces cAMP production, but surprisingly, β1-adrenergic receptor activation of adenylyl cyclase was not functionally linked to rapid CREB phosphorylation. These findings demonstrate that activation of β1-adrenergic receptors on striatal neurons can stimulate two distinct signaling pathways. These adrenergic actions can produce long-term changes in gene expression, as well as rapidly modulate cellular physiology. By elucidating the mechanisms by which norepinephrine and β1-adrenergic receptor activation affects striatal physiology, we provide the means to more fully understand the role of monoamines in modulating striatal function, specifically how norepinephrine and β1-adrenergic receptors may affect striatal physiology. PMID:21143600

  1. ITAM-like signalling for efficient phagocytosis : The paradigm of the granulocyte receptor CEACAM3

    OpenAIRE

    Pils, Stefan

    2010-01-01

    Human CEACAM3 is a tailor-made receptor of the innate immune system to fight pathogens exploiting epithelial CEACAM-family members for colonisation and invasion of their host. Previous studies established CEACAM3 as the receptor facilitating rapid phagocytosis and elimination of N. gonorrhoeae by human granulocytes. The studies reported here set out to shed light on the evolution of this highly specialised receptor and the associated signalling machinery.CEACAM3 arose from exon shuffling afte...

  2. Analysis of receptor signaling pathways by mass spectrometry: identification of vav-2 as a substrate of the epidermal and platelet-derived growth factor receptors

    DEFF Research Database (Denmark)

    Pandey, A; Podtelejnikov, A V; Blagoev, B

    2000-01-01

    Oligomerization of receptor protein tyrosine kinases such as the epidermal growth factor receptor (EGFR) by their cognate ligands leads to activation of the receptor. Transphosphorylation of the receptor subunits is followed by the recruitment of signaling molecules containing src homology 2 (SH2...

  3. Fluorescence resonance energy transfer (FRET-based subcellular visualization of pathogen-induced host receptor signaling

    Directory of Open Access Journals (Sweden)

    Zimmermann Timo

    2009-11-01

    Full Text Available Abstract Background Bacteria-triggered signaling events in infected host cells are key elements in shaping the host response to pathogens. Within the eukaryotic cell, signaling complexes are spatially organized. However, the investigation of protein-protein interactions triggered by bacterial infection in the cellular context is technically challenging. Here, we provide a methodological approach to exploit fluorescence resonance energy transfer (FRET to visualize pathogen-initiated signaling events in human cells. Results Live-cell microscopy revealed the transient recruitment of the Src family tyrosine kinase Hck upon bacterial engagement of the receptor carcinoembryonic antigen-related cell adhesion molecule 3 (CEACAM3. In cells expressing a CEACAM3 variant lacking the cytoplasmic domain, the Src homology 2 (SH2 domain of Hck (Hck-SH2 was not recruited, even though bacteria still bound to the receptor. FRET measurements on the basis of whole cell lysates revealed intimate binding between Hck-SH2 (using enhanced yellow fluorescent protein (YPet-Hck-SH2 and the tyrosine-phosphorylated enhanced cyan fluorescent protein-labeled cytoplasmic domain of wild-type CEACAM3 (CEACAM3 WT-CyPet and a flow cytometry-based FRET approach verified this association in intact cells. Using confocal microscopy and acceptor photobleaching, FRET between Hck-SH2 and CEACAM3 was localized to the sites of bacteria-host cell contact. Conclusion These data demonstrate not only the intimate binding of the SH2 domain of Hck to the tyrosine-phosphorylated cytoplasmic domain of CEACAM3 in intact cells, but furthermore, FRET measurements allow the subcellular localization of this process during bacterial infection. FRET-based assays are valuable tools to resolve bacteria-induced protein-protein interactions in the context of the intact host cell.

  4. The role of Ryk and Ror receptor tyrosine kinases in Wnt signal transduction

    NARCIS (Netherlands)

    Green, J.; Nusse, R.; van Amerongen, R.

    2014-01-01

    Receptor tyrosine kinases of the Ryk and Ror families were initially classified as orphan receptors because their ligands were unknown. They are now known to contain functional extracellular Wnt-binding domains and are implicated in Wnt-signal transduction in multiple species. Although their

  5. PKCζ regulates Notch receptor routing and activity in a Notch signaling-dependent manner

    NARCIS (Netherlands)

    Sjöqvist, M.; Antfolk, D.; Ferraris, S.; Rraklli, V.; Haga, C.; Antila, C.; Mutvei, A.; Imanishi, S.Y.; Holmberg, J.; Jin, S.; Eriksson, J.E.; Lendahl, U.; Sahlgren, C.M.

    Activation of Notch signaling requires intracellular routing of the receptor, but the mechanisms controlling the distinct steps in the routing process is poorly understood. We identify PKCζ as a key regulator of Notch receptor intracellular routing. When PKCζ was inhibited in the developing chick

  6. Clathrin-dependent internalization, signaling, and metabolic processing of guanylyl cyclase/natriuretic peptide receptor-A.

    Science.gov (United States)

    Somanna, Naveen K; Mani, Indra; Tripathi, Satyabha; Pandey, Kailash N

    2018-04-01

    Cardiac hormones, atrial and brain natriuretic peptides (ANP and BNP), have pivotal roles in renal hemodynamics, neuroendocrine signaling, blood pressure regulation, and cardiovascular homeostasis. Binding of ANP and BNP to the guanylyl cyclase/natriuretic peptide receptor-A (GC-A/NPRA) induces rapid internalization and trafficking of the receptor via endolysosomal compartments, with concurrent generation of cGMP. However, the mechanisms of the endocytotic processes of NPRA are not well understood. The present study, using 125 I-ANP binding assay and confocal microscopy, examined the function of dynamin in the internalization of NPRA in stably transfected human embryonic kidney-293 (HEK-293) cells. Treatment of recombinant HEK-293 cells with ANP time-dependently accelerated the internalization of receptor from the cell surface to the cell interior. However, the internalization of ligand-receptor complexes of NPRA was drastically decreased by the specific inhibitors of clathrin- and dynamin-dependent receptor internalization, almost 85% by monodansylcadaverine, 80% by chlorpromazine, and 90% by mutant dynamin, which are specific blockers of endocytic vesicle formation. Visualizing the internalization of NPRA and enhanced GFP-tagged NPRA in HEK-293 cells by confocal microscopy demonstrated the formation of endocytic vesicles after 5 min of ANP treatment; this effect was blocked by the inhibitors of clathrin and by mutant dynamin construct. Our results suggest that NPRA undergoes internalization via clathrin-mediated endocytosis as part of its normal itinerary, including trafficking, signaling, and metabolic degradation.

  7. In vivo characterization of high Basal signaling from the ghrelin receptor

    DEFF Research Database (Denmark)

    Petersen, Pia Steen; Woldbye, David P D; Madsen, Andreas Nygaard

    2009-01-01

    The receptor for the orexigenic peptide, ghrelin, is one of the most constitutively active 7TM receptors known, as demonstrated under in vitro conditions. Change in expression of a constitutively active receptor is associated with change in signaling independent of the endogenous ligand. In the f......The receptor for the orexigenic peptide, ghrelin, is one of the most constitutively active 7TM receptors known, as demonstrated under in vitro conditions. Change in expression of a constitutively active receptor is associated with change in signaling independent of the endogenous ligand....... In the following study, we found that the expression of the ghrelin receptor in the hypothalamus was up-regulated approximately 2-fold in rats both during 48-h fasting and by streptozotocin-induced hyperphagia. In a separate experiment, to probe for the effect of the high basal signaling of the ghrelin receptor...... in vivo, we used intracerebroventricular administration by osmotic pumps of a peptide [D-Arg(1), D-Phe(5), D-Trp(7,9), Leu(11)]-substance P. This peptide selectively displays inverse agonism at the ghrelin receptor as compared with an inactive control peptide with just a single amino acid substitution...

  8. Predominant membrane localization is an essential feature of the bacterial signal recognition particle receptor

    Directory of Open Access Journals (Sweden)

    Graumann Peter

    2009-11-01

    Full Text Available Abstract Background The signal recognition particle (SRP receptor plays a vital role in co-translational protein targeting, because it connects the soluble SRP-ribosome-nascent chain complex (SRP-RNCs to the membrane bound Sec translocon. The eukaryotic SRP receptor (SR is a heterodimeric protein complex, consisting of two unrelated GTPases. The SRβ subunit is an integral membrane protein, which tethers the SRP-interacting SRα subunit permanently to the endoplasmic reticulum membrane. The prokaryotic SR lacks the SRβ subunit and consists of only the SRα homologue FtsY. Strikingly, although FtsY requires membrane contact for functionality, cell fractionation studies have localized FtsY predominantly to the cytosolic fraction of Escherichia coli. So far, the exact function of the soluble SR in E. coli is unknown, but it has been suggested that, in contrast to eukaryotes, the prokaryotic SR might bind SRP-RNCs already in the cytosol and only then initiates membrane targeting. Results In the current study we have determined the contribution of soluble FtsY to co-translational targeting in vitro and have re-analysed the localization of FtsY in vivo by fluorescence microscopy. Our data show that FtsY can bind to SRP-ribosome nascent chains (RNCs in the absence of membranes. However, these soluble FtsY-SRP-RNC complexes are not efficiently targeted to the membrane. In contrast, we observed effective targeting of SRP-RNCs to membrane-bond FtsY. These data show that soluble FtsY does not contribute significantly to cotranslational targeting in E. coli. In agreement with this observation, our in vivo analyses of FtsY localization in bacterial cells by fluorescence microscopy revealed that the vast majority of FtsY was localized to the inner membrane and that soluble FtsY constituted only a negligible species in vivo. Conclusion The exact function of the SRP receptor (SR in bacteria has so far been enigmatic. Our data show that the bacterial SR is

  9. Complex Interplay of Hormonal Signals during Grape Berry Ripening

    Directory of Open Access Journals (Sweden)

    Ana Margarida Fortes

    2015-05-01

    Full Text Available Grape and wine production and quality is extremely dependent on the fruit ripening process. Sensory and nutritional characteristics are important aspects for consumers and their development during fruit ripening involves complex hormonal control. In this review, we explored data already published on grape ripening and compared it with the hormonal regulation of ripening of other climacteric and non-climacteric fruits. The roles of abscisic acid, ethylene, and brassinosteroids as promoters of ripening are discussed, as well as the role of auxins, cytokinins, gibberellins, jasmonates, and polyamines as inhibitors of ripening. In particular, the recently described role of polyamine catabolism in grape ripening is discussed, together with its putative interaction with other hormones. Furthermore, other recent examples of cross-talk among the different hormones are presented, revealing a complex interplay of signals during grape development and ripening.

  10. CD25 and CD69 induction by α4β1 outside-in signalling requires TCR early signalling complex proteins

    Science.gov (United States)

    Cimo, Ann-Marie; Ahmed, Zamal; McIntyre, Bradley W.; Lewis, Dorothy E.; Ladbury, John E.

    2013-01-01

    Distinct signalling pathways producing diverse cellular outcomes can utilize similar subsets of proteins. For example, proteins from the TCR (T-cell receptor) ESC (early signalling complex) are also involved in interferon-α receptor signalling. Defining the mechanism for how these proteins function within a given pathway is important in understanding the integration and communication of signalling networks with one another. We investigated the contributions of the TCR ESC proteins Lck (lymphocyte-specific kinase), ZAP-70 (ζ-chain-associated protein of 70 kDa), Vav1, SLP-76 [SH2 (Src homology 2)-domain-containing leukocyte protein of 76 kDa] and LAT (linker for activation of T-cells) to integrin outside-in signalling in human T-cells. Lck, ZAP-70, SLP-76, Vav1 and LAT were activated by α4β1 outside-in signalling, but in a manner different from TCR signalling. TCR stimulation recruits ESC proteins to activate the mitogen-activated protein kinase ERK (extracellular-signal-regulated kinase). α4β1 outside-in-mediated ERK activation did not require TCR ESC proteins. However, α4β1 outside-in signalling induced CD25 and co-stimulated CD69 and this was dependent on TCR ESC proteins. TCR and α4β1 outside-in signalling are integrated through the common use of TCR ESC proteins; however, these proteins display functionally distinct roles in these pathways. These novel insights into the cross-talk between integrin outside-in and TCR signalling pathways are highly relevant to the development of therapeutic strategies to overcome disease associated with T-cell deregulation. PMID:23758320

  11. Crystal structure of mouse coronavirus receptor-binding domain complexed with its murine receptor

    Energy Technology Data Exchange (ETDEWEB)

    Peng, Guiqing; Sun, Dawei; Rajashankar, Kanagalaghatta R.; Qian, Zhaohui; Holmes, Kathryn V.; Li, Fang (Cornell); (UMM-MED); (Colorado)

    2011-09-28

    Coronaviruses have evolved diverse mechanisms to recognize different receptors for their cross-species transmission and host-range expansion. Mouse hepatitis coronavirus (MHV) uses the N-terminal domain (NTD) of its spike protein as its receptor-binding domain. Here we present the crystal structure of MHV NTD complexed with its receptor murine carcinoembryonic antigen-related cell adhesion molecule 1a (mCEACAM1a). Unexpectedly, MHV NTD contains a core structure that has the same {beta}-sandwich fold as human galectins (S-lectins) and additional structural motifs that bind to the N-terminal Ig-like domain of mCEACAM1a. Despite its galectin fold, MHV NTD does not bind sugars, but instead binds mCEACAM1a through exclusive protein-protein interactions. Critical contacts at the interface have been confirmed by mutagenesis, providing a structural basis for viral and host specificities of coronavirus/CEACAM1 interactions. Sugar-binding assays reveal that galectin-like NTDs of some coronaviruses such as human coronavirus OC43 and bovine coronavirus bind sugars. Structural analysis and mutagenesis localize the sugar-binding site in coronavirus NTDs to be above the {beta}-sandwich core. We propose that coronavirus NTDs originated from a host galectin and retained sugar-binding functions in some contemporary coronaviruses, but evolved new structural features in MHV for mCEACAM1a binding.

  12. Ruthenium complexes with phenylterpyridine derivatives target cell membrane and trigger death receptors-mediated apoptosis in cancer cells.

    Science.gov (United States)

    Deng, Zhiqin; Gao, Pan; Yu, Lianling; Ma, Bin; You, Yuanyuan; Chan, Leung; Mei, Chaoming; Chen, Tianfeng

    2017-06-01

    Elucidation of the communication between metal complexes and cell membrane may provide useful information for rational design of metal-based anticancer drugs. Herein we synthesized a novel class of ruthenium (Ru) complexes containing phtpy derivatives (phtpy = phenylterpyridine), analyzed their structure-activity relationship and revealed their action mechanisms. The result showed that, the increase in the planarity of hydrophobic Ru complexes significantly enhanced their lipophilicity and cellular uptake. Meanwhile, the introduction of nitro group effectively improved their anticancer efficacy. Further mechanism studies revealed that, complex (2c), firstly accumulated on cell membrane and interacted with death receptors to activate extrinsic apoptosis signaling pathway. The complex was then transported into cell cytoplasm through transferrin receptor-mediated endocytosis. Most of the intracellular 2c accumulated in cell plasma, decreasing the level of cellular ROS, inducing the activation of caspase-9 and thus intensifying the apoptosis. At the same time, the residual 2c can translocate into cell nucleus to interact with DNA, induce DNA damage, activate p53 pathway and enhance apoptosis. Comparing with cisplatin, 2c possesses prolonged circulation time in blood, comparable antitumor ability and importantly, much lower toxicity in vivo. Taken together, this study uncovers the role of membrane receptors in the anticancer actions of Ru complexes, and provides fundamental information for rational design of membrane receptor targeting anticancer drugs. Copyright © 2017 Elsevier Ltd. All rights reserved.

  13. Association between increased EEG signal complexity and cannabis dependence.

    Science.gov (United States)

    Laprevote, Vincent; Bon, Laura; Krieg, Julien; Schwitzer, Thomas; Bourion-Bedes, Stéphanie; Maillard, Louis; Schwan, Raymund

    2017-12-01

    Both acute and regular cannabis use affects the functioning of the brain. While several studies have demonstrated that regular cannabis use can impair the capacity to synchronize neural assemblies during specific tasks, less is known about spontaneous brain activity. This can be explored by measuring EEG complexity, which reflects the spontaneous variability of human brain activity. A recent study has shown that acute cannabis use can affect that complexity. Since the characteristics of cannabis use can affect the impact on brain functioning, this study sets out to measure EEG complexity in regular cannabis users with or without dependence, in comparison with healthy controls. We recruited 26 healthy controls, 25 cannabis users without cannabis dependence and 14 cannabis users with cannabis dependence, based on DSM IV TR criteria. The EEG signal was extracted from at least 250 epochs of the 500ms pre-stimulation phase during a visual evoked potential paradigm. Brain complexity was estimated using Lempel-Ziv Complexity (LZC), which was compared across groups by non-parametric Kruskall-Wallis ANOVA. The analysis revealed a significant difference between the groups, with higher LZC in participants with cannabis dependence than in non-dependent cannabis users. There was no specific localization of this effect across electrodes. We showed that cannabis dependence is associated to an increased spontaneous brain complexity in regular users. This result is in line with previous results in acute cannabis users. It may reflect increased randomness of neural activity in cannabis dependence. Future studies should explore whether this effect is permanent or diminishes with cannabis cessation. Copyright © 2017 Elsevier B.V. and ECNP. All rights reserved.

  14. Age-Related Changes in Electroencephalographic Signal Complexity

    Science.gov (United States)

    Zappasodi, Filippo; Marzetti, Laura; Olejarczyk, Elzbieta; Tecchio, Franca; Pizzella, Vittorio

    2015-01-01

    The study of active and healthy aging is a primary focus for social and neuroscientific communities. Here, we move a step forward in assessing electrophysiological neuronal activity changes in the brain with healthy aging. To this end, electroencephalographic (EEG) resting state activity was acquired in 40 healthy subjects (age 16–85). We evaluated Fractal Dimension (FD) according to the Higuchi algorithm, a measure which quantifies the presence of statistical similarity at different scales in temporal fluctuations of EEG signals. Our results showed that FD increases from age twenty to age fifty and then decreases. The curve that best fits the changes in FD values across age over the whole sample is a parabola, with the vertex located around age fifty. Moreover, FD changes are site specific, with interhemispheric FD asymmetry being pronounced in elderly individuals in the frontal and central regions. The present results indicate that fractal dimension well describes the modulations of brain activity with age. Since fractal dimension has been proposed to be related to the complexity of the signal dynamics, our data demonstrate that the complexity of neuronal electric activity changes across the life span of an individual, with a steady increase during young adulthood and a decrease in the elderly population. PMID:26536036

  15. Enabling complex genetic circuits to respond to extrinsic environmental signals.

    Science.gov (United States)

    Hoynes-O'Connor, Allison; Shopera, Tatenda; Hinman, Kristina; Creamer, John Philip; Moon, Tae Seok

    2017-07-01

    Genetic circuits have the potential to improve a broad range of metabolic engineering processes and address a variety of medical and environmental challenges. However, in order to engineer genetic circuits that can meet the needs of these real-world applications, genetic sensors that respond to relevant extrinsic and intrinsic signals must be implemented in complex genetic circuits. In this work, we construct the first AND and NAND gates that respond to temperature and pH, two signals that have relevance in a variety of real-world applications. A previously identified pH-responsive promoter and a temperature-responsive promoter were extracted from the E. coli genome, characterized, and modified to suit the needs of the genetic circuits. These promoters were combined with components of the type III secretion system in Salmonella typhimurium and used to construct a set of AND gates with up to 23-fold change. Next, an antisense RNA was integrated into the circuit architecture to invert the logic of the AND gate and generate a set of NAND gates with up to 1168-fold change. These circuits provide the first demonstration of complex pH- and temperature-responsive genetic circuits, and lay the groundwork for the use of similar circuits in real-world applications. Biotechnol. Bioeng. 2017;114: 1626-1631. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

  16. GABA(A)-benzodiazepine receptor complex sensitivity in 5-HT(1A) receptor knockout mice on a 129/Sv background.

    NARCIS (Netherlands)

    Pattij, T.; Groenink, L.; Oosting, R.S.; Gugten, J. van der; Maes, R.A.A.; Olivier, B.

    2002-01-01

    Previous studies in 5-HT(1A) receptor knockout (1AKO) mice on a mixed Swiss Websterx129/Sv (SWx129/Sv) and a pure 129/Sv genetic background suggest a differential gamma-aminobutyric acid (GABA(A))-benzodiazepine receptor complex sensitivity in both strains, independent from the anxious phenotype. To

  17. Molecular, pharmacological, and signaling properties of octopamine receptors from honeybee (Apis mellifera) brain.

    Science.gov (United States)

    Balfanz, Sabine; Jordan, Nadine; Langenstück, Teresa; Breuer, Johanna; Bergmeier, Vera; Baumann, Arnd

    2014-04-01

    G protein-coupled receptors are important regulators of cellular signaling processes. Within the large family of rhodopsin-like receptors, those binding to biogenic amines form a discrete subgroup. Activation of biogenic amine receptors leads to transient changes of intracellular Ca²⁺-([Ca²⁺](i)) or 3',5'-cyclic adenosine monophosphate ([cAMP](i)) concentrations. Both second messengers modulate cellular signaling processes and thereby contribute to long-lasting behavioral effects in an organism. In vivo pharmacology has helped to reveal the functional effects of different biogenic amines in honeybees. The phenolamine octopamine is an important modulator of behavior. Binding of octopamine to its receptors causes elevation of [Ca²⁺](i) or [cAMP](i). To date, only one honeybee octopamine receptor that induces Ca²⁺ signals has been molecularly and pharmacologically characterized. Here, we examined the pharmacological properties of four additional honeybee octopamine receptors. When heterologously expressed, all receptors induced cAMP production after binding to octopamine with EC₅₀(s) in the nanomolar range. Receptor activity was most efficiently blocked by mianserin, a substance with antidepressant activity in vertebrates. The rank order of inhibitory potency for potential receptor antagonists was very similar on all four honeybee receptors with mianserin > cyproheptadine > metoclopramide > chlorpromazine > phentolamine. The subroot of octopamine receptors activating adenylyl cyclases is the largest that has so far been characterized in arthropods, and it should now be possible to unravel the contribution of individual receptors to the physiology and behavior of honeybees. © 2013 International Society for Neurochemistry.

  18. A peptide targeting an interaction interface disrupts the dopamine D1-D2 receptor heteromer to block signaling and function in vitro and in vivo: effective selective antagonism

    Science.gov (United States)

    Hasbi, Ahmed; Perreault, Melissa L.; Shen, Maurice Y. F.; Zhang, Lucia; To, Ryan; Fan, Theresa; Nguyen, Tuan; Ji, Xiaodong; O'Dowd, Brian F.; George, Susan R.

    2014-01-01

    Although the dopamine D1-D2 receptor heteromer has emerging physiological relevance and a postulated role in different neuropsychiatric disorders, such as drug addiction, depression, and schizophrenia, there is a need for pharmacological tools that selectively target such receptor complexes in order to analyze their biological and pathophysiological functions. Since no selective antagonists for the D1-D2 heteromer are available, serial deletions and point mutations were used to precisely identify the amino acids involved in an interaction interface between the receptors, residing within the carboxyl tail of the D1 receptor that interacted with the D2 receptor to form the D1-D2 receptor heteromer. It was determined that D1 receptor carboxyl tail residues 404Glu and 405Glu were critical in mediating the interaction with the D2 receptor. Isolated mutation of these residues in the D1 receptor resulted in the loss of agonist activation of the calcium signaling pathway mediated through the D1-D2 receptor heteromer. The physical interaction between the D1 and D2 receptor could be disrupted, as shown by coimmunoprecipitation and BRET analysis, by a small peptide generated from the D1 receptor sequence that contained these amino acids, leading to a switch in G-protein affinities and loss of calcium signaling, resulting in the inhibition of D1-D2 heteromer function. The use of the D1-D2 heteromer-disrupting peptide in vivo revealed a pathophysiological role for the D1-D2 heteromer in the modulation of behavioral despair. This peptide may represent a novel pharmacological tool with potential therapeutic benefits in depression treatment.—Hasbi, A., Perreault, M. L., Shen, M. Y. F., Zhang, L., To, R., Fan, T., Nguyen, T., Ji, X., O'Dowd, B. F., George, S. R. A peptide targeting an interaction interface disrupts the dopamine D1-D2 receptor heteromer to block signaling and function in vitro and in vivo: effective selective antagonism. PMID:25063849

  19. Orphan nuclear receptor TR3 acts in autophagic cell death via mitochondrial signaling pathway.

    Science.gov (United States)

    Wang, Wei-jia; Wang, Yuan; Chen, Hang-zi; Xing, Yong-zhen; Li, Feng-wei; Zhang, Qian; Zhou, Bo; Zhang, Hong-kui; Zhang, Jie; Bian, Xue-li; Li, Li; Liu, Yuan; Zhao, Bi-xing; Chen, Yan; Wu, Rong; Li, An-zhong; Yao, Lu-ming; Chen, Ping; Zhang, Yi; Tian, Xu-yang; Beermann, Friedrich; Wu, Mian; Han, Jiahuai; Huang, Pei-qiang; Lin, Tianwei; Wu, Qiao

    2014-02-01

    Autophagy is linked to cell death, yet the associated mechanisms are largely undercharacterized. We discovered that melanoma, which is generally resistant to drug-induced apoptosis, can undergo autophagic cell death with the participation of orphan nuclear receptor TR3. A sequence of molecular events leading to cellular demise is launched by a specific chemical compound, 1-(3,4,5-trihydroxyphenyl)nonan-1-one, newly acquired from screening a library of TR3-targeting compounds. The autophagic cascade comprises TR3 translocation to mitochondria through interaction with the mitochondrial outer membrane protein Nix, crossing into the mitochondrial inner membrane through Tom40 and Tom70 channel proteins, dissipation of mitochondrial membrane potential by the permeability transition pore complex ANT1-VDAC1 and induction of autophagy. This process leads to excessive mitochondria clearance and irreversible cell death. It implicates a new approach to melanoma therapy through activation of a mitochondrial signaling pathway that integrates a nuclear receptor with autophagy for cell death.

  20. STC1 interference on calcitonin family of receptors signaling during osteoblastogenesis via adenylate cyclase inhibition.

    Science.gov (United States)

    Terra, Silvia R; Cardoso, João Carlos R; Félix, Rute C; Martins, Leo Anderson M; Souza, Diogo Onofre G; Guma, Fatima C R; Canário, Adelino Vicente M; Schein, Vanessa

    2015-03-05

    Stanniocalcin 1 (STC1) and calcitonin gene-related peptide (CGRP) are involved in bone formation/remodeling. Here we investigate the effects of STC1 on functional heterodimer complex CALCRL/RAMP1, expression and activity during osteoblastogenesis. STC1 did not modify CALCRL and ramp1 gene expression during osteoblastogenesis when compared to controls. However, plasma membrane spatial distribution of CALCRL/RAMP1 was modified in 7-day pre-osteoblasts exposed to either CGRP or STC1, and both peptides induced CALCRL and RAMP1 assembly. CGRP, but not STC1 stimulated cAMP accumulation in 7-day osteoblasts and in CALCRL/RAMP1 transfected HEK293 cells. Furthermore, STC1 inhibited forskolin stimulated cAMP accumulation of HEK293 cells, but not in CALCRL/RAMP1 transfected HEK293 cells. However, STC1 inhibited cAMP accumulation in calcitonin receptor (CTR) HEK293 transfected cells stimulated by calcitonin. In conclusion, STC1 signals through inhibitory G-protein modulates CGRP receptor spatial localization during osteoblastogenesis and may function as a regulatory factor interacting with calcitonin peptide members during bone formation. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

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

    DEFF Research Database (Denmark)

    Christensen, M D; Geisler, C

    2000-01-01

    Receptors expressing the immunoreceptor tyrosine-based inhibitory motif (ITIM) in their cytoplasmic tail play an important role in the negative regulation of natural killer and B-cell activation. A subpopulation of T cells expresses the ITIM containing killer cell inhibitory receptor (KIR), which...... recognize MHC class I molecules. Following coligation of KIR with an activating receptor, the tyrosine in the ITIM is phosphorylated and the cytoplasmic protein tyrosine phosphatase SHP-1 is recruited to the ITIM via its SH2 domains. It is still not clear how SHP-1 affects T-cell receptor (TCR) signalling...... regarding total protein tyrosine phosphorylation, TCR down-regulation, mobilization of intracellular free calcium, or induction of the activation markers CD69 and CD25....

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

    International Nuclear Information System (INIS)

    Christopoulos, G.; Morfis, M.; Sexton, P.M.; Christopoulos, A.; Laburthe, M.; Couvineau, A.

    2001-01-01

    Full text: Receptor activity modifying proteins (RAMP) constitute a family of three accessory proteins that affect the expression and/or phenotype of the calcitonin receptor (CTR) or CTR-like receptor (CRLR). In this study we screened a range of class II G protein-coupled receptors (PTH1, PTH2, GHRH, VPAC1, VPAC2 receptors) for possible RAMP interactions by measurement of receptor-induced translocation of c-myc tagged RAMP1 or HA tagged RAMP3. Of these, only the VPAC1 receptor caused significant translocation of c-myc-RAMP1 or HA-RAMP3 to the cell surface. Co-transfection of VPAC1 and RAMPs did not alter 125 I-VIP binding and specificity. VPAC1 receptor function was subsequently analyzed through parallel determinations of cAMP accumulation and phosphoinositide (PI) hydrolysis in the presence and absence of each of the three RAMPs. In contrast to CTR-RAMP interaction, where there was an increase in cAMP Pharmacologisand a decrease in PI hydrolysis, VPAC1-RAMP interaction was characterized by a specific increase in agonist-mediated PI hydrolysis when co-transfected with RAMP2. This change was due to an enhancement of Emax with no change in EC 50 value for VIP. No significant change in cAMP accumulation was observed. This is the first demonstration of an interaction of RAMPs with a G protein-coupled receptor outside the CTR family and may suggest a more generalized role for RAMPs in modulating G protein-coupled receptor signaling. Copyright (2001) Australasian Society of Clinical and Experimental Pharmacologists and Toxicologists

  3. MicroRNAs regulate B-cell receptor signaling-induced apoptosis

    NARCIS (Netherlands)

    Kluiver, J. L.; Chen, C-Z

    Apoptosis induced by B-cell receptor (BCR) signaling is critical for antigen-driven selection, a process critical to tolerance and immunity. Here, we examined the roles of microRNAs (miRNAs) in BCR signaling-induced apoptosis using the widely applied WEHI-231 model. Comparison of miRNA levels in

  4. Stochastic effects as a force to increase the complexity of signaling networks

    KAUST Repository

    Kuwahara, Hiroyuki; Gao, Xin

    2013-01-01

    Cellular signaling networks are complex and appear to include many nonfunctional elements. Recently, it was suggested that nonfunctional interactions of proteins cause signaling noise, which, perhaps, shapes the signal transduction mechanism

  5. Assembly of Slx4 signaling complexes behind DNA replication forks.

    Science.gov (United States)

    Balint, Attila; Kim, TaeHyung; Gallo, David; Cussiol, Jose Renato; Bastos de Oliveira, Francisco M; Yimit, Askar; Ou, Jiongwen; Nakato, Ryuichiro; Gurevich, Alexey; Shirahige, Katsuhiko; Smolka, Marcus B; Zhang, Zhaolei; Brown, Grant W

    2015-08-13

    Obstructions to replication fork progression, referred to collectively as DNA replication stress, challenge genome stability. In Saccharomyces cerevisiae, cells lacking RTT107 or SLX4 show genome instability and sensitivity to DNA replication stress and are defective in the completion of DNA replication during recovery from replication stress. We demonstrate that Slx4 is recruited to chromatin behind stressed replication forks, in a region that is spatially distinct from that occupied by the replication machinery. Slx4 complex formation is nucleated by Mec1 phosphorylation of histone H2A, which is recognized by the constitutive Slx4 binding partner Rtt107. Slx4 is essential for recruiting the Mec1 activator Dpb11 behind stressed replication forks, and Slx4 complexes are important for full activity of Mec1. We propose that Slx4 complexes promote robust checkpoint signaling by Mec1 by stably recruiting Dpb11 within a discrete domain behind the replication fork, during DNA replication stress. © 2015 The Authors.

  6. Signaling by purinergic receptors and channels in the pituitary gland

    Czech Academy of Sciences Publication Activity Database

    Stojilkovic, S. S.; He, M. L.; Koshimizu, T.; Balík, A.; Zemková, Hana

    2010-01-01

    Roč. 314, č. 2 (2010), s. 184-191 ISSN 0303-7207 R&D Projects: GA ČR(CZ) GA305/07/0681 Institutional research plan: CEZ:AV0Z50110509 Keywords : purinergic receptors * ATP * anterior pituitary Subject RIV: ED - Physiology Impact factor: 4.119, year: 2010

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

    Lifescience Database Archive (English)

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

  8. Intercellular calcium signaling occurs between human osteoblasts and osteoclasts and requires activation of osteoclast P2X7 receptors

    DEFF Research Database (Denmark)

    Jørgensen, Niklas R; Henriksen, Zanne; Sørensen, Ole

    2002-01-01

    that human osteoclasts expressed functional P2Y1 receptors, but, unexpectedly, desensitization of P2Y1 did not block calcium signaling to osteoclasts. We also found that osteoclasts expressed functional P2X7 receptors and showed that pharmacological inhibition of these receptors blocked calcium signaling...

  9. Purinergic receptors have different effects in rat exocrine pancreas. Calcium signals monitored by fura-2 using confocal microscopy

    DEFF Research Database (Denmark)

    Novak, Ivana; Nitschke, Roland; Amstrup, Jan

    2002-01-01

    Pancreatic ducts have several types of purinergic P2 receptors, however, nothing is known about P2 receptors in acini. The aim was to establish whether acini express functional P2 receptors coupled to intracellular Ca2+ signals and to measure the signals ratiometrically in a confocal laser scanning...

  10. The Hrs/Stam complex acts as a positive and negative regulator of RTK signaling during Drosophila development.

    Directory of Open Access Journals (Sweden)

    Hélène Chanut-Delalande

    Full Text Available BACKGROUND: Endocytosis is a key regulatory step of diverse signalling pathways, including receptor tyrosine kinase (RTK signalling. Hrs and Stam constitute the ESCRT-0 complex that controls the initial selection of ubiquitinated proteins, which will subsequently be degraded in lysosomes. It has been well established ex vivo and during Drosophila embryogenesis that Hrs promotes EGFR down regulation. We have recently isolated the first mutations of stam in flies and shown that Stam is required for air sac morphogenesis, a larval respiratory structure whose formation critically depends on finely tuned levels of FGFR activity. This suggest that Stam, putatively within the ESCRT-0 complex, modulates FGF signalling, a possibility that has not been examined in Drosophila yet. PRINCIPAL FINDINGS: Here, we assessed the role of the Hrs/Stam complex in the regulation of signalling activity during Drosophila development. We show that stam and hrs are required for efficient FGFR signalling in the tracheal system, both during cell migration in the air sac primordium and during the formation of fine cytoplasmic extensions in terminal cells. We find that stam and hrs mutant cells display altered FGFR/Btl localisation, likely contributing to impaired signalling levels. Electron microscopy analyses indicate that endosome maturation is impaired at distinct steps by hrs and stam mutations. These somewhat unexpected results prompted us to further explore the function of stam and hrs in EGFR signalling. We show that while stam and hrs together downregulate EGFR signalling in the embryo, they are required for full activation of EGFR signalling during wing development. CONCLUSIONS/SIGNIFICANCE: Our study shows that the ESCRT-0 complex differentially regulates RTK signalling, either positively or negatively depending on tissues and developmental stages, further highlighting the importance of endocytosis in modulating signalling pathways during development.

  11. IL-4 and IL-13 Receptor Signaling From 4PS to Insulin Receptor Substrate 2: There and Back Again, a Historical View.

    Science.gov (United States)

    Keegan, Achsah D; Zamorano, Jose; Keselman, Aleksander; Heller, Nicola M

    2018-01-01

    In this historical perspective, written in honor of Dr. William E. Paul, we describe the initial discovery of one of the dominant substrates for tyrosine phosphorylation stimulated by IL-4. We further describe how this "IL-4-induced phosphorylated substrate" (4PS) was characterized as a member of the insulin receptor substrate (IRS) family of large adaptor proteins that link IL-4 and insulin receptors to activation of the phosphatidyl-inositol 3' kinase pathway as well as other downstream signaling pathways. The relative contribution of the 4PS/IRS pathway to the early models of IL-4-induced proliferation and suppression of apoptosis are compared to our more recent understanding of the complex interplay between positive and negative regulatory pathways emanating from members of the IRS family that impact allergic responses.

  12. IL-4 and IL-13 Receptor Signaling From 4PS to Insulin Receptor Substrate 2: There and Back Again, a Historical View

    Directory of Open Access Journals (Sweden)

    Achsah D. Keegan

    2018-05-01

    Full Text Available In this historical perspective, written in honor of Dr. William E. Paul, we describe the initial discovery of one of the dominant substrates for tyrosine phosphorylation stimulated by IL-4. We further describe how this “IL-4-induced phosphorylated substrate” (4PS was characterized as a member of the insulin receptor substrate (IRS family of large adaptor proteins that link IL-4 and insulin receptors to activation of the phosphatidyl-inositol 3′ kinase pathway as well as other downstream signaling pathways. The relative contribution of the 4PS/IRS pathway to the early models of IL-4-induced proliferation and suppression of apoptosis are compared to our more recent understanding of the complex interplay between positive and negative regulatory pathways emanating from members of the IRS family that impact allergic responses.

  13. MET receptor tyrosine kinase controls dendritic complexity, spine morphogenesis, and glutamatergic synapse maturation in the hippocampus.

    Science.gov (United States)

    Qiu, Shenfeng; Lu, Zhongming; Levitt, Pat

    2014-12-03

    The MET receptor tyrosine kinase (RTK), implicated in risk for autism spectrum disorder (ASD) and in functional and structural circuit integrity in humans, is a temporally and spatially regulated receptor enriched in dorsal pallial-derived structures during mouse forebrain development. Here we report that loss or gain of function of MET in vitro or in vivo leads to changes, opposite in nature, in dendritic complexity, spine morphogenesis, and the timing of glutamatergic synapse maturation onto hippocampus CA1 neurons. Consistent with the morphological and biochemical changes, deletion of Met in mutant mice results in precocious maturation of excitatory synapse, as indicated by a reduction of the proportion of silent synapses, a faster GluN2A subunit switch, and an enhanced acquisition of AMPA receptors at synaptic sites. Thus, MET-mediated signaling appears to serve as a mechanism for controlling the timing of neuronal growth and functional maturation. These studies suggest that mistimed maturation of glutamatergic synapses leads to the aberrant neural circuits that may be associated with ASD risk. Copyright © 2014 the authors 0270-6474/14/3416166-14$15.00/0.

  14. Blind identification and separation of complex-valued signals

    CERN Document Server

    Moreau, Eric

    2013-01-01

    Blind identification consists of estimating a multi-dimensional system only through the use of its output, and source separation, the blind estimation of the inverse of the system. Estimation is generally carried out using different statistics of the output. The authors of this book consider the blind identification and source separation problem in the complex-domain, where the available statistical properties are richer and include non-circularity of the sources - underlying components. They define identifiability conditions and present state-of-the-art algorithms that are based on algebraic methods as well as iterative algorithms based on maximum likelihood theory. Contents 1. Mathematical Preliminaries. 2. Estimation by Joint Diagonalization. 3. Maximum Likelihood ICA. About the Authors Eric Moreau is Professor of Electrical Engineering at the University of Toulon, France. His research interests concern statistical signal processing, high order statistics and matrix/tensor decompositions with applic...

  15. Evaluation of Intracellular Signaling Downstream Chimeric Antigen Receptors.

    Directory of Open Access Journals (Sweden)

    Hannah Karlsson

    Full Text Available CD19-targeting CAR T cells have shown potency in clinical trials targeting B cell leukemia. Although mainly second generation (2G CARs carrying CD28 or 4-1BB have been investigated in patients, preclinical studies suggest that third generation (3G CARs with both CD28 and 4-1BB have enhanced capacity. However, little is known about the intracellular signaling pathways downstream of CARs. In the present work, we have analyzed the signaling capacity post antigen stimulation in both 2G and 3G CARs. 3G CAR T cells expanded better than 2G CAR T cells upon repeated stimulation with IL-2 and autologous B cells. An antigen-driven accumulation of CAR+ cells was evident post antigen stimulation. The cytotoxicity of both 2G and 3G CAR T cells was maintained by repeated stimulation. The phosphorylation status of intracellular signaling proteins post antigen stimulation showed that 3G CAR T cells had a higher activation status than 2G. Several proteins involved in signaling downstream the TCR were activated, as were proteins involved in the cell cycle, cell adhesion and exocytosis. In conclusion, 3G CAR T cells had a higher degree of intracellular signaling activity than 2G CARs which may explain the increased proliferative capacity seen in 3G CAR T cells. The study also indicates that there may be other signaling pathways to consider when designing or evaluating new generations of CARs.

  16. Microscopic insight into thermodynamics of conformational changes of SAP-SLAM complex in signal transduction cascade

    Science.gov (United States)

    Samanta, Sudipta; Mukherjee, Sanchita

    2017-04-01

    The signalling lymphocytic activation molecule (SLAM) family of receptors, expressed by an array of immune cells, associate with SLAM-associated protein (SAP)-related molecules, composed of single SH2 domain architecture. SAP activates Src-family kinase Fyn after SLAM ligation, resulting in a SLAM-SAP-Fyn complex, where, SAP binds the Fyn SH3 domain that does not involve canonical SH3 or SH2 interactions. This demands insight into this SAP mediated signalling cascade. Thermodynamics of the conformational changes are extracted from the histograms of dihedral angles obtained from the all-atom molecular dynamics simulations of this structurally well characterized SAP-SLAM complex. The results incorporate the binding induced thermodynamic changes of individual amino acid as well as the secondary structural elements of the protein and the solvent. Stabilization of the peptide partially comes through a strong hydrogen bonding network with the protein, while hydrophobic interactions also play a significant role where the peptide inserts itself into a hydrophobic cavity of the protein. SLAM binding widens SAP's second binding site for Fyn, which is the next step in the signal transduction cascade. The higher stabilization and less fluctuation of specific residues of SAP in the Fyn binding site, induced by SAP-SLAM complexation, emerge as the key structural elements to trigger the recognition of SAP by the SH3 domain of Fyn. The thermodynamic quantification of the protein due to complexation not only throws deeper understanding in the established mode of SAP-SLAM interaction but also assists in the recognition of the relevant residues of the protein responsible for alterations in its activity.

  17. Arabidopsis CPR5 regulates ethylene signaling via molecular association with the ETR1 receptor.

    Science.gov (United States)

    Wang, Feifei; Wang, Lijuan; Qiao, Longfei; Chen, Jiacai; Pappa, Maria Belen; Pei, Haixia; Zhang, Tao; Chang, Caren; Dong, Chun-Hai

    2017-11-01

    The plant hormone ethylene plays various functions in plant growth, development and response to environmental stress. Ethylene is perceived by membrane-bound ethylene receptors, and among the homologous receptors in Arabidopsis, the ETR1 ethylene receptor plays a major role. The present study provides evidence demonstrating that Arabidopsis CPR5 functions as a novel ETR1 receptor-interacting protein in regulating ethylene response and signaling. Yeast split ubiquitin assays and bi-fluorescence complementation studies in plant cells indicated that CPR5 directly interacts with the ETR1 receptor. Genetic analyses indicated that mutant alleles of cpr5 can suppress ethylene insensitivity in both etr1-1 and etr1-2, but not in other dominant ethylene receptor mutants. Overexpression of Arabidopsis CPR5 either in transgenic Arabidopsis plants, or ectopically in tobacco, significantly enhanced ethylene sensitivity. These findings indicate that CPR5 plays a critical role in regulating ethylene signaling. CPR5 is localized to endomembrane structures and the nucleus, and is involved in various regulatory pathways, including pathogenesis, leaf senescence, and spontaneous cell death. This study provides evidence for a novel regulatory function played by CPR5 in the ethylene receptor signaling pathway in Arabidopsis. © 2017 Institute of Botany, Chinese Academy of Sciences.

  18. Interplay between TGF-β signaling and receptor tyrosine kinases in tumor development.

    Science.gov (United States)

    Shi, Qiaoni; Chen, Ye-Guang

    2017-10-01

    Transforming growth factor-β (TGF-β) signaling regulates cell proliferation, differentiation, migration and death, and plays a critical role in embryogenesis and tissue homeostasis. Its deregulation results in various diseases including tumor formation. Receptor tyrosine kinases (RTKs), such as epidermal growth factor receptor (EGFR), fibroblast growth factor receptor (FGFR), vascular endothelial growth factor receptor (VEGFR) and platelet-derived growth factor receptor (PDGFR), also play key roles in the development and progression of many types of tumors. It has been realized that TGF-β signaling and RTK pathways interact with each other and their interplay is important for cancer development. They are mutually regulated and cooperatively modulate cell survival and migration, epithelial-mesenchymal transition, and tumor microenvironment to accelerate tumorigenesis and tumor metastasis. RTKs can modulate Smad-dependent transcription or cooperate with TGF-β to potentiate its oncogenic activity, while TGF-β signaling can in turn control RTK signaling by regulating their activities or expression. This review summarizes current understandings of the interplay between TGF-β signaling and RTKs and its influence on tumor development.

  19. Terbutaline causes immobilization of single β2-adrenergic receptor-ligand complexes in the plasma membrane of living A549 cells as revealed by single-molecule microscopy

    Science.gov (United States)

    Sieben, Anne; Kaminski, Tim; Kubitscheck, Ulrich; Häberlein, Hanns

    2011-02-01

    G-protein-coupled receptors are important targets for various drugs. After signal transduction, regulatory processes, such as receptor desensitization and internalization, change the lateral receptor mobility. In order to study the lateral diffusion of β2-adrenergic receptors (β2AR) complexed with fluorescently labeled noradrenaline (Alexa-NA) in plasma membranes of A549 cells, trajectories of single receptor-ligand complexes were monitored using single-particle tracking. We found that a fraction of 18% of all β2ARs are constitutively immobile. About 2/3 of the β2ARs moved with a diffusion constant of D2 = 0.03+/-0.001 μm2/s and about 17% were diffusing five-fold faster (D3 = 0.15+/-0.02 μm2/s). The mobile receptors moved within restricted domains and also showed a discontinuous diffusion behavior. Analysis of the trajectory lengths revealed two different binding durations with τ1 = 77+/-1 ms and τ2 = 388+/-11 ms. Agonistic stimulation of the β2AR-Alexa-NA complexes with 1 μM terbutaline caused immobilization of almost 50% of the receptors within 35 min. Simultaneously, the mean area covered by the mobile receptors decreased significantly. Thus, we demonstrated that agonistic stimulation followed by cell regulatory processes results in a change in β2AR mobility suggesting that different receptor dynamics characterize different receptor states.

  20. Singular Location and Signaling Profile of Adenosine A2A-Cannabinoid CB1 Receptor Heteromers in the Dorsal Striatum.

    Science.gov (United States)

    Moreno, Estefanía; Chiarlone, Anna; Medrano, Mireia; Puigdellívol, Mar; Bibic, Lucka; Howell, Lesley A; Resel, Eva; Puente, Nagore; Casarejos, María J; Perucho, Juan; Botta, Joaquín; Suelves, Nuria; Ciruela, Francisco; Ginés, Silvia; Galve-Roperh, Ismael; Casadó, Vicent; Grandes, Pedro; Lutz, Beat; Monory, Krisztina; Canela, Enric I; Lluís, Carmen; McCormick, Peter J; Guzmán, Manuel

    2018-04-01

    The dorsal striatum is a key node for many neurobiological processes such as motor activity, cognitive functions, and affective processes. The proper functioning of striatal neurons relies critically on metabotropic receptors. Specifically, the main adenosine and endocannabinoid receptors present in the striatum, ie, adenosine A 2A receptor (A 2A R) and cannabinoid CB 1 receptor (CB 1 R), are of pivotal importance in the control of neuronal excitability. Facilitatory and inhibitory functional interactions between striatal A 2A R and CB 1 R have been reported, and evidence supports that this cross-talk may rely, at least in part, on the formation of A 2A R-CB 1 R heteromeric complexes. However, the specific location and properties of these heteromers have remained largely unknown. Here, by using techniques that allowed a precise visualization of the heteromers in situ in combination with sophisticated genetically modified animal models, together with biochemical and pharmacological approaches, we provide a high-resolution expression map and a detailed functional characterization of A 2A R-CB 1 R heteromers in the dorsal striatum. Specifically, our data unveil that the A 2A R-CB 1 R heteromer (i) is essentially absent from corticostriatal projections and striatonigral neurons, and, instead, is largely present in striatopallidal neurons, (ii) displays a striking G protein-coupled signaling profile, where co-stimulation of both receptors leads to strongly reduced downstream signaling, and (iii) undergoes an unprecedented dysfunction in Huntington's disease, an archetypal disease that affects striatal neurons. Altogether, our findings may open a new conceptual framework to understand the role of coordinated adenosine-endocannabinoid signaling in the indirect striatal pathway, which may be relevant in motor function and neurodegenerative diseases.

  1. Distinct signalling properties of insulin receptor substrate (IRS)-1 and IRS-2 in mediating insulin/IGF-1 action

    DEFF Research Database (Denmark)

    Rabiee, Atefeh; Krüger, Marcus; Ardenkjær-Larsen, Jacob

    2018-01-01

    Insulin/IGF-1 action is driven by a complex and highly integrated signalling network. Loss-of-function studies indicate that the major insulin/IGF-1 receptor substrate (IRS) proteins, IRS-1 and IRS-2, mediate different biological functions in vitro and in vivo, suggesting specific signalling...... properties despite their high degree of homology. To identify mechanisms contributing to the differential signalling properties of IRS-1 and IRS-2 in the mediation of insulin/IGF-1 action, we performed comprehensive mass spectrometry (MS)-based phosphoproteomic profiling of brown preadipocytes from wild type......, IRS-1-/-and IRS-2-/-mice in the basal and IGF-1-stimulated states. We applied stable isotope labeling by amino acids in cell culture (SILAC) for the accurate quantitation of changes in protein phosphorylation. We found ~10% of the 6262 unique phosphorylation sites detected to be regulated by IGF-1...

  2. A model for the biosynthesis and transport of plasma membrane-associated signaling receptors to the cell surface

    Directory of Open Access Journals (Sweden)

    Sorina Claudia Popescu

    2012-04-01

    Full Text Available Intracellular protein transport is emerging as critical in determining the outcome of receptor-activated signal transduction pathways. In plants, relatively little is known about the nature of the molecular components and mechanisms involved in coordinating receptor synthesis and transport to the cell surface. Recent advances in this field indicate that signaling pathways and intracellular transport machinery converge and coordinate to render receptors competent for signaling at their plasma membrane activity sites. The biogenesis and transport to the cell surface of signaling receptors appears to require both general trafficking and receptor-specific factors. Several molecular determinants, residing or associated with compartments of the secretory pathway and known to influence aspects in receptor biogenesis, are discussed and integrated into a predictive cooperative model for the functional expression of signaling receptors at the plasma membrane.

  3. Association of cannabis use during adolescence, prefrontal CB1 receptor signaling and schizophrenia

    Directory of Open Access Journals (Sweden)

    Adriana eCaballero

    2012-05-01

    Full Text Available The cannabinoid receptor 1 (CB1R is the G-protein coupled receptor responsible for the majority of the endocannabinoid signaling in the human brain. It is widely distributed in the limbic system, basal ganglia, and cerebellum, which are areas responsible for cognition, memory, and motor control. Because of this widespread distribution, it is not surprising that drugs that co-opt CB1R have expected behavioral outcomes consistent with dysregulated signaling from these areas (e.g. memory loss, cognitive deficits, etc. In the context of this review, we present evidence for the role of CB1R signaling in the prefrontal cortex (PFC, an area involved in executive functions, with emphasis on the developmental regulation of CB1R signaling in the acquisition of mature PFC function. We further hypothesize how alterations of CB1R signaling specifically during adolescent maturation might confer liability to psychiatric disorders.

  4. Total Lipopolysaccharide from the Human Gut Microbiome Silences Toll-Like Receptor Signaling.

    Science.gov (United States)

    d'Hennezel, Eva; Abubucker, Sahar; Murphy, Leon O; Cullen, Thomas W

    2017-01-01

    Cohabitation of microbial communities with the host enables the formation of a symbiotic relationship that maintains homeostasis in the gut and beyond. One prevailing model suggests that this relationship relies on the capacity of host cells and tissues to remain tolerant to the strong immune stimulation generated by the microbiota such as the activation of Toll-like receptor 4 (TLR4) pathways by lipopolysaccharide (LPS). Indeed, gut microbial LPS is thought to be one of the most potent activators of innate immune signaling and an important mediator of the microbiome's influence on host physiology. In this study, we performed computational and experimental analyses of healthy human fecal samples to examine the TLR4 signaling capacity of the gut microbiota. These analyses revealed that an immunoinhibitory activity of LPS, conserved across the members of the order Bacteroidales and derived from an underacylated structural feature, silences TLR4 signaling for the entire consortium of organisms inhabiting the human gut. Comparative analysis of metagenomic data from the Human Microbiome Project and healthy-donor samples indicates that immune silencing via LPS is a microbe-intrinsic feature in all healthy adults. These findings challenge the current belief that robust TLR4 signaling is a feature of the microbiome and demonstrate that microbiome-derived LPS has the ability to facilitate host tolerance of gut microbes. These findings have broad implications for how we model host-microbe interactions and for our understanding of microbiome-linked disease. IMPORTANCE While the ability for humans to host a complex microbial ecosystem is an essential property of life, the mechanisms allowing for immune tolerance of such a large microbial load are not completely understood and are currently the focus of intense research. This study shows that an important proinflammatory pathway that is commonly triggered by pathogenic bacteria upon interaction with the host is, in fact

  5. Dangerous mating systems: signal complexity, signal content and neural capacity in spiders.

    Science.gov (United States)

    Herberstein, M E; Wignall, A E; Hebets, E A; Schneider, J M

    2014-10-01

    Spiders are highly efficient predators in possession of exquisite sensory capacities for ambushing prey, combined with machinery for launching rapid and determined attacks. As a consequence, any sexually motivated approach carries a risk of ending up as prey rather than as a mate. Sexual selection has shaped courtship to effectively communicate the presence, identity, motivation and/or quality of potential mates, which help ameliorate these risks. Spiders communicate this information via several sensory channels, including mechanical (e.g. vibrational), visual and/or chemical, with examples of multimodal signalling beginning to emerge in the literature. The diverse environments that spiders inhabit have further shaped courtship content and form. While our understanding of spider neurobiology remains in its infancy, recent studies are highlighting the unique and considerable capacities of spiders to process and respond to complex sexual signals. As a result, the dangerous mating systems of spiders are providing important insights into how ecology shapes the evolution of communication systems, with future work offering the potential to link this complex communication with its neural processes. Copyright © 2014 Elsevier Ltd. All rights reserved.

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

    Science.gov (United States)

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

    2010-07-01

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

  7. Evidence for cooperative signal triggering at the extracellular loops of the TSH receptor.

    Science.gov (United States)

    Kleinau, Gunnar; Jaeschke, Holger; Mueller, Sandra; Raaka, Bruce M; Neumann, Susanne; Paschke, Ralf; Krause, Gerd

    2008-08-01

    The mechanisms governing transition of the thyroid stimulating hormone (TSH) receptor (TSHR) from basal to active conformations are poorly understood. Considering that constitutively activating mutations (CAMs) and inactivating mutations in each of the extracellular loops (ECLs) trigger only partial TSHR activation or inactivation, respectively, we hypothesized that full signaling occurs via multiple extracellular signal propagation events. Therefore, individual CAMs in the extracellular region were combined to create double and triple mutants. In support of our hypothesis, combinations of mutants in the ECLs are in some cases additive, while in others they are even synergistic, with triple mutant I486A/I568V/V656F exhibiting a 70-fold increase in TSH-independent signaling. The proximity but likely different spatial orientation of the residues of activating and inactivating mutations in each ECL supports a dual functionality to facilitate signal induction and conduction, respectively. This is the first report for G-protein coupled receptors, suggesting that multiple and cooperative signal propagating events at all three ECLs are required for full receptor activation. Our findings provide new insights concerning molecular signal transmission from extracellular domains toward the transmembrane helix bundle of the glycoprotein hormone receptors.

  8. ATAR, a novel tumor necrosis factor receptor family member, signals through TRAF2 and TRAF5.

    Science.gov (United States)

    Hsu, H; Solovyev, I; Colombero, A; Elliott, R; Kelley, M; Boyle, W J

    1997-05-23

    Members of tumor necrosis factor receptor (TNFR) family signal largely through interactions with death domain proteins and TRAF proteins. Here we report the identification of a novel TNFR family member ATAR. Human and mouse ATAR contain 283 and 276 amino acids, respectively, making them the shortest known members of the TNFR superfamily. The receptor is expressed mainly in spleen, thymus, bone marrow, lung, and small intestine. The intracellular domains of human and mouse ATAR share only 25% identity, yet both interact with TRAF5 and TRAF2. This TRAF interaction domain resides at the C-terminal 20 amino acids. Like most other TRAF-interacting receptors, overexpression of ATAR activates the transcription factor NF-kappaB. Co-expression of ATAR with TRAF5, but not TRAF2, results in synergistic activation of NF-kappaB, suggesting potentially different roles for TRAF2 and TRAF5 in post-receptor signaling.

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

    OpenAIRE

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

    2016-01-01

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

  10. Signal Transduction of Sphingosine-1-Phosphate G Protein—Coupled Receptors

    Directory of Open Access Journals (Sweden)

    Nicholas Young

    2006-01-01

    Full Text Available Sphingosine-1-phosphate (S1P is a bioactive lipid capable of eliciting dramatic effects in a variety of cell types. Signaling by this molecule is by a family of five G protein—coupled receptors named S1P1–5 that signal through a variety of pathways to regulate cell proliferation, migration, cytoskeletal organization, and differentiation. These receptors are expressed in a wide variety of tissues and cell types, and their cellular effects contribute to important biological and pathological functions of S1P in many processes, including angiogenesis, vascular development, lymphocyte trafficking, and cancer. This review will focus on the current progress in the field of S1P receptor signaling and biology.

  11. Non-genomic actions of aldosterone: From receptors and signals to membrane targets.

    LENUS (Irish Health Repository)

    2012-02-01

    In tissues which express the mineralocorticoid receptor (MR), aldosterone modulates the expression of membrane targets such as the subunits of the epithelial Na(+) channel, in combination with important signalling intermediates such as serum and glucocorticoid-regulated kinase-1. In addition, the rapid \\'non-genomic\\' activation of protein kinases and secondary messenger signalling cascades has also been detected in aldosterone-sensitive tissues of the nephron, distal colon and cardiovascular system. These rapid actions are variously described as being coupled to MR or to an as yet unidentified, membrane-associated aldosterone receptor. The rapidly activated signalling cascades add a level of fine-tuning to the activity of aldosterone-responsive membrane transporters and also modulate the aldosterone-induced changes in gene expression through receptor and transcription factor phosphorylation.

  12. Non-genomic actions of aldosterone: From receptors and signals to membrane targets.

    LENUS (Irish Health Repository)

    Dooley, Ruth

    2011-07-26

    In tissues which express the mineralocorticoid receptor (MR), aldosterone modulates the expression of membrane targets such as the subunits of the epithelial Na(+) channel, in combination with important signalling intermediates such as serum and glucocorticoid-regulated kinase-1. In addition, the rapid \\'non-genomic\\' activation of protein kinases and secondary messenger signalling cascades has also been detected in aldosterone-sensitive tissues of the nephron, distal colon and cardiovascular system. These rapid actions are variously described as being coupled to MR or to an as yet unidentified, membrane-associated aldosterone receptor. The rapidly activated signalling cascades add a level of fine-tuning to the activity of aldosterone-responsive membrane transporters and also modulate the aldosterone-induced changes in gene expression through receptor and transcription factor phosphorylation.

  13. Delineation of the GPRC6A Receptor Signaling Pathways Using a Mammalian Cell Line Stably Expressing the Receptor

    DEFF Research Database (Denmark)

    Jacobsen, Stine Engesgaard; Nørskov-Lauritsen, Lenea; Thomsen, Alex Rojas Bie

    2013-01-01

    receptor has been suggested to couple to multiple G protein classes albeit via indirect methods. Thus, the exact ligand preferences and signaling pathways are yet to be elucidated. In the present study, we generated a Chinese hamster ovary (CHO) cell line that stably expresses mouse GPRC6A. In an effort...... and divalent cations, and for the first time, we conclusively show that these responses are mediated through the Gq pathway. We were not able to confirm previously published data demonstrating Gi- and Gs-mediated signaling; neither could we detect agonistic activity of testosterone and osteocalcin. Generation...... of the stable CHO cell line with robust receptor responsiveness and optimization of the highly sensitive homogeneous time resolved fluorescence technology allow fast assessment of Gq activation without previous manipulations like cotransfection of mutated G proteins. This cell-based assay system for GPRC6A...

  14. A Significant Role of the Truncated Ghrelin Receptor GHS-R1b in Ghrelin-induced Signaling in Neurons.

    Science.gov (United States)

    Navarro, Gemma; Aguinaga, David; Angelats, Edgar; Medrano, Mireia; Moreno, Estefanía; Mallol, Josefa; Cortés, Antonio; Canela, Enric I; Casadó, Vicent; McCormick, Peter J; Lluís, Carme; Ferré, Sergi

    2016-06-17

    The truncated non-signaling ghrelin receptor growth hormone secretagogue R1b (GHS-R1b) has been suggested to simply exert a dominant negative role in the trafficking and signaling of the full and functional ghrelin receptor GHS-R1a. Here we reveal a more complex modulatory role of GHS-R1b. Differential co-expression of GHS-R1a and GHS-R1b, both in HEK-293T cells and in striatal and hippocampal neurons in culture, demonstrates that GHS-R1b acts as a dual modulator of GHS-R1a function: low relative GHS-R1b expression potentiates and high relative GHS-R1b expression inhibits GHS-R1a function by facilitating GHS-R1a trafficking to the plasma membrane and by exerting a negative allosteric effect on GHS-R1a signaling, respectively. We found a preferential Gi/o coupling of the GHS-R1a-GHS-R1b complex in HEK-293T cells and, unexpectedly, a preferential Gs/olf coupling in both striatal and hippocampal neurons in culture. A dopamine D1 receptor (D1R) antagonist blocked ghrelin-induced cAMP accumulation in striatal but not hippocampal neurons, indicating the involvement of D1R in the striatal GHS-R1a-Gs/olf coupling. Experiments in HEK-293T cells demonstrated that D1R co-expression promotes a switch in GHS-R1a-G protein coupling from Gi/o to Gs/olf, but only upon co-expression of GHS-R1b. Furthermore, resonance energy transfer experiments showed that D1R interacts with GHS-R1a, but only in the presence of GHS-R1b. Therefore, GHS-R1b not only determines the efficacy of ghrelin-induced GHS-R1a-mediated signaling but also determines the ability of GHS-R1a to form oligomeric complexes with other receptors, promoting profound qualitative changes in ghrelin-induced signaling. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  15. A Significant Role of the Truncated Ghrelin Receptor GHS-R1b in Ghrelin-induced Signaling in Neurons*

    Science.gov (United States)

    Navarro, Gemma; Aguinaga, David; Angelats, Edgar; Medrano, Mireia; Moreno, Estefanía; Mallol, Josefa; Cortés, Antonio; Canela, Enric I.; Casadó, Vicent; McCormick, Peter J.; Lluís, Carme; Ferré, Sergi

    2016-01-01

    The truncated non-signaling ghrelin receptor growth hormone secretagogue R1b (GHS-R1b) has been suggested to simply exert a dominant negative role in the trafficking and signaling of the full and functional ghrelin receptor GHS-R1a. Here we reveal a more complex modulatory role of GHS-R1b. Differential co-expression of GHS-R1a and GHS-R1b, both in HEK-293T cells and in striatal and hippocampal neurons in culture, demonstrates that GHS-R1b acts as a dual modulator of GHS-R1a function: low relative GHS-R1b expression potentiates and high relative GHS-R1b expression inhibits GHS-R1a function by facilitating GHS-R1a trafficking to the plasma membrane and by exerting a negative allosteric effect on GHS-R1a signaling, respectively. We found a preferential Gi/o coupling of the GHS-R1a-GHS-R1b complex in HEK-293T cells and, unexpectedly, a preferential Gs/olf coupling in both striatal and hippocampal neurons in culture. A dopamine D1 receptor (D1R) antagonist blocked ghrelin-induced cAMP accumulation in striatal but not hippocampal neurons, indicating the involvement of D1R in the striatal GHS-R1a-Gs/olf coupling. Experiments in HEK-293T cells demonstrated that D1R co-expression promotes a switch in GHS-R1a-G protein coupling from Gi/o to Gs/olf, but only upon co-expression of GHS-R1b. Furthermore, resonance energy transfer experiments showed that D1R interacts with GHS-R1a, but only in the presence of GHS-R1b. Therefore, GHS-R1b not only determines the efficacy of ghrelin-induced GHS-R1a-mediated signaling but also determines the ability of GHS-R1a to form oligomeric complexes with other receptors, promoting profound qualitative changes in ghrelin-induced signaling. PMID:27129257

  16. Activation of GABAB receptors inhibits protein kinase B /Glycogen Synthase Kinase 3 signaling

    Directory of Open Access Journals (Sweden)

    Lu Frances Fangjia

    2012-11-01

    Full Text Available Abstract Accumulated evidence has suggested that potentiation of cortical GABAergic inhibitory neurotransmission may be a key mechanism in the treatment of schizophrenia. However, the downstream molecular mechanisms related to GABA potentiation remain unexplored. Recent studies have suggested that dopamine D2 receptor antagonists, which are used in the clinical treatment of schizophrenia, modulate protein kinase B (Akt/glycogen synthase kinase (GSK-3 signaling. Here we report that activation of GABAB receptors significantly inhibits Akt/GSK-3 signaling in a β-arrestin-dependent pathway. Agonist stimulation of GABAB receptors enhances the phosphorylation of Akt (Thr-308 and enhances the phosphorylation of GSK-3α (Ser-21/β (Ser-9 in both HEK-293T cells expressing GABAB receptors and rat hippocampal slices. Furthermore, knocking down the expression of β-arrestin2 using siRNA abolishes the GABAB receptor-mediated modulation of GSK-3 signaling. Our data may help to identify potentially novel targets through which GABAB receptor agents may exert therapeutic effects in the treatment of schizophrenia.

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

    Science.gov (United States)

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

    2018-04-27

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

  18. Quantitative properties and receptor reserve of the IP(3) and calcium branch of G(q)-coupled receptor signaling.

    Science.gov (United States)

    Dickson, Eamonn J; Falkenburger, Björn H; Hille, Bertil

    2013-05-01

    Gq-coupled plasma membrane receptors activate phospholipase C (PLC), which hydrolyzes membrane phosphatidylinositol 4,5-bisphosphate (PIP2) into the second messengers inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DAG). This leads to calcium release, protein kinase C (PKC) activation, and sometimes PIP2 depletion. To understand mechanisms governing these diverging signals and to determine which of these signals is responsible for the inhibition of KCNQ2/3 (KV7.2/7.3) potassium channels, we monitored levels of PIP2, IP3, and calcium in single living cells. DAG and PKC are monitored in our companion paper (Falkenburger et al. 2013. J. Gen. Physiol. http://dx.doi.org/10.1085/jgp.201210887). The results extend our previous kinetic model of Gq-coupled receptor signaling to IP3 and calcium. We find that activation of low-abundance endogenous P2Y2 receptors by a saturating concentration of uridine 5'-triphosphate (UTP; 100 µM) leads to calcium release but not to PIP2 depletion. Activation of overexpressed M1 muscarinic receptors by 10 µM Oxo-M leads to a similar calcium release but also depletes PIP2. KCNQ2/3 channels are inhibited by Oxo-M (by 85%), but not by UTP (calcium responses can be elicited even after PIP2 was partially depleted by overexpressed inducible phosphatidylinositol 5-phosphatases, suggesting that very low amounts of IP3 suffice to elicit a full calcium release. Hence, weak PLC activation can elicit robust calcium signals without net PIP2 depletion or KCNQ2/3 channel inhibition.

  19. Anxiolytic-Like Effects of Increased Ghrelin Receptor Signaling in the Amygdala

    DEFF Research Database (Denmark)

    Jensen, Morten; Ratner, Cecilia; Rudenko, Olga

    2016-01-01

    BACKGROUND: Besides the well-known effects of ghrelin on adiposity and food intake regulation, the ghrelin system has been shown to regulate aspects of behavior including anxiety and stress. However, the effect of virus-mediated overexpression of the ghrelin receptor in the amygdala has...... not previously been addressed directly. METHOD: First, we examined the acute effect of peripheral ghrelin administration on anxiety- and depression-like behavior using the open field, elevated plus maze, forced swim and tail suspension tests. Next, we examined the effect of peripheral ghrelin administration...... and ghrelin receptor deficiency on stress in a familiar and social environment using the Intellicage system. Importantly, we also used a novel approach to study ghrelin receptor signaling in the brain by overexpressing the ghrelin receptor in the amygdala. We examined the effect of ghrelin receptor...

  20. A single amino acid residue controls Ca2+ signaling by an octopamine receptor from Drosophila melanogaster.

    Science.gov (United States)

    Hoff, Max; Balfanz, Sabine; Ehling, Petra; Gensch, Thomas; Baumann, Arnd

    2011-07-01

    Rhythmic activity of cells and cellular networks plays an important role in physiology. In the nervous system oscillations of electrical activity and/or second messenger concentrations are important to synchronize neuronal activity. At the molecular level, rhythmic activity can be initiated by different routes. We have recently shown that an octopamine-activated G-protein-coupled receptor (GPCR; DmOctα1Rb, CG3856) from Drosophila initiates Ca(2+) oscillations. Here, we have unraveled the molecular basis of cellular Ca(2+) signaling controlled by the DmOctα1Rb receptor using a combination of pharmacological intervention, site-directed mutagenesis, and functional cellular Ca(2+) imaging on heterologously expressed receptors. Phosphorylation of a single amino acid residue in the third intracellular loop of the GPCR by PKC is necessary and sufficient to desensitize the receptor. From its desensitized state, DmOctα1Rb is resensitized by dephosphorylation, and a new Ca(2+) signal occurs on octopamine stimulation. Our findings show that transient changes of the receptor's surface profile have a strong effect on its physiological signaling properties. We expect that the detailed knowledge of DmOctα1Rb-dependent signal transduction fosters the identification of specific drugs that can be used for GPCR-mediated pest control, since octopamine serves important physiological and behavioral functions in arthropods.

  1. Negative Regulation of Receptor Tyrosine Kinase (RTK Signaling: A Developing Field

    Directory of Open Access Journals (Sweden)

    Fernanda Ledda

    2007-01-01

    Full Text Available ophic factors control cellular physiology by activating specific receptor tyrosine kinases (RTKs. While the over activation of RTK signaling pathways is associated with cell growth and cancer, recent findings support the concept that impaired down-regulation or deactivation of RTKs may also be a mechanism involved in tumor formation. Under this perspective, the molecular determinants of RTK signaling inhibition may act as tumor-suppressor genes and have a potential role as tumor markers to monitor and predict disease progression. Here, we review the current understanding of the physiological mechanisms that attenuate RTK signaling and discuss evidence that implicates deregulation of these events in cancer.Abbreviations: BDP1: Brain-derived phosphatase 1; Cbl: Casitas B-lineage lymphoma; CIN-85: Cbl-interacting protein of 85 kDa; DER: Drosophila EGFR; EGFR: Epidermal growth factor receptor; ERK 1/2: Extracellular signal-regulated kinase 1/2; Grb2: Growth factor receptor-bound protein 2; HER2: Human epidermal growth factor receptor 2; LRIG: Leucine-rich repeats and immunoglobulin-like domain 1; MAPK: Mitogen-activated protein kinase; Mig 6: Mitogen-inducible gene 6; PTEN: Phosphatase and tensin homologue; RET: Rearranged in transformation; RTK: Receptor tyrosine kinase. SH2 domain: Src-homology 2 domain; SH3 domain: Src-homology 3 domain; Spry: Sprouty.

  2. Death receptor Fas (CD95) signaling in the central nervous system: tuning neuroplasticity?

    Science.gov (United States)

    Reich, Arno; Spering, Christopher; Schulz, Jörg B

    2008-09-01

    For over a decade, neuroscientific research has focused on processes of apoptosis and its contribution to the pathophysiology of neurological diseases. In the central nervous system, the degree of intrinsic mitochondrial-mediated apoptotic signaling expresses a cell's individual metabolic stress, whereas activation of the extrinsic death receptor-induced cascade is regarded as a sign of imbalanced cellular networks. Under physiological conditions, most neurons possess death receptors without being sensitive to receptor-mediated apoptosis. This paradox raises two questions: what is the evolutionary advantage of expressing potentially harmful proteins? How is their signaling controlled? This review summarizes the functional relevance of FasL-Fas signaling--a quintessential death ligand/receptor system--in different neurological disease models ranging from traumatic, inflammatory and ischemic to neurodegenerative processes. Furthermore, it outlines alternative non-apoptotic Fas signaling, shedding new light on its neuroplastic capacity. Finally, receptor-proximal regulatory proteins are introduced and identified as potential protagonists of disease-modifying neurological therapies.

  3. Molecular Mechanisms of SH2- and PTB-Domain-Containing Proteins in Receptor Tyrosine Kinase Signaling

    Science.gov (United States)

    Wagner, Melany J.; Stacey, Melissa M.; Liu, Bernard A.; Pawson, Tony

    2013-01-01

    Intracellular signaling is mediated by reversible posttranslational modifications (PTMs) that include phosphorylation, ubiquitination, and acetylation, among others. In response to extracellular stimuli such as growth factors, receptor tyrosine kinases (RTKs) typically dimerize and initiate signaling through phosphorylation of their cytoplasmic tails and downstream scaffolds. Signaling effectors are recruited to these phosphotyrosine (pTyr) sites primarily through Src homology 2 (SH2) domains and pTyr-binding (PTB) domains. This review describes how these conserved domains specifically recognize pTyr residues and play a major role in mediating precise downstream signaling events. PMID:24296166

  4. Molecular mechanisms of SH2- and PTB-domain-containing proteins in receptor tyrosine kinase signaling.

    Science.gov (United States)

    Wagner, Melany J; Stacey, Melissa M; Liu, Bernard A; Pawson, Tony

    2013-12-01

    Intracellular signaling is mediated by reversible posttranslational modifications (PTMs) that include phosphorylation, ubiquitination, and acetylation, among others. In response to extracellular stimuli such as growth factors, receptor tyrosine kinases (RTKs) typically dimerize and initiate signaling through phosphorylation of their cytoplasmic tails and downstream scaffolds. Signaling effectors are recruited to these phosphotyrosine (pTyr) sites primarily through Src homology 2 (SH2) domains and pTyr-binding (PTB) domains. This review describes how these conserved domains specifically recognize pTyr residues and play a major role in mediating precise downstream signaling events.

  5. Rapid Phospho-Turnover by Receptor Tyrosine Kinases Impacts Downstream Signaling and Drug Binding

    OpenAIRE

    Kleiman, Laura B.; Maiwald, Thomas; Conzelmann, Holger; Lauffenburger, Douglas A.; Sorger, Peter K.

    2011-01-01

    Epidermal growth factor receptors (ErbB1–4) are oncogenic receptor tyrosine kinases (RTKs) that regulate diverse cellular processes. In this study, we combine measurement and mathematical modeling to quantify phospho-turnover at ErbB receptors in human cells and to determine the consequences for signaling and drug binding. We find that phosphotyrosine residues on ErbB1 have half-lives of a few seconds and therefore turn over 100–1000 times in the course of a typical immediate-early response t...

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

    Science.gov (United States)

    Alvaro, Christopher G; Thorner, Jeremy

    2016-04-08

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

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

    Science.gov (United States)

    Dunn, Henry A; Ferguson, Stephen S G

    2015-10-01

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

  8. Normalization of TAM post-receptor signaling reveals a cell invasive signature for Axl tyrosine kinase.

    Science.gov (United States)

    Kimani, Stanley G; Kumar, Sushil; Davra, Viralkumar; Chang, Yun-Juan; Kasikara, Canan; Geng, Ke; Tsou, Wen-I; Wang, Shenyan; Hoque, Mainul; Boháč, Andrej; Lewis-Antes, Anita; De Lorenzo, Mariana S; Kotenko, Sergei V; Birge, Raymond B

    2016-09-06

    Tyro3, Axl, and Mertk (TAMs) are a family of three conserved receptor tyrosine kinases that have pleiotropic roles in innate immunity and homeostasis and when overexpressed in cancer cells can drive tumorigenesis. In the present study, we engineered EGFR/TAM chimeric receptors (EGFR/Tyro3, EGFR/Axl, and EGF/Mertk) with the goals to interrogate post-receptor functions of TAMs, and query whether TAMs have unique or overlapping post-receptor activation profiles. Stable expression of EGFR/TAMs in EGFR-deficient CHO cells afforded robust EGF inducible TAM receptor phosphorylation and activation of downstream signaling. Using a series of unbiased screening approaches, that include kinome-view analysis, phosphor-arrays, RNAseq/GSEA analysis, as well as cell biological and in vivo readouts, we provide evidence that each TAM has unique post-receptor signaling platforms and identify an intrinsic role for Axl that impinges on cell motility and invasion compared to Tyro3 and Mertk. These studies demonstrate that TAM show unique post-receptor signatures that impinge on distinct gene expression profiles and tumorigenic outcomes.

  9. Unsaturated free fatty acids increase benzodiazepine receptor agonist binding depending on the subunit composition of the GABAA receptor complex.

    Science.gov (United States)

    Witt, M R; Westh-Hansen, S E; Rasmussen, P B; Hastrup, S; Nielsen, M

    1996-11-01

    It has been shown previously that unsaturated free fatty acids (FFAs) strongly enhance the binding of agonist benzodiazepine receptor ligands and GABAA receptor ligands in the CNS in vitro. To investigate the selectivity of this effect, recombinant human GABAA/benzodiazepine receptor complexes formed by different subunit compositions (alpha x beta y gamma 2, x = 1, 2, 3, and 5; y = 1, 2, and 3) were expressed using the baculovirus-transfected Sf9 insect cell system. At 10(-4) M, unsaturated FFAs, particularly arachidonic (20:4) and docosahexaenoic (22:6) acids, strongly stimulated (> 200% of control values) the binding of [3H]flunitrazepam ([3H]FNM) to the alpha 3 beta 2 gamma 2 receptor combination in whole cell preparations. No effect or small increases in levels of unsaturated FFAs on [3H]FNM binding to alpha 1 beta x gamma 2 and alpha 2 beta x gamma 2 receptor combinations were observed, and weak effects (130% of control values) were detected using the alpha 5 beta 2 gamma 2 receptor combination. The saturated FFAs, stearic and palmitic acids, were without effect on [3H]FNM binding to any combination of receptor complexes. The hydroxylated unsaturated FFAs, ricinoleic and ricinelaidic acids, were shown to decrease the binding of [3H]FNM only if an alpha 1 beta 2 gamma 2 receptor combination was used. Given the heterogeneity of the GABAA/ benzodiazepine receptor subunit distribution in the CNS, the effects of FFAs on the benzodiazepine receptor can be assumed to vary at both cellular and regional levels.

  10. Increased GABAB receptor signaling in a rat model for schizophrenia

    NARCIS (Netherlands)

    Selten, M.M.; Meyer, F.; Ba, W.; Valles, A.; Maas, D.A.; Negwer, M.J.; Eijsink, V.D.; Vugt, R.W.M. van; Hulten, J.A; Bakel, N.H.M. van; Roosen, J.; Linden, R.J. van der; Schubert, D.; Verheij, M.M.M.; Nadif Kasri, N.; Martens, G.J.M.

    2016-01-01

    Schizophrenia is a complex disorder that affects cognitive function and has been linked, both in patients and animal models, to dysfunction of the GABAergic system. However, the pathophysiological consequences of this dysfunction are not well understood. Here, we examined the GABAergic system in an

  11. Cross-regulation of signaling pathways: An example of nuclear hormone receptors and the canonical Wnt pathway

    Energy Technology Data Exchange (ETDEWEB)

    Beildeck, Marcy E. [Lombardi Comprehensive Cancer Center, Georgetown University, 3970 Reservoir Road, NW, Washington, DC 20057 (United States); Gelmann, Edward P. [Columbia University, Department of Medicine, New York, NY (United States); Byers, Stephen W., E-mail: byerss@georgetown.edu [Lombardi Comprehensive Cancer Center, Georgetown University, 3970 Reservoir Road, NW, Washington, DC 20057 (United States)

    2010-07-01

    Predicting the potential physiological outcome(s) of any given molecular pathway is complex because of cross-talk with other pathways. This is particularly evident in the case of the nuclear hormone receptor and canonical Wnt pathways, which regulate cell growth and proliferation, differentiation, apoptosis, and metastatic potential in numerous tissues. These pathways are known to intersect at many levels: in the intracellular space, at the membrane, in the cytoplasm, and within the nucleus. The outcomes of these interactions are important in the control of stem cell differentiation and maintenance, feedback loops, and regulating oncogenic potential. The aim of this review is to demonstrate the importance of considering pathway cross-talk when predicting functional outcomes of signaling, using nuclear hormone receptor/canonical Wnt pathway cross-talk as an example.

  12. Cross-regulation of signaling pathways: An example of nuclear hormone receptors and the canonical Wnt pathway

    International Nuclear Information System (INIS)

    Beildeck, Marcy E.; Gelmann, Edward P.; Byers, Stephen W.

    2010-01-01

    Predicting the potential physiological outcome(s) of any given molecular pathway is complex because of cross-talk with other pathways. This is particularly evident in the case of the nuclear hormone receptor and canonical Wnt pathways, which regulate cell growth and proliferation, differentiation, apoptosis, and metastatic potential in numerous tissues. These pathways are known to intersect at many levels: in the intracellular space, at the membrane, in the cytoplasm, and within the nucleus. The outcomes of these interactions are important in the control of stem cell differentiation and maintenance, feedback loops, and regulating oncogenic potential. The aim of this review is to demonstrate the importance of considering pathway cross-talk when predicting functional outcomes of signaling, using nuclear hormone receptor/canonical Wnt pathway cross-talk as an example.

  13. Vitamin D controls T cell antigen receptor signaling and activation of human T cells

    DEFF Research Database (Denmark)

    von Essen, Marina Rode; Kongsbak-Wismann, Martin; Schjerling, Peter

    2010-01-01

    Phospholipase C (PLC) isozymes are key signaling proteins downstream of many extracellular stimuli. Here we show that naive human T cells had very low expression of PLC-gamma1 and that this correlated with low T cell antigen receptor (TCR) responsiveness in naive T cells. However, TCR triggering...... led to an upregulation of approximately 75-fold in PLC-gamma1 expression, which correlated with greater TCR responsiveness. Induction of PLC-gamma1 was dependent on vitamin D and expression of the vitamin D receptor (VDR). Naive T cells did not express VDR, but VDR expression was induced by TCR...... signaling via the alternative mitogen-activated protein kinase p38 pathway. Thus, initial TCR signaling via p38 leads to successive induction of VDR and PLC-gamma1, which are required for subsequent classical TCR signaling and T cell activation....

  14. Heparanase augments insulin receptor signaling in breast carcinoma

    Science.gov (United States)

    Goldberg, Rachel; Sonnenblick, Amir; Hermano, Esther; Hamburger, Tamar; Meirovitz, Amichay; Peretz, Tamar; Elkin, Michael

    2017-01-01

    Recently, growing interest in the potential link between metabolic disorders (i.e., diabetes, obesity, metabolic syndrome) and breast cancer has mounted, including studies which indicate that diabetic/hyperinsulinemic women have a significantly higher risk of bearing breast tumors that are more aggressive and associated with higher death rates. Insulin signaling is regarded as a major contributor to this phenomenon; much less is known about the role of heparan sulfate-degrading enzyme heparanase in the link between metabolic disorders and cancer. In the present study we analyzed clinical samples of breast carcinoma derived from diabetic/non-diabetic patients, and investigated effects of heparanase on insulin signaling in breast carcinoma cell lines, as well as insulin-driven growth of breast tumor cells. We demonstrate that heparanase activity leads to enhanced insulin signaling and activation of downstream tumor-promoting pathways in breast carcinoma cells. In agreement, heparanase enhances insulin-induced proliferation of breast tumor cells in vitro. Moreover, analyzing clinical data from diabetic breast carcinoma patients, we found that concurrent presence of both diabetic state and heparanase in tumor tissue (as opposed to either condition alone) was associated with more aggressive phenotype of breast tumors in the patient cohort analyzed in our study (two-sided Fisher's exact test; p=0.04). Our findings highlight the emerging role of heparanase in powering effect of hyperinsulinemic state on breast tumorigenesis and imply that heparanase targeting, which is now under intensive development/clinical testing, could be particularly efficient in a growing fraction of breast carcinoma patients suffering from metabolic disorders. PMID:28038446

  15. Signaling-sensitive amino acids surround the allosteric ligand binding site of the thyrotropin receptor.

    Science.gov (United States)

    Kleinau, Gunnar; Haas, Ann-Karin; Neumann, Susanne; Worth, Catherine L; Hoyer, Inna; Furkert, Jens; Rutz, Claudia; Gershengorn, Marvin C; Schülein, Ralf; Krause, Gerd

    2010-07-01

    The thyrotropin receptor [thyroid-stimulating hormone receptor (TSHR)], a G-protein-coupled receptor (GPCR), is endogenously activated by thyrotropin, which binds to the extracellular region of the receptor. We previously identified a low-molecular-weight (LMW) agonist of the TSHR and predicted its allosteric binding pocket within the receptor's transmembrane domain. Because binding of the LMW agonist probably disrupts interactions or leads to formation of new interactions among amino acid residues surrounding the pocket, we tested whether mutation of residues at these positions would lead to constitutive signaling activity. Guided by molecular modeling, we performed site-directed mutagenesis of 24 amino acids in this spatial region, followed by functional characterization of the mutant receptors in terms of expression and signaling, measured as cAMP accumulation. We found that mutations V421I, Y466A, T501A, L587V, M637C, M637W, S641A, Y643F, L645V, and Y667A located in several helices exhibit constitutive activity. Of note is mutation M637W at position 6.48 in transmembrane helix 6, which has a significant effect on the interaction of the receptor with the LMW agonist. In summary, we found that a high proportion of residues in several helices surrounding the allosteric binding site of LMW ligands in the TSHR when mutated lead to constitutively active receptors. Our findings of signaling-sensitive residues in this region of the transmembrane bundle may be of general importance as this domain appears to be evolutionarily retained among GPCRs.

  16. E3 Ubiquitin Ligase RNF125 Activates Interleukin-36 Receptor Signaling and Contributes to Its Turnover.

    Science.gov (United States)

    Saha, Siddhartha S; Caviness, Gary; Yi, Guanghui; Raymond, Ernest L; Mbow, M Lamine; Kao, C Cheng

    2018-01-01

    Signaling by the interleukin-36 receptor (IL-36R) is linked to inflammatory diseases such as psoriasis. However, the regulation of IL-36R signaling is poorly understood. Activation of IL-36R signaling in cultured cells results in an increased polyubiquitination of the receptor subunit, IL-1Rrp2. Treatment with deubiquitinases shows that the receptor subunit of IL-36R, IL-1Rrp2, is primarily polyubiquitinated at the K63 position, which is associated with endocytic trafficking and signal transduction. A minor amount of ubiquitination is at the K48 position that is associated with protein degradation. A focused siRNA screen identified RNF125, an E3 ubiquitin ligase, to ubiquitinate IL-1Rrp2 upon activation of IL-36R signaling while not affecting the activated IL-1 receptor. Knockdown of RNF125 decreases signal transduction by the IL-36R. Overexpression of RNF125 in HEK293T cells activates IL-36R signaling and increases the ubiquitination of IL-1Rrp2 and its subsequent turnover. RNF125 can coimmunoprecipitate with the IL-36R, and it traffics with IL-1Rrp2 from the cell surface to lysosomes. Mutations of Lys568 and Lys569 in the C-terminal tail of IL-1Rrp2 decrease ubiquitination by RNF125 and increase the steady-state levels of IL-1Rrp2. These results demonstrate that RNF125 has multiple regulatory roles in the signaling, trafficking, and turnover of the IL-36R. © 2017 S. Karger AG, Basel.

  17. Subcellular Localization of Patched and Smoothened, the Receptors for Sonic Hedgehog Signaling, in the Hippocampal Neuron

    OpenAIRE

    Petralia, Ronald S.; Schwartz, Catherine M.; Wang, Ya-Xian; Mattson, Mark P.; Yao, Pamela J.

    2011-01-01

    Cumulative evidence suggests that, aside from patterning the embryonic neural tube, Sonic hedgehog (Shh) signaling plays important roles in the mature nervous system. In this study, we investigate the expression and localization of the Shh signaling receptors, Patched (Ptch) and Smoothened (Smo), in the hippocampal neurons of young and mature rats. Reverse transcriptase-polymerase chain reaction and immunoblotting analyses show that the expression of Ptch and Smo remains at a moderate level i...

  18. DESENSITIZATION PROPERTIES OF P2X3 RECEPTORS SHAPING PAIN SIGNALLING

    Directory of Open Access Journals (Sweden)

    Rashid eGiniatullin

    2013-12-01

    Full Text Available ATP-gated P2X3 receptors are mostly expressed by nociceptive sensory neurons and participate in transduction of pain signals. P2X3 receptors show a combination of fast desensitization onset and slow recovery. Moreover, even low nanomolar agonist concentrations unable to evoke a response, can induce desensitization via a phenomenon called ‘high affinity desensitization’. We have also observed that recovery from desensitization is agonist-specific and can range from seconds to minutes. The recovery process displays unusually high temperature dependence. Likewise, recycling of P2X3 receptors in peri-membrane regions shows unexpectedly large temperature sensitivity. By applying kinetic modeling, we have previously shown that desensitization characteristics of P2X3 receptor are best explained with a cyclic model of receptor operation involving three agonist molecules binding a single receptor and that desensitization is primarily developing from the open receptor state. Mutagenesis experiments suggested that desensitization depends on a certain conformation of the ATP binding pocket and on the structure of the transmembrane domains forming the ion pore. Further molecular determinants of desensitization have been identified by mutating the intracellular N- and C-termini of P2X3 receptor. Unlike other P2X receptors, the P2X3 subtype is facilitated by extracellular calcium that acts via specific sites in the ectodomain neighboring the ATP binding pocket. Thus, substitution of serine275 in this region (called ‘left flipper’ converts the natural facilitation induced by extracellular calcium to receptor inhibition. Given such their strategic location in nociceptive neurons and unique desensitization properties, P2X3 receptors represent an attractive target for development of new analgesic drugs via promotion of desensitization aimed at suppressing chronic pain.

  19. Role of Receptor Tyrosine Kinase Signaling in Renal Fibrosis

    Directory of Open Access Journals (Sweden)

    Feng Liu

    2016-06-01

    Full Text Available Renal fibrosis can be induced in different renal diseases, but ultimately progresses to end stage renal disease. Although the pathophysiologic process of renal fibrosis have not been fully elucidated, it is characterized by glomerulosclerosis and/or tubular interstitial fibrosis, and is believed to be caused by the proliferation of renal inherent cells, including glomerular epithelial cells, mesangial cells, and endothelial cells, along with defective kidney repair, renal interstitial fibroblasts activation, and extracellular matrix deposition. Receptor tyrosine kinases (RTKs regulate a variety of cell physiological processes, including metabolism, growth, differentiation, and survival. Many studies from in vitro and animal models have provided evidence that RTKs play important roles in the pathogenic process of renal fibrosis. It is also showed that tyrosine kinases inhibitors (TKIs have anti-fibrotic effects in basic research and clinical trials. In this review, we summarize the evidence for involvement of specific RTKs in renal fibrosis process and the employment of TKIs as a therapeutic approach for renal fibrosis.

  20. 14-3-3 Proteins Buffer Intracellular Calcium Sensing Receptors to Constrain Signaling.

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    Michael P Grant

    Full Text Available Calcium sensing receptors (CaSR interact with 14-3-3 binding proteins at a carboxyl terminal arginine-rich motif. Mutations identified in patients with familial hypocalciuric hypercalcemia, autosomal dominant hypocalcemia, pancreatitis or idiopathic epilepsy support the functional importance of this motif. We combined total internal reflection fluorescence microscopy and biochemical approaches to determine the mechanism of 14-3-3 protein regulation of CaSR signaling. Loss of 14-3-3 binding caused increased basal CaSR signaling and plasma membrane levels, and a significantly larger signaling-evoked increase in plasma membrane receptors. Block of core glycosylation with tunicamycin demonstrated that changes in plasma membrane CaSR levels were due to differences in exocytic rate. Western blotting to quantify time-dependent changes in maturation of expressed wt CaSR and a 14-3-3 protein binding-defective mutant demonstrated that signaling increases synthesis to maintain constant levels of the immaturely and maturely glycosylated forms. CaSR thus operates by a feed-forward mechanism, whereby signaling not only induces anterograde trafficking of nascent receptors but also increases biosynthesis to maintain steady state levels of net cellular CaSR. Overall, these studies suggest that 14-3-3 binding at the carboxyl terminus provides an important buffering mechanism to increase the intracellular pool of CaSR available for signaling-evoked trafficking, but attenuates trafficking to control the dynamic range of responses to extracellular calcium.

  1. Non-Ligand-Induced Dimerization is Sufficient to Initiate the Signalling and Endocytosis of EGF Receptor

    Directory of Open Access Journals (Sweden)

    George Kourouniotis

    2016-07-01

    Full Text Available The binding of epidermal growth factor (EGF to EGF receptor (EGFR stimulates cell mitogenesis and survival through various signalling cascades. EGF also stimulates rapid EGFR endocytosis and its eventual degradation in lysosomes. The immediate events induced by ligand binding include receptor dimerization, activation of intrinsic tyrosine kinase and autophosphorylation. However, in spite of intensified efforts, the results regarding the roles of these events in EGFR signalling and internalization is still very controversial. In this study, we constructed a chimeric EGFR by replacing its extracellular domain with leucine zipper (LZ and tagged a green fluorescent protein (GFP at its C-terminus. We showed that the chimeric LZ-EGFR-GFP was constitutively dimerized. The LZ-EGFR-GFP dimer autophosphorylated each of its five well-defined C-terminal tyrosine residues as the ligand-induced EGFR dimer does. Phosphorylated LZ-EGFR-GFP was localized to both the plasma membrane and endosomes, suggesting it is capable of endocytosis. We also showed that LZ-EGFR-GFP activated major signalling proteins including Src homology collagen-like (Shc, extracellular signal-regulated kinase (ERK and Akt. Moreover, LZ-EGFR-GFP was able to stimulate cell proliferation. These results indicate that non-ligand induced dimerization is sufficient to activate EGFR and initiate cell signalling and EGFR endocytosis. We conclude that receptor dimerization is a critical event in EGF-induced cell signalling and EGFR endocytosis.

  2. The receptor kinase CERK1 has dual functions in symbiosis and immunity signalling.

    Science.gov (United States)

    Zhang, Xiaowei; Dong, Wentao; Sun, Jongho; Feng, Feng; Deng, Yiwen; He, Zuhua; Oldroyd, Giles E D; Wang, Ertao

    2015-01-01

    The establishment of symbiotic interactions between mycorrhizal fungi, rhizobial bacteria and their legume hosts involves a common symbiosis signalling pathway. This signalling pathway is activated by Nod factors produced by rhizobia and these are recognised by the Nod factor receptors NFR1/LYK3 and NFR5/NFP. Mycorrhizal fungi produce lipochitooligosaccharides (LCOs) similar to Nod factors, as well as short-chain chitin oligomers (CO4/5), implying commonalities in signalling during mycorrhizal and rhizobial associations. Here we show that NFR1/LYK3, but not NFR5/NFP, is required for the establishment of the mycorrhizal interaction in legumes. NFR1/LYK3 is necessary for the recognition of mycorrhizal fungi and the activation of the symbiosis signalling pathway leading to induction of calcium oscillations and gene expression. Chitin oligosaccharides also act as microbe associated molecular patterns that promote plant immunity via similar LysM receptor-like kinases. CERK1 in rice has the highest homology to NFR1 and we show that this gene is also necessary for the establishment of the mycorrhizal interaction as well as for resistance to the rice blast fungus. Our results demonstrate that NFR1/LYK3/OsCERK1 represents a common receptor for chitooligosaccharide-based signals produced by mycorrhizal fungi, rhizobial bacteria (in legumes) and fungal pathogens. It would appear that mycorrhizal recognition has been conserved in multiple receptors across plant species, but additional diversification in certain plant species has defined other signals that this class of receptors can perceive. © 2014 The Authors The Plant Journal © 2014 John Wiley & Sons Ltd.

  3. The Peroxisomal Targeting Signal 1 in sterol carrier protein 2 is autonomous and essential for receptor recognition

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    Bond Charles S

    2011-03-01

    Full Text Available Abstract Background The majority of peroxisomal matrix proteins destined for translocation into the peroxisomal lumen are recognised via a C-terminal Peroxisomal Target Signal type 1 by the cycling receptor Pex5p. The only structure to date of Pex5p in complex with a cargo protein is that of the C-terminal cargo-binding domain of the receptor with sterol carrier protein 2, a small, model peroxisomal protein. In this study, we have tested the contribution of a second, ancillary receptor-cargo binding site, which was found in addition to the characterised Peroxisomal Target Signal type 1. Results To investigate the function of this secondary interface we have mutated two key residues from the ancillary binding site and analyzed the level of binding first by a yeast-two-hybrid assay, followed by quantitative measurement of the binding affinity and kinetics of purified protein components and finally, by in vivo measurements, to determine translocation capability. While a moderate but significant reduction of the interaction was found in binding assays, we were not able to measure any significant defects in vivo. Conclusions Our data therefore suggest that at least in the case of sterol carrier protein 2 the contribution of the second binding site is not essential for peroxisomal import. At this stage, however, we cannot rule out that other cargo proteins may require this ancillary binding site.

  4. Sigma-1 receptor chaperones at the ER-mitochondrion interface regulate Ca(2+) signaling and cell survival.

    Science.gov (United States)

    Hayashi, Teruo; Su, Tsung-Ping

    2007-11-02

    Communication between the endoplasmic reticulum (ER) and mitochondrion is important for bioenergetics and cellular survival. The ER supplies Ca(2+) directly to mitochondria via inositol 1,4,5-trisphosphate receptors (IP3Rs) at close contacts between the two organelles referred to as mitochondrion-associated ER membrane (MAM). We found here that the ER protein sigma-1 receptor (Sig-1R), which is implicated in neuroprotection, carcinogenesis, and neuroplasticity, is a Ca(2+)-sensitive and ligand-operated receptor chaperone at MAM. Normally, Sig-1Rs form a complex at MAM with another chaperone, BiP. Upon ER Ca(2+) depletion or via ligand stimulation, Sig-1Rs dissociate from BiP, leading to a prolonged Ca(2+) signaling into mitochondria via IP3Rs. Sig-1Rs can translocate under chronic ER stress. Increasing Sig-1Rs in cells counteracts ER stress response, whereas decreasing them enhances apoptosis. These results reveal that the orchestrated ER chaperone machinery at MAM, by sensing ER Ca(2+) concentrations, regulates ER-mitochondrial interorganellar Ca(2+) signaling and cell survival.

  5. The matricellular receptor LRP1 forms an interface for signaling and endocytosis in modulation of the extracellular tumor environment

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    Bart eVan Gool

    2015-11-01

    Full Text Available The membrane protein low-density lipoprotein receptor related-protein 1 (LRP1 has been attributed a role in cancer. However, its presumably often indirect involvement is far from understood. LRP1 has both endocytic and signaling activities. As a matricellular receptor it is involved in regulation, mostly by clearing, of various extracellular matrix degrading enzymes including matrix metalloproteinases, serine proteases, protease-inhibitor complexes and the endoglycosidase heparanase. Furthermore, by binding extracellular ligands including growth factors and subsequent intracellular interaction with scaffolding and adaptor proteins it is involved in regulation of various signaling cascades. LRP1 expression levels are often downregulated in cancer and some studies consider low LRP1 levels a poor prognostic factor. On the contrary, upregulation in brain cancers has been noted and clinical trials explore the use of LRP1 as cargo receptor to deliver cytotoxic agents.This mini-review focuses on LRP1’s role in tumor growth and metastasis especially by modulation of the extracellular tumor environment. In relation to this role its diagnostic, prognostic and therapeutic potential will be discussed.

  6. The angiotensin II type 1 receptor antagonist Losartan binds and activates bradykinin B2 receptor signaling

    DEFF Research Database (Denmark)

    Bonde, Marie Mi; Olsen, Kristine Boisen; Erikstrup, Niels

    2011-01-01

    The angiotensin II type 1 receptor (AT1R) blocker (ARB) Losartan has cardioprotective effects during ischemia-reperfusion injury and inhibits reperfusion arrhythmias -effects that go beyond the benefits of lowering blood pressure. The renin-angiotensin and kallikrein-kinin systems are intricately...

  7. Bicaudal-D1 regulates the intracellular sorting and signalling of neurotrophin receptors.

    Science.gov (United States)

    Terenzio, Marco; Golding, Matthew; Russell, Matthew R G; Wicher, Krzysztof B; Rosewell, Ian; Spencer-Dene, Bradley; Ish-Horowicz, David; Schiavo, Giampietro

    2014-07-17

    We have identified a new function for the dynein adaptor Bicaudal D homolog 1 (BICD1) by screening a siRNA library for genes affecting the dynamics of neurotrophin receptor-containing endosomes in motor neurons (MNs). Depleting BICD1 increased the intracellular accumulation of brain-derived neurotrophic factor (BDNF)-activated TrkB and p75 neurotrophin receptor (p75(NTR)) by disrupting the endosomal sorting, reducing lysosomal degradation and increasing the co-localisation of these neurotrophin receptors with retromer-associated sorting nexin 1. The resulting re-routing of active receptors increased their recycling to the plasma membrane and altered the repertoire of signalling-competent TrkB isoforms and p75(NTR) available for ligand binding on the neuronal surface. This resulted in attenuated, but more sustained, AKT activation in response to BDNF stimulation. These data, together with our observation that Bicd1 expression is restricted to the developing nervous system when neurotrophin receptor expression peaks, indicate that BICD1 regulates neurotrophin signalling by modulating the endosomal sorting of internalised ligand-activated receptors. © 2014 The Authors.

  8. Histone H2A.Z is essential for estrogen receptor signaling

    Science.gov (United States)

    Gévry, Nicolas; Hardy, Sara; Jacques, Pierre-Étienne; Laflamme, Liette; Svotelis, Amy; Robert, François; Gaudreau, Luc

    2009-01-01

    Incorporation of H2A.Z into the chromatin of inactive promoters has been shown to poise genes for their expression. Here we provide strong evidence that H2A.Z is incorporated into the promoter regions of estrogen receptor (ERα) target genes only upon gene induction, and that, in a cyclic pattern. Moreover, members of the human H2A.Z-depositing complex, p400, also follow the same gene recruitment kinetics as H2A.Z. Importantly, cellular depletion of H2A.Z or p400 leads to a severe defect in estrogen signaling, including loss of estrogen-specific cell proliferation. We find that incorporation of H2A.Z within TFF1 promoter chromatin allows nucleosomes to adopt preferential positions along the DNA translational axis. Finally, we provide evidence that H2A.Z is essential to allow estrogen-responsive enhancer function. Taken together, our results provide strong mechanistic insight into how H2A.Z regulates ERα-mediated gene expression and provide a novel link between H2A.Z–p400 and ERα-dependent gene regulation and enhancer function. PMID:19515975

  9. Role of ERK/MAPK in endothelin receptor signaling in human aortic smooth muscle cells

    DEFF Research Database (Denmark)

    Chen, Qing-wen; Edvinsson, Lars; Xu, Cang-Bao

    2009-01-01

    muscle cells (VSMCs) through activation of endothelin type A (ETA) and type B (ETB) receptors. The extracellular signal-regulated kinase 1 and 2 (ERK1/2) mitogen-activated protein kinases (MAPK) are involved in ET-1-induced VSMC contraction and proliferation. This study was designed to investigat...

  10. Legume receptors perceive the rhizobial lipochitin oligosaccharide signal molecules by direct binding

    DEFF Research Database (Denmark)

    Broghammer, Angelique; Krusell, Lene; Blaise, Mickael

    2012-01-01

    Lipochitin oligosaccharides called Nod factors function as primary rhizobial signal molecules triggering legumes to develop new plant organs: root nodules that host the bacteria as nitrogen-fixing bacteroids. Here, we show that the Lotus japonicus Nod factor receptor 5 (NFR5) and Nod factor recep...

  11. In Vivo Phosphoproteomics Analysis Reveals the Cardiac Targets of β-Adrenergic Receptor Signaling

    DEFF Research Database (Denmark)

    Lundby, Alicia; Andersen, Martin N; Steffensen, Annette B

    2013-01-01

    β-Blockers are widely used to prevent cardiac arrhythmias and to treat hypertension by inhibiting β-adrenergic receptors (βARs) and thus decreasing contractility and heart rate. βARs initiate phosphorylation-dependent signaling cascades, but only a small number of the target proteins are known. We...

  12. Fluorescence analysis of the Hansenula polymorpha peroxisomal targeting signal-1 receptor, Pex5p

    NARCIS (Netherlands)

    Boteva, R.; Koek, A.; Visser, N.V.; Visser, A.J.W.G.; Krieger, E.; Zlateva, T.; Veenhuis, M.; Klei, van der I.

    2003-01-01

    Correct sorting of newly synthesized peroxisomal matrix proteins is dependent on a peroxisomal targeting signal (PTS). So far two PTSs are known. PTS1 consists of a tripeptide that is located at the extreme C terminus of matrix proteins and is specifically recognized by the PTS1-receptor Pex5p. We

  13. What do we really know about 5-HT1A receptor signaling in neuronal cells?

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    JENNY LUCY FIEDLER

    2016-11-01

    Full Text Available Serotonin (5-HT is a neurotransmitter that plays an important role in neuronal plasticity. Variations in the levels of 5-HT at the synaptic cleft, expression or dysfunction of serotonin receptors may alter brain development and predispose to various mental diseases. Here, we review the transduction pathways described in various cell types transfected with recombinant 5-HT1A receptor (5-HT1AR, specially contrasting with those findings obtained in neuronal cells. The 5-HT1AR is detected in early stages of neural development and is located in the soma, dendrites and spines of hippocampal neurons. The 5-HT1AR differs from other serotonin receptors because it is coupled to different pathways, depending on the targeted cell. The signaling pathway associated with this receptor is determined by Gα isoforms and some cascades involve βγ signaling. The activity of 5-HT1AR usually promotes a reduction in neuronal excitability and firing, provokes a variation in cAMP and Ca2+, levels which may be linked to specific types of behavior and cognition. Furthermore, evidence indicates that 5-HT1AR induces neuritogesis and synapse formation, probably by modulation of the neuronal cytoskeleton through MAPK and PI3K-Akt signaling pathways. Advances in understanding the actions of 5-HT1AR and its association with different signaling pathways in the central nervous system will reveal their pivotal role in health and disease.

  14. Activation of Toll-like receptors nucleates assembly of the MyDDosome signaling hub.

    Science.gov (United States)

    Latty, Sarah Louise; Sakai, Jiro; Hopkins, Lee; Verstak, Brett; Paramo, Teresa; Berglund, Nils A; Cammorota, Eugenia; Cicuta, Pietro; Gay, Nicholas J; Bond, Peter J; Klenerman, David; Bryant, Clare E

    2018-01-24

    Infection and tissue damage induces assembly of supramolecular organizing centres (SMOCs)), such as the Toll-like receptor (TLR) MyDDosome, to co-ordinate inflammatory signaling. SMOC assembly is thought to drive digital all-or-none responses, yet TLR activation by diverse microbes induces anything from mild to severe inflammation. Using single-molecule imaging of TLR4-MyDDosome signaling in living macrophages, we find that MyDDosomes assemble within minutes of TLR4 stimulation. TLR4/MD2 activation leads only to formation of TLR4/MD2 heterotetramers, but not oligomers, suggesting a stoichiometric mismatch between activated receptors and MyDDosomes. The strength of TLR4 signalling depends not only on the number and size of MyDDosomes formed but also how quickly these structures assemble. Activated TLR4, therefore, acts transiently nucleating assembly of MyDDosomes, a process that is uncoupled from receptor activation. These data explain how the oncogenic mutation of MyD88 (L265P) assembles MyDDosomes in the absence of receptor activation to cause constitutive activation of pro-survival NF-κB signalling. © 2018, Latty et al.

  15. Growth Hormone Receptor Signaling Pathways and its Negative Regulation by SOCS2

    DEFF Research Database (Denmark)

    Fernández Pérez, Leandro; Flores-Morales, Amilcar; Guerra, Borja

    2016-01-01

    Growth hormone (GH) is a critical regulator of linear body growth during childhood but continues to have important metabolic actions throughout life. The GH receptor (GHR) is ubiquitously expressed, and deficiency of GHR signaling causes a dramatic impact on normal physiology during somatic devel...

  16. Integration of Nuclear- and Extranuclear-Initiated Estrogen Receptor Signaling in Breast Cancer Cells

    Science.gov (United States)

    Madak Erdogan, Zeynep

    2009-01-01

    Estrogenic hormones exert their effects through binding to Estrogen Receptors (ERs), which work in concert with coregulators and extranuclear signaling pathways to control gene expression in normal as well as cancerous states, including breast tumors. In this thesis, we have used multiple genome-wide analysis tools to elucidate various ways that…

  17. Disruption of glucagon receptor signaling causes hyperaminoacidemia exposing a possible liver - alpha-cell axis

    DEFF Research Database (Denmark)

    Galsgaard, Katrine D; Winther-Sørensen, Marie; Ørskov, Cathrine

    2018-01-01

    Glucagon secreted from the pancreatic alpha-cells is essential for regulation of blood glucose levels. However, glucagon may play an equally important role in the regulation of amino acid metabolism by promoting ureagenesis. We hypothesized that disruption of glucagon receptor signaling would lead...

  18. High constitutive signaling of the ghrelin receptor--identification of a potent inverse agonist

    DEFF Research Database (Denmark)

    Holst, Birgitte; Cygankiewicz, Adam; Jensen, Tine Halkjaer

    2003-01-01

    Ghrelin is a GH-releasing peptide that also has an important role as an orexigenic hormone-stimulating food intake. By measuring inositol phosphate turnover or by using a reporter assay for transcriptional activity controlled by cAMP-responsive elements, the ghrelin receptor showed strong, ligand......-independent signaling in transfected COS-7 or human embryonic kidney 293 cells. Ghrelin and a number of the known nonpeptide GH secretagogues acted as agonists stimulating inositol phosphate turnover further. In contrast, the low potency ghrelin antagonist, [D-Arg1,D-Phe5,D-Trp7,9,Leu11]-substance P was surprisingly...... found to be a high potency (EC50 = 5.2 nm) full inverse agonist as it decreased the constitutive signaling of the ghrelin receptor down to that observed in untransfected cells. The homologous motilin receptor functioned as a negative control as it did not display any sign of constitutive activity...

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

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    Ritva Tikkanen

    2012-10-01

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

  20. Vitamin D receptor–retinoid X receptor heterodimer signaling regulates oligodendrocyte progenitor cell differentiation

    Science.gov (United States)

    de la Fuente, Alerie Guzman; Errea, Oihana; van Wijngaarden, Peter; Gonzalez, Ginez A.; Kerninon, Christophe; Jarjour, Andrew A.; Lewis, Hilary J.; Jones, Clare A.; Nait-Oumesmar, Brahim; Zhao, Chao; Huang, Jeffrey K.; ffrench-Constant, Charles

    2015-01-01

    The mechanisms regulating differentiation of oligodendrocyte (OLG) progenitor cells (OPCs) into mature OLGs are key to understanding myelination and remyelination. Signaling via the retinoid X receptor γ (RXR-γ) has been shown to be a positive regulator of OPC differentiation. However, the nuclear receptor (NR) binding partner of RXR-γ has not been established. In this study we show that RXR-γ binds to several NRs in OPCs and OLGs, one of which is vitamin D receptor (VDR). Using pharmacological and knockdown approaches we show that RXR–VDR signaling induces OPC differentiation and that VDR agonist vitamin D enhances OPC differentiation. We also show expression of VDR in OLG lineage cells in multiple sclerosis. Our data reveal a role for vitamin D in the regenerative component of demyelinating disease and identify a new target for remyelination medicines. PMID:26644513

  1. The cannabinoid CB1 receptor and mTORC1 signalling pathways interact to modulate glucose homeostasis in mice

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    Francisco J. Bermudez-Silva

    2016-01-01

    Full Text Available The endocannabinoid system (ECS is an intercellular signalling mechanism that is present in the islets of Langerhans and plays a role in the modulation of insulin secretion and expansion of the β-cell mass. The downstream signalling pathways mediating these effects are poorly understood. Mammalian target of rapamycin complex 1 (mTORC1 signalling is a key intracellular pathway involved in energy homeostasis and is known to importantly affect the physiology of pancreatic islets. We investigated the possible relationship between cannabinoid type 1 (CB1 receptor signalling and the mTORC1 pathway in the endocrine pancreas of mice by using pharmacological analysis as well as mice genetically lacking the CB1 receptor or the downstream target of mTORC1, the kinase p70S6K1. In vitro static secretion experiments on islets, western blotting, and in vivo glucose and insulin tolerance tests were performed. The CB1 receptor antagonist rimonabant decreased glucose-stimulated insulin secretion (GSIS at 0.1 µM while increasing phosphorylation of p70S6K1 and ribosomal protein S6 (rpS6 within the islets. Specific pharmacological blockade of mTORC1 by 3 nM rapamycin, as well as genetic deletion of p70S6K1, impaired the CB1-antagonist-mediated decrease in GSIS. In vivo experiments showed that 3 mg/kg body weight rimonabant decreased insulin levels and induced glucose intolerance in lean mice without altering peripheral insulin sensitivity; this effect was prevented by peripheral administration of low doses of rapamycin (0.1 mg/kg body weight, which increased insulin sensitivity. These findings suggest a functional interaction between the ECS and the mTORC1 pathway within the endocrine pancreas and at the whole-organism level, which could have implications for the development of new therapeutic approaches for pancreatic β-cell diseases.

  2. Hypocretin/Orexin regulation of dopamine signaling and cocaine self-administration is mediated predominantly by hypocretin receptor 1.

    Science.gov (United States)

    Prince, Courtney D; Rau, Andrew R; Yorgason, Jordan T; España, Rodrigo A

    2015-01-21

    Extensive evidence suggests that the hypocretins/orexins influence cocaine reinforcement and dopamine signaling via actions at hypocretin receptor 1. By comparison, the involvement of hypocretin receptor 2 in reward and reinforcement processes has received relatively little attention. Thus, although there is some evidence that hypocretin receptor 2 regulates intake of some drugs of abuse, it is currently unclear to what extent hypocretin receptor 2 participates in the regulation of dopamine signaling or cocaine self-administration, particularly under high effort conditions. To address this, we examined the effects of hypocretin receptor 1, and/or hypocretin receptor 2 blockade on dopamine signaling and cocaine reinforcement. We used in vivo fast scan cyclic voltammetry to test the effects of hypocretin antagonists on dopamine signaling in the nucleus accumbens core and a progressive ratio schedule to examine the effects of these antagonists on cocaine self-administration. Results demonstrate that blockade of either hypocretin receptor 1 or both hypocretin receptor 1 and 2 significantly reduces the effects of cocaine on dopamine signaling and decreases the motivation to take cocaine. In contrast, blockade of hypocretin receptor 2 alone had no significant effects on dopamine signaling or self-administration. These findings suggest a differential involvement of the two hypocretin receptors, with hypocretin receptor 1 appearing to be more involved than hypocretin receptor 2 in the regulation of dopamine signaling and cocaine self-administration. When considered with the existing literature, these data support the hypothesis that hypocretins exert a permissive influence on dopamine signaling and motivated behavior via preferential actions on hypocretin receptor 1.

  3. Sphingosine-1-phosphate (S1P) displays sustained S1P1 receptor agonism and signaling through S1P lyase-dependent receptor recycling.

    Science.gov (United States)

    Gatfield, John; Monnier, Lucile; Studer, Rolf; Bolli, Martin H; Steiner, Beat; Nayler, Oliver

    2014-07-01

    The sphingosine-1-phosphate (S1P) type 1 receptor (S1P1R) is a novel therapeutic target in lymphocyte-mediated autoimmune diseases. S1P1 receptor desensitization caused by synthetic S1P1 receptor agonists prevents T-lymphocyte egress from secondary lymphoid organs into the circulation. The selective S1P1 receptor agonist ponesimod, which is in development for the treatment of autoimmune diseases, efficiently reduces peripheral lymphocyte counts and displays efficacy in animal models of autoimmune disease. Using ponesimod and the natural ligand S1P, we investigated the molecular mechanisms leading to different signaling, desensitization and trafficking behavior of S1P1 receptors. In recombinant S1P1 receptor-expressing cells, ponesimod and S1P triggered Gαi protein-mediated signaling and β-arrestin recruitment with comparable potency and efficiency, but only ponesimod efficiently induced intracellular receptor accumulation. In human umbilical vein endothelial cells (HUVEC), ponesimod and S1P triggered translocation of the endogenous S1P1 receptor to the Golgi compartment. However, only ponesimod treatment caused efficient surface receptor depletion, receptor accumulation in the Golgi and degradation. Impedance measurements in HUVEC showed that ponesimod induced only short-lived Gαi protein-mediated signaling followed by resistance to further stimulation, whereas S1P induced sustained Gαi protein-mediated signaling without desensitization. Inhibition of S1P lyase activity in HUVEC rendered S1P an efficient S1P1 receptor internalizing compound and abrogated S1P-mediated sustained signaling. This suggests that S1P lyase - by facilitating S1P1 receptor recycling - is essential for S1P-mediated sustained signaling, and that synthetic agonists are functional antagonists because they are not S1P lyase substrates. Copyright © 2014 Elsevier Inc. All rights reserved.

  4. Bovine lactoferrin counteracts Toll-like receptor mediated activation signals in antigen presenting cells.

    Directory of Open Access Journals (Sweden)

    Patrizia Puddu

    Full Text Available Lactoferrin (LF, a key element in mammalian immune system, plays pivotal roles in host defence against infection and excessive inflammation. Its protective effects range from direct antimicrobial activities against a large panel of microbes, including bacteria, viruses, fungi and parasites, to antinflammatory and anticancer activities. In this study, we show that monocyte-derived dendritic cells (MD-DCs generated in the presence of bovine LF (bLF fail to undergo activation by up-modulating CD83, co-stimulatory and major histocompatibility complex molecules, and cytokine/chemokine secretion. Moreover, these cells are weak activators of T cell proliferation and retain antigen uptake activity. Consistent with an impaired maturation, bLF-MD-DC primed T lymphocytes exhibit a functional unresponsiveness characterized by reduced expression of CD154 and impaired expression of IFN-γ and IL-2. The observed imunosuppressive effects correlate with an increased expression of molecules with negative regulatory functions (i.e. immunoglobulin-like transcript 3 and programmed death ligand 1, indoleamine 2,3-dioxygenase, and suppressor of cytokine signaling-3. Interestingly, bLF-MD-DCs produce IL-6 and exhibit constitutive signal transducer and activator of transcription 3 activation. Conversely, bLF exposure of already differentiated MD-DCs completely fails to induce IL-6, and partially inhibits Toll-like receptor (TLR agonist-induced activation. Cell-specific differences in bLF internalization likely account for the distinct response elicited by bLF in monocytes versus immature DCs, providing a mechanistic base for its multiple effects. These results indicate that bLF exerts a potent anti-inflammatory activity by skewing monocyte differentiation into DCs with impaired capacity to undergo activation and to promote Th1 responses. Overall, these bLF-mediated effects may represent a strategy to block excessive DC activation upon TLR-induced inflammation, adding

  5. Engineering of PDMS surfaces for use in microsystems for capture and isolation of complex and biomedically important proteins: epidermal growth factor receptor as a model system.

    Science.gov (United States)

    Lowe, Aaron M; Ozer, Byram H; Wiepz, Gregory J; Bertics, Paul J; Abbott, Nicholas L

    2008-08-01

    Elastomers based on poly(dimethylsiloxane) (PDMS) are promising materials for fabrication of a wide range of microanalytical systems due to their mechanical and optical properties and ease of processing. To date, however, quantitative studies that demonstrate reliable and reproducible methods for attachment of binding groups that capture complex receptor proteins of relevance to biomedical applications of PDMS microsystems have not been reported. Herein we describe methods that lead to the reproducible capture of a transmembrane protein, the human epidermal growth factor (EGF) receptor, onto PDMS surfaces presenting covalently immobilized antibodies for EGF receptor, and subsequent isolation of the captured receptor by mechanical transfer of the receptor onto a chemically functionalized surface of a gold film for detection. This result is particularly significant because the physical properties of transmembrane proteins make this class of proteins a difficult one to analyze. We benchmark the performance of antibodies to the human EGF receptor covalently immobilized on PDMS against the performance of the same antibodies physisorbed to conventional surfaces utilized in ELISA assays through the use of EGF receptor that was (32)P-radiolabeled in its autophosphorylation domain. These results reveal that two pan-reactive antibodies for the EGF receptor (clones H11 and 111.6) and one phosphospecific EGF receptor antibody (clone pY1068) capture the receptor on both PDMS and ELISA plates. When using H11 antibody to capture EGF receptor and subsequent treatment with a stripping buffer (NaOH and sodium dodecylsulfate) to isolate the receptor, the signal-to-background obtained using the PDMS surface was 82 : 1, exceeding the signal-to-background measured on the ELISA plate (<48 : 1). We also characterized the isolation of captured EGF receptor by mechanical contact of the PDMS surface with a chemically functionalized gold film. The efficiency of mechanical transfer of the

  6. Effect of spatial inhomogeneities on the membrane surface on receptor dimerization and signal initiation

    Directory of Open Access Journals (Sweden)

    Romica Kerketta

    2016-08-01

    Full Text Available Important signal transduction pathways originate on the plasma membrane, where microdomains may transiently entrap diffusing receptors. This results in a non-random distribution of receptors even in the resting state, which can be visualized as clusters by high resolution imaging methods. Here, we explore how spatial in-homogeneities in the plasma membrane might influence the dimerization and phosphorylation status of ErbB2 and ErbB3, two receptor tyrosine kinases that preferentially heterodimerize and are often co-expressed in cancer. This theoretical study is based upon spatial stochastic simulations of the two-dimensional membrane landscape, where variables include differential distributions and overlap of transient confinement zones (domains for the two receptor species. The in silico model is parameterized and validated using data from single particle tracking experiments. We report key differences in signaling output based on the degree of overlap between domains and the relative retention of receptors in such domains, expressed as escape probability. Results predict that a high overlap of domains, which favors transient co-confinement of both receptor species, will enhance the rate of hetero-interactions. Where domains do not overlap, simulations confirm expectations that homo-interactions are favored. Since ErbB3 is uniquely dependent on ErbB2 interactions for activation of its catalytic activity, variations in domain overlap or escape probability markedly alter the predicted patterns and time course of ErbB3 and ErbB2 phosphorylation. Taken together, these results implicate membrane domain organization as an important modulator of signal initiation, motivating the design of novel experimental approaches to measure these important parameters across a wider range of receptor systems.

  7. Signaling Properties of Chemerin Receptors CMKLR1, GPR1 and CCRL2.

    Science.gov (United States)

    De Henau, Olivier; Degroot, Gaetan-Nagim; Imbault, Virginie; Robert, Virginie; De Poorter, Cédric; Mcheik, Saria; Galés, Céline; Parmentier, Marc; Springael, Jean-Yves

    2016-01-01

    Chemerin is a small chemotactic protein originally identified as the natural ligand of CMKLR1. More recently, two other receptors, GPR1 and CCRL2, have been reported to bind chemerin but their functional relevance remains poorly understood. In this study, we compared the binding and signaling properties of the three human chemerin receptors and showed differences in mode of chemerin binding and receptor signaling. Chemerin binds to all three receptors with low nanomolar affinities. However, the contribution of the chemerin C-terminus to binding efficiency varies greatly amongst receptors. By using BRET-based biosensors monitoring the activation of various G proteins, we showed that binding of chemerin and the chemerin 9 nonapeptide (149YFPGQFAFS157) to CMKLR1 activates the three Gαi subtypes (Gαi1, Gαi2 and Gαi3) and the two Gαo isoforms (Gαoa and Gαob) with potencies correlated to binding affinities. In contrast, no significant activation of G proteins was detected upon binding of chemerin to GPR1 or CCRL2. Binding of chemerin and the chemerin 9 peptide also induced the recruitment of β-arrestin1 and 2 to CMKLR1 and GPR1, though to various degree, but not to CCRL2. However, the propensity of chemerin 9 to activate β-arrestins relative to chemerin is higher when bound to GPR1. Finally, we showed that binding of chemerin to CMKLR1 and GPR1 promotes also the internalization of the two receptors and the phosphorylation of ERK1/2 MAP kinases, although with a different efficiency, and that phosphorylation of ERK1/2 requires both Gαi/o and β-arrestin2 activation but not β-arrestin1. Collectively, these data support a model in which each chemerin receptor displays selective signaling properties.

  8. JNK Signaling: Regulation and Functions Based on Complex Protein-Protein Partnerships

    Science.gov (United States)

    Zeke, András; Misheva, Mariya

    2016-01-01

    SUMMARY The c-Jun N-terminal kinases (JNKs), as members of the mitogen-activated protein kinase (MAPK) family, mediate eukaryotic cell responses to a wide range of abiotic and biotic stress insults. JNKs also regulate important physiological processes, including neuronal functions, immunological actions, and embryonic development, via their impact on gene expression, cytoskeletal protein dynamics, and cell death/survival pathways. Although the JNK pathway has been under study for >20 years, its complexity is still perplexing, with multiple protein partners of JNKs underlying the diversity of actions. Here we review the current knowledge of JNK structure and isoforms as well as the partnerships of JNKs with a range of intracellular proteins. Many of these proteins are direct substrates of the JNKs. We analyzed almost 100 of these target proteins in detail within a framework of their classification based on their regulation by JNKs. Examples of these JNK substrates include a diverse assortment of nuclear transcription factors (Jun, ATF2, Myc, Elk1), cytoplasmic proteins involved in cytoskeleton regulation (DCX, Tau, WDR62) or vesicular transport (JIP1, JIP3), cell membrane receptors (BMPR2), and mitochondrial proteins (Mcl1, Bim). In addition, because upstream signaling components impact JNK activity, we critically assessed the involvement of signaling scaffolds and the roles of feedback mechanisms in the JNK pathway. Despite a clarification of many regulatory events in JNK-dependent signaling during the past decade, many other structural and mechanistic insights are just beginning to be revealed. These advances open new opportunities to understand the role of JNK signaling in diverse physiological and pathophysiological states. PMID:27466283

  9. Therapeutic Targeting of the IL-6 Trans-Signaling/Mechanistic Target of Rapamycin Complex 1 Axis in Pulmonary Emphysema.

    Science.gov (United States)

    Ruwanpura, Saleela M; McLeod, Louise; Dousha, Lovisa F; Seow, Huei J; Alhayyani, Sultan; Tate, Michelle D; Deswaerte, Virginie; Brooks, Gavin D; Bozinovski, Steven; MacDonald, Martin; Garbers, Christoph; King, Paul T; Bardin, Philip G; Vlahos, Ross; Rose-John, Stefan; Anderson, Gary P; Jenkins, Brendan J

    2016-12-15

    The potent immunomodulatory cytokine IL-6 is consistently up-regulated in human lungs with emphysema and in mouse emphysema models; however, the mechanisms by which IL-6 promotes emphysema remain obscure. IL-6 signals using two distinct modes: classical signaling via its membrane-bound IL-6 receptor (IL-6R), and trans-signaling via a naturally occurring soluble IL-6R. To identify whether IL-6 trans-signaling and/or classical signaling contribute to the pathogenesis of emphysema. We used the gp130 F/F genetic mouse model for spontaneous emphysema and cigarette smoke-induced emphysema models. Emphysema in mice was quantified by various methods including in vivo lung function and stereology, and terminal deoxynucleotidyl transferase dUTP nick end labeling assay was used to assess alveolar cell apoptosis. In mouse and human lung tissues, the expression level and location of IL-6 signaling-related genes and proteins were measured, and the levels of IL-6 and related proteins in sera from emphysematous mice and patients were also assessed. Lung tissues from patients with emphysema, and from spontaneous and cigarette smoke-induced emphysema mouse models, were characterized by excessive production of soluble IL-6R. Genetic blockade of IL-6 trans-signaling in emphysema mouse models and therapy with the IL-6 trans-signaling antagonist sgp130Fc ameliorated emphysema by suppressing augmented alveolar type II cell apoptosis. Furthermore, IL-6 trans-signaling-driven emphysematous changes in the lung correlated with mechanistic target of rapamycin complex 1 hyperactivation, and treatment of emphysema mouse models with the mechanistic target of rapamycin complex 1 inhibitor rapamycin attenuated emphysematous changes. Collectively, our data reveal that specific targeting of IL-6 trans-signaling may represent a novel treatment strategy for emphysema.

  10. UDP/P2Y6 receptor signaling regulates IgE-dependent degranulation in human basophils

    Directory of Open Access Journals (Sweden)

    Manabu Nakano

    2017-10-01

    Conclusions: This study showed that UDP/P2Y6 receptor signaling is involved in the regulation of IgE-dependent degranulation in basophils, which might stimulate the P2Y6 receptor via the autocrine secretion of UTP. Thus, this receptor represents a potential target to regulate IgE-dependent degranulation in basophils during allergic diseases.

  11. Large-Scale Phosphoproteomics Reveals Shp-2 Phosphatase-Dependent Regulators of Pdgf Receptor Signaling

    DEFF Research Database (Denmark)

    Batth, Tanveer S; Papetti, Moreno; Pfeiffer, Anamarija

    2018-01-01

    Despite its low cellular abundance, phosphotyrosine (pTyr) regulates numerous cell signaling pathways in health and disease. We applied comprehensive phosphoproteomics to unravel differential regulators of receptor tyrosine kinase (RTK)-initiated signaling networks upon activation by Pdgf-ββ, Fgf-2...... of Pdgfr pTyr signaling. Application of a recently introduced allosteric Shp-2 inhibitor revealed global regulation of the Pdgf-dependent tyrosine phosphoproteome, which significantly impaired cell migration. In addition, we present a list of hundreds of Shp-2-dependent targets and putative substrates...

  12. The selectivity of receptor tyrosine kinase signaling is controlled by a secondary SH2 domain binding site.

    Science.gov (United States)

    Bae, Jae Hyun; Lew, Erin Denise; Yuzawa, Satoru; Tomé, Francisco; Lax, Irit; Schlessinger, Joseph

    2009-08-07

    SH2 domain-mediated interactions represent a crucial step in transmembrane signaling by receptor tyrosine kinases. SH2 domains recognize phosphotyrosine (pY) in the context of particular sequence motifs in receptor phosphorylation sites. However, the modest binding affinity of SH2 domains to pY containing peptides may not account for and likely represents an oversimplified mechanism for regulation of selectivity of signaling pathways in living cells. Here we describe the crystal structure of the activated tyrosine kinase domain of FGFR1 in complex with a phospholipase Cgamma fragment. The structural and biochemical data and experiments with cultured cells show that the selectivity of phospholipase Cgamma binding and signaling via activated FGFR1 are determined by interactions between a secondary binding site on an SH2 domain and a region in FGFR1 kinase domain in a phosphorylation independent manner. These experiments reveal a mechanism for how SH2 domain selectivity is regulated in vivo to mediate a specific cellular process.

  13. DMPD: Signaling to NF-kappaB by Toll-like receptors. [Dynamic Macrophage Pathway CSML Database

    Lifescience Database Archive (English)

    Full Text Available l Med. 2007 Nov;13(11):460-9. Epub 2007 Oct 29. (.png) (.svg) (.html) (.csml) Show Signaling to NF-kappaB by Toll-like receptors. Pub...medID 18029230 Title Signaling to NF-kappaB by Toll-like receptors. Authors Kawai T

  14. S1P receptor signalling and RGS proteins; expression and function in vascular smooth muscle cells and transfected CHO cells

    NARCIS (Netherlands)

    Hendriks-Balk, Mariëlle C.; van Loenen, Pieter B.; Hajji, Najat; Michel, Martin C.; Peters, Stephan L. M.; Alewijnse, Astrid E.

    2009-01-01

    Sphingosine-1-phosphate (S1P) signalling via G protein-coupled receptors is important for the regulation of cell function and differentiation. Specific Regulators of G protein Signalling (RGS) proteins modulate the function of these receptors in many cell types including vascular smooth muscle cells

  15. Cardiac Alpha1-Adrenergic Receptors: Novel Aspects of Expression, Signaling Mechanisms, Physiologic Function, and Clinical Importance

    Science.gov (United States)

    O’Connell, Timothy D.; Jensen, Brian C.; Baker, Anthony J.

    2014-01-01

    Adrenergic receptors (AR) are G-protein-coupled receptors (GPCRs) that have a crucial role in cardiac physiology in health and disease. Alpha1-ARs signal through Gαq, and signaling through Gq, for example, by endothelin and angiotensin receptors, is thought to be detrimental to the heart. In contrast, cardiac alpha1-ARs mediate important protective and adaptive functions in the heart, although alpha1-ARs are only a minor fraction of total cardiac ARs. Cardiac alpha1-ARs activate pleiotropic downstream signaling to prevent pathologic remodeling in heart failure. Mechanisms defined in animal and cell models include activation of adaptive hypertrophy, prevention of cardiac myocyte death, augmentation of contractility, and induction of ischemic preconditioning. Surprisingly, at the molecular level, alpha1-ARs localize to and signal at the nucleus in cardiac myocytes, and, unlike most GPCRs, activate “inside-out” signaling to cause cardioprotection. Contrary to past opinion, human cardiac alpha1-AR expression is similar to that in the mouse, where alpha1-AR effects are seen most convincingly in knockout models. Human clinical studies show that alpha1-blockade worsens heart failure in hypertension and does not improve outcomes in heart failure, implying a cardioprotective role for human alpha1-ARs. In summary, these findings identify novel functional and mechanistic aspects of cardiac alpha1-AR function and suggest that activation of cardiac alpha1-AR might be a viable therapeutic strategy in heart failure. PMID:24368739

  16. Spatiotemporal intracellular dynamics of neurotrophin and its receptors. Implications for neurotrophin signaling and neuronal function.

    Science.gov (United States)

    Bronfman, F C; Lazo, O M; Flores, C; Escudero, C A

    2014-01-01

    Neurons possess a polarized morphology specialized to contribute to neuronal networks, and this morphology imposes an important challenge for neuronal signaling and communication. The physiology of the network is regulated by neurotrophic factors that are secreted in an activity-dependent manner modulating neuronal connectivity. Neurotrophins are a well-known family of neurotrophic factors that, together with their cognate receptors, the Trks and the p75 neurotrophin receptor, regulate neuronal plasticity and survival and determine the neuronal phenotype in healthy and regenerating neurons. Is it now becoming clear that neurotrophin signaling and vesicular transport are coordinated to modify neuronal function because disturbances of vesicular transport mechanisms lead to disturbed neurotrophin signaling and to diseases of the nervous system. This chapter summarizes our current understanding of how the regulated secretion of neurotrophin, the distribution of neurotrophin receptors in different locations of neurons, and the intracellular transport of neurotrophin-induced signaling in distal processes are achieved to allow coordinated neurotrophin signaling in the cell body and axons.

  17. Connexins and M3 Muscarinic Receptors Contribute to Heterogeneous Ca2+ Signaling in Mouse Aortic Endothelium

    Directory of Open Access Journals (Sweden)

    François-Xavier Boittin

    2013-02-01

    Full Text Available Background/Aims: Smooth muscle tone is controlled by Ca2+ signaling in the endothelial layer. Mouse endothelial cells are interconnected by gap junctions made of Connexin40 (Cx40 and Cx37, which allow the exchange of signaling molecules to coordinate their activity. Here, we investigated the role of Cx40 in the endothelial Ca2+ signaling of the mouse aorta. Methods: Ca2+ imaging was performed on intact aortic endothelium from both wild type (Cx40+/+ and Connexin40-deficient (Cx40 -/- mice. Results: Acetylcholine (ACh induced early fast and high amplitude Ca2+ transients in a fraction of endothelial cells expressing the M3 muscarinic receptors. Inhibition of intercellular communication using carbenoxolone or octanol fully blocked the propagation of ACh-induced Ca2+ transients toward adjacent cells in WT and Cx40-/- mice. As compared to WT, Cx40-/- mice displayed a reduced propagation of ACh-induced Ca2+ waves, indicating that Cx40 contributes to the spreading of Ca2+ signals. The propagation of those Ca2+ responses was not blocked by suramin, a blocker of purinergic ATP receptors, indicating that there is no paracrine effect of ATP release on the Ca2+ waves. Conclusions: Altogether our data show that Cx40 and Cx37 contribute to the propagation and amplification of the Ca2+ signaling triggered by ACh in endothelial cells expressing the M3 muscarinic receptors.

  18. Expression of G(alpha)(s) proteins and TSH receptor signalling in hyperfunctioning thyroid nodules with TSH receptor mutations.

    Science.gov (United States)

    Holzapfel, Hans-Peter; Bergner, Beate; Wonerow, Peter; Paschke, Ralf

    2002-07-01

    Constitutively activating mutations of the thyrotrophin receptor (TSHR) are the main molecular cause of hyperfunctioning thyroid nodules (HTNs). The G protein coupling is an important and critical step in the TSHR signalling which mainly includes G(alpha)(s), G(alpha)(i) and G(alpha)(q)/11 proteins. We investigated the in vitro consequences of overexpressing G(alpha) proteins on signalling of the wild-type (WT) or mutated TSHR. Moreover, we investigated whether changes in G(alpha) protein expression are pathophysiologically relevant in HTNs or cold thyroid nodules (CTNs). Wild-type TSH receptor and mutated TSH receptors were coexpressed with G(alpha)(s), G(alpha)(i) or G(alpha)(q)/11, and cAMP and inositol phosphate (IP) production was measured after stimulation with TSH. The expression of G(alpha)(s), G(alpha)(i) and G(alpha)(q)/11 proteins was examined by Western blotting in 28 HTNs and 14 CTNs. Coexpression of G(alpha)(s) with the WT TSH receptor in COS 7 cells significantly increased the basal and TSH-stimulated cAMP accumulation while coexpression of the G(alpha)(q) or G(alpha)11 protein significantly increased the production of cAMP and inositol triphosphate (IP(3)). The coexpression of the TSH receptor mutants (I486F, DEL613-621), known to couple constitutively to G(alpha)(s) and G(alpha)(q) with G(alpha)(s) and G(alpha)(q)/11, significantly increased the basal and stimulated cAMP and IP(3) accumulation. Coexpression of the TSH receptor mutant V556F with G(alpha)(s) only increased the basal and stimulated cAMP production while its coexpression with G(alpha)(q)/11 increased the basal and stimulated IP(3) signalling. The expression of G(alpha)(s) protein subunits determined by Western blotting was significantly decreased in 14 HTNs with a constitutively activating TSH receptor mutation in comparison with the corresponding surrounding tissue, while in 14 HTNs without TSH receptor or G(alpha)(s) protein mutation and in 14 CTNs the expression of G

  19. Signaling by myeloid C-type lectin receptors in immunity and homeostasis.

    Science.gov (United States)

    Sancho, David; Reis e Sousa, Caetano

    2012-01-01

    Myeloid cells are key drivers of physiological responses to pathogen invasion or tissue damage. Members of the C-type lectin receptor (CLR) family stand out among the specialized receptors utilized by myeloid cells to orchestrate these responses. CLR ligands include carbohydrate, protein, and lipid components of both pathogens and self, which variably trigger endocytic, phagocytic, proinflammatory, or anti-inflammatory reactions. These varied outcomes rely on a versatile system for CLR signaling that includes tyrosine-based motifs that recruit kinases, phosphatases, or endocytic adaptors as well as nontyrosine-based signals that modulate the activation of other pathways or couple to the uptake machinery. Here, we review the signaling properties of myeloid CLRs and how they impact the role of myeloid cells in innate and adaptive immunity.

  20. Regulation of Akt and Wnt signaling by the group II metabotropic glutamate receptor antagonist LY341495 and agonist LY379268.

    Science.gov (United States)

    Sutton, Laurie P; Rushlow, Walter J

    2011-06-01

    Metabotropic glutamate receptors 2/3 (mGlu(2/3)) have been implicated in schizophrenia and as a novel treatment target for schizophrenia. The current study examined whether mGlu(2/3) regulates Akt (protein kinase B) and Wnt (Wingless/Int-1) signaling, two cascades associated with schizophrenia and modified by antipsychotics. Western blotting revealed increases in phosphorylated Akt (pAkt) and phosphorylated glycogen synthase kinase-3 (pGSK-3) following acute and repeated treatment of LY379268 (mGlu(2/3) agonist), whereas increases in dishevelled-2 (Dvl-2), dishevelled-3 (Dvl-3), GSK-3 and β-catenin were only observed following repeated treatment. LY341495 (mGlu(2/3) antagonist) induced the opposite response compared with LY379268. Co-immunoprecipitation experiments showed an association between the mGlu(2/3) complex and Dvl-2 providing a possible mechanism to explain how the mGlu(2/3) can mediate changes in Wnt signaling. However, there was no association between the mGlu(2/3) complex and Akt suggesting that changes in Akt signaling following LY341495 and LY379268 treatments may not be directly mediated by the mGlu(2/3) . Finally, an increase in locomotor activity induced by LY341495 treatment correlated with increased pAkt and pGSK-3 levels and was attenuated by the administration of the GSK-3 inhibitor, SB216763. Overall, the results suggest that mGlu(2/3) regulates Akt and Wnt signaling and LY379268 treatment has overlapping effects with D(2) dopamine receptor antagonists (antipsychotic drugs). © 2011 The Authors. Journal of Neurochemistry © 2011 International Society for Neurochemistry.

  1. Androgen Receptor: A Complex Therapeutic Target for Breast Cancer

    Science.gov (United States)

    Narayanan, Ramesh; Dalton, James T.

    2016-01-01

    Molecular and histopathological profiling have classified breast cancer into multiple sub-types empowering precision treatment. Although estrogen receptor (ER) and human epidermal growth factor receptor (HER2) are the mainstay therapeutic targets in breast cancer, the androgen receptor (AR) is evolving as a molecular target for cancers that have developed resistance to conventional treatments. The high expression of AR in breast cancer and recent discovery and development of new nonsteroidal drugs targeting the AR provide a strong rationale for exploring it again as a therapeutic target in this disease. Ironically, both nonsteroidal agonists and antagonists for the AR are undergoing clinical trials, making AR a complicated target to understand in breast cancer. This review provides a detailed account of AR’s therapeutic role in breast cancer. PMID:27918430

  2. Androgen Receptor: A Complex Therapeutic Target for Breast Cancer

    Directory of Open Access Journals (Sweden)

    Ramesh Narayanan

    2016-12-01

    Full Text Available Molecular and histopathological profiling have classified breast cancer into multiple sub-types empowering precision treatment. Although estrogen receptor (ER and human epidermal growth factor receptor (HER2 are the mainstay therapeutic targets in breast cancer, the androgen receptor (AR is evolving as a molecular target for cancers that have developed resistance to conventional treatments. The high expression of AR in breast cancer and recent discovery and development of new nonsteroidal drugs targeting the AR provide a strong rationale for exploring it again as a therapeutic target in this disease. Ironically, both nonsteroidal agonists and antagonists for the AR are undergoing clinical trials, making AR a complicated target to understand in breast cancer. This review provides a detailed account of AR’s therapeutic role in breast cancer.

  3. Predicting chemical environments of bacteria from receptor signaling.

    Directory of Open Access Journals (Sweden)

    Diana Clausznitzer

    2014-10-01

    Full Text Available Sensory systems have evolved to respond to input stimuli of certain statistical properties, and to reliably transmit this information through biochemical pathways. Hence, for an experimentally well-characterized sensory system, one ought to be able to extract valuable information about the statistics of the stimuli. Based on dose-response curves from in vivo fluorescence resonance energy transfer (FRET experiments of the bacterial chemotaxis sensory system, we predict the chemical gradients chemotactic Escherichia coli cells typically encounter in their natural environment. To predict average gradients cells experience, we revaluate the phenomenological Weber's law and its generalizations to the Weber-Fechner law and fold-change detection. To obtain full distributions of gradients we use information theory and simulations, considering limitations of information transmission from both cell-external and internal noise. We identify broad distributions of exponential gradients, which lead to log-normal stimuli and maximal drift velocity. Our results thus provide a first step towards deciphering the chemical nature of complex, experimentally inaccessible cellular microenvironments, such as the human intestine.

  4. Amyloid β production is regulated by β2-adrenergic signaling-mediated post-translational modifications of the ryanodine receptor.

    Science.gov (United States)

    Bussiere, Renaud; Lacampagne, Alain; Reiken, Steven; Liu, Xiaoping; Scheuerman, Valerie; Zalk, Ran; Martin, Cécile; Checler, Frederic; Marks, Andrew R; Chami, Mounia

    2017-06-16

    Alteration of ryanodine receptor (RyR)-mediated calcium (Ca 2+ ) signaling has been reported in Alzheimer disease (AD) models. However, the molecular mechanisms underlying altered RyR-mediated intracellular Ca 2+ release in AD remain to be fully elucidated. We report here that RyR2 undergoes post-translational modifications (phosphorylation, oxidation, and nitrosylation) in SH-SY5Y neuroblastoma cells expressing the β-amyloid precursor protein (βAPP) harboring the familial double Swedish mutations (APPswe). RyR2 macromolecular complex remodeling, characterized by depletion of the regulatory protein calstabin2, resulted in increased cytosolic Ca 2+ levels and mitochondrial oxidative stress. We also report a functional interplay between amyloid β (Aβ), β-adrenergic signaling, and altered Ca 2+ signaling via leaky RyR2 channels. Thus, post-translational modifications of RyR occur downstream of Aβ through a β2-adrenergic signaling cascade that activates PKA. RyR2 remodeling in turn enhances βAPP processing. Importantly, pharmacological stabilization of the binding of calstabin2 to RyR2 channels, which prevents Ca 2+ leakage, or blocking the β2-adrenergic signaling cascade reduced βAPP processing and the production of Aβ in APPswe-expressing SH-SY5Y cells. We conclude that targeting RyR-mediated Ca 2+ leakage may be a therapeutic approach to treat AD. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  5. Dopamine receptors modulate cytotoxicity of natural killer cells via cAMP-PKA-CREB signaling pathway.

    Directory of Open Access Journals (Sweden)

    Wei Zhao

    Full Text Available Dopamine (DA, a neurotransmitter in the nervous system, has been shown to modulate immune function. We have previously reported that five subtypes of DA receptors, including D1R, D2R, D3R, D4R and D5R, are expressed in T lymphocytes and they are involved in regulation of T cells. However, roles of these DA receptor subtypes and their coupled signal-transduction pathway in modulation of natural killer (NK cells still remain to be clarified. The spleen of mice was harvested and NK cells were isolated and purified by negative selection using magnetic activated cell sorting. After NK cells were incubated with various drugs for 4 h, flow cytometry measured cytotoxicity of NK cells against YAC-1 lymphoma cells. NK cells expressed the five subtypes of DA receptors at mRNA and protein levels. Activation of D1-like receptors (including D1R and D5R with agonist SKF38393 enhanced NK cell cytotoxicity, but activation of D2-like receptors (including D2R, D3R and D4R with agonist quinpirole attenuated NK cells. Simultaneously, SKF38393 elevated D1R and D5R expression, cAMP content, and phosphorylated cAMP-response element-binding (CREB level in NK cells, while quinpirole reduced D3R and D4R expression, cAMP content, and phosphorylated CREB level in NK cells. These effects of SKF38393 were blocked by SCH23390, an antagonist of D1-like receptors, and quinpirole effects were abolished by haloperidol, an antagonist of D2-like receptors. In support these results, H89, an inhibitor of phosphokinase A (PKA, prevented the SKF38393-dependent enhancement of NK cells and forskolin, an activator of adenylyl cyclase (AC, counteracted the quinpirole-dependent suppression of NK cells. These findings show that DA receptor subtypes are involved in modulation of NK cells and suggest that D1-like receptors facilitate NK cells by stimulating D1R/D5R-cAMP-PKA-CREB signaling pathway and D2-like receptors suppress NK cells by inhibiting D3R/D4R-cAMP-PKA-CREB signaling pathway. The

  6. The plant natriuretic peptide receptor is a guanylyl cyclase and enables cGMP-dependent signaling

    KAUST Repository

    Turek, Ilona; Gehring, Christoph A

    2016-01-01

    and water balance and responses to biotrophic plant pathogens. Although there is increasing understanding of the complex roles of PNPs in plant responses at the systems level, little is known about the underlying signaling mechanisms. Here we report

  7. New twist on neuronal insulin receptor signaling in health, disease, and therapeutics.

    Science.gov (United States)

    Wada, Akihiko; Yokoo, Hiroki; Yanagita, Toshihiko; Kobayashi, Hideyuki

    2005-10-01

    Long after the pioneering studies documenting the existence of insulin (year 1967) and insulin receptor (year 1978) in brain, the last decade has witnessed extraordinary progress in the understanding of brain region-specific multiple roles of insulin receptor signalings in health and disease. In the hypothalamus, insulin regulates food intake, body weight, peripheral fat deposition, hepatic gluconeogenesis, reproductive endocrine axis, and compensatory secretion of counter-regulatory hormones to hypoglycemia. In the hippocampus, insulin promotes learning and memory, independent of the glucoregulatory effect of insulin. Defective insulin receptor signalings are associated with the dementia in normal aging and patients with age-related neurodegenerative diseases (e.g., Alzheimer's disease); the cognitive impairment can be reversed with systemic administration of insulin in the euglycemic condition. Intranasal administration of insulin enhances memory and mood and decreases body weight in healthy humans, without causing hypoglycemia. In the hypothalamus, insulin-induced activation of the phosphoinositide 3-kinase pathway followed by opening of ATP-sensitive K+ channel has been shown to be related to multiple effects of insulin. However, the precise molecular mechanisms of insulin's pleiotropic effects still remain obscure. More importantly, much remains unknown about the quality control mechanisms ensuring correct conformational maturation of the insulin receptor, and the cellular mechanisms regulating density of cell surface functional insulin receptors.

  8. Signal transduction by VEGF receptors in regulation of angiogenesis and lymphangiogenesis

    International Nuclear Information System (INIS)

    Shibuya, Masabumi; Claesson-Welsh, Lena

    2006-01-01

    The VEGF/VPF (vascular endothelial growth factor/vascular permeability factor) ligands and receptors are crucial regulators of vasculogenesis, angiogenesis, lymphangiogenesis and vascular permeability in vertebrates. VEGF-A, the prototype VEGF ligand, binds and activates two tyrosine kinase receptors: VEGFR1 (Flt-1) and VEGFR2 (KDR/Flk-1). VEGFR1, which occurs in transmembrane and soluble forms, negatively regulates vasculogenesis and angiogenesis during early embryogenesis, but it also acts as a positive regulator of angiogenesis and inflammatory responses, playing a role in several human diseases such as rheumatoid arthritis and cancer. The soluble VEGFR1 is overexpressed in placenta in preeclampsia patients. VEGFR2 has critical functions in physiological and pathological angiogenesis through distinct signal transduction pathways regulating proliferation and migration of endothelial cells. VEGFR3, a receptor for the lymphatic growth factors VEGF-C and VEGF-D, but not for VEGF-A, regulates vascular and lymphatic endothelial cell function during embryogenesis. Loss-of-function variants of VEGFR3 have been identified in lymphedema. Formation of tumor lymphatics may be stimulated by tumor-produced VEGF-C, allowing increased spread of tumor metastases through the lymphatics. Mapping the signaling system of these important receptors may provide the knowledge necessary to suppress specific signaling pathways in major human diseases

  9. N-wasp is essential for the negative regulation of B cell receptor signaling.

    Directory of Open Access Journals (Sweden)

    Chaohong Liu

    2013-11-01

    Full Text Available Negative regulation of receptor signaling is essential for controlling cell activation and differentiation. In B-lymphocytes, the down-regulation of B-cell antigen receptor (BCR signaling is critical for suppressing the activation of self-reactive B cells; however, the mechanism underlying the negative regulation of signaling remains elusive. Using genetically manipulated mouse models and total internal reflection fluorescence microscopy, we demonstrate that neuronal Wiskott-Aldrich syndrome protein (N-WASP, which is coexpressed with WASP in all immune cells, is a critical negative regulator of B-cell signaling. B-cell-specific N-WASP gene deletion causes enhanced and prolonged BCR signaling and elevated levels of autoantibodies in the mouse serum. The increased signaling in N-WASP knockout B cells is concurrent with increased accumulation of F-actin at the B-cell surface, enhanced B-cell spreading on the antigen-presenting membrane, delayed B-cell contraction, inhibition in the merger of signaling active BCR microclusters into signaling inactive central clusters, and a blockage of BCR internalization. Upon BCR activation, WASP is activated first, followed by N-WASP in mouse and human primary B cells. The activation of N-WASP is suppressed by Bruton's tyrosine kinase-induced WASP activation, and is restored by the activation of SH2 domain-containing inositol 5-phosphatase that inhibits WASP activation. Our results reveal a new mechanism for the negative regulation of BCR signaling and broadly suggest an actin-mediated mechanism for signaling down-regulation.

  10. Frizzled7: A Promising Achilles’ Heel for Targeting the Wnt Receptor Complex to Treat Cancer

    Science.gov (United States)

    Phesse, Toby; Flanagan, Dustin; Vincan, Elizabeth

    2016-01-01

    Frizzled7 is arguably the most studied member of the Frizzled family, which are the cognate Wnt receptors. Frizzled7 is highly conserved through evolution, from Hydra through to humans, and is expressed in diverse organisms, tissues and human disease contexts. Frizzled receptors can homo- or hetero-polymerise and associate with several co-receptors to transmit Wnt signalling. Notably, Frizzled7 can transmit signalling via multiple Wnt transduction pathways and bind to several different Wnt ligands, Frizzled receptors and co-receptors. These promiscuous binding and functional properties are thought to underlie the pivotal role Frizzled7 plays in embryonic developmental and stem cell function. Recent studies have identified that Frizzled7 is upregulated in diverse human cancers, and promotes proliferation, progression and invasion, and orchestrates cellular transitions that underscore cancer metastasis. Importantly, Frizzled7 is able to regulate Wnt signalling activity even in cancer cells which have mutations to down-stream signal transducers. In this review we discuss the various aspects of Frizzled7 signalling and function, and the implications these have for therapeutic targeting of Frizzled7 in cancer. PMID:27196929

  11. Hypocretin/Orexin Regulation of Dopamine Signaling and Cocaine Self-Administration Is Mediated Predominantly by Hypocretin Receptor 1

    OpenAIRE

    Prince, Courtney D.; Rau, Andrew R.; Yorgason, Jordan T.; Espa?a, Rodrigo A.

    2014-01-01

    Extensive evidence suggests that the hypocretins/orexins influence cocaine reinforcement and dopamine signaling via actions at hypocretin receptor 1. By comparison, the involvement of hypocretin receptor 2 in reward and reinforcement processes has received relatively little attention. Thus, although there is some evidence that hypocretin receptor 2 regulates intake of some drugs of abuse, it is currently unclear to what extent hypocretin receptor 2 participates in the regulation of dopamine s...

  12. Opposing roles of Toll-like receptor and cytosolic DNA-STING signaling pathways for Staphylococcus aureus cutaneous host defense.

    Directory of Open Access Journals (Sweden)

    Philip O Scumpia

    2017-07-01

    Full Text Available Successful host defense against pathogens requires innate immune recognition of the correct pathogen associated molecular patterns (PAMPs by pathogen recognition receptors (PRRs to trigger the appropriate gene program tailored to the pathogen. While many PRR pathways contribute to the innate immune response to specific pathogens, the relative importance of each pathway for the complete transcriptional program elicited has not been examined in detail. Herein, we used RNA-sequencing with wildtype and mutant macrophages to delineate the innate immune pathways contributing to the early transcriptional response to Staphylococcus aureus, a ubiquitous microorganism that can activate a wide variety of PRRs. Unexpectedly, two PRR pathways-the Toll-like receptor (TLR and Stimulator of Interferon Gene (STING pathways-were identified as dominant regulators of approximately 95% of the genes that were potently induced within the first four hours of macrophage infection with live S. aureus. TLR signaling predominantly activated a pro-inflammatory program while STING signaling activated an antiviral/type I interferon response with live but not killed S. aureus. This STING response was largely dependent on the cytosolic DNA sensor cyclic guanosine-adenosine synthase (cGAS. Using a cutaneous infection model, we found that the TLR and STING pathways played opposite roles in host defense to S. aureus. TLR signaling was required for host defense, with its absence reducing interleukin (IL-1β production and neutrophil recruitment, resulting in increased bacterial growth. In contrast, absence of STING signaling had the opposite effect, enhancing the ability to restrict the infection. These results provide novel insights into the complex interplay of innate immune signaling pathways triggered by S. aureus and uncover opposing roles of TLR and STING in cutaneous host defense to S. aureus.

  13. Endogenous GAS6 and Mer receptor signaling regulate prostate cancer stem cells in bone marrow.

    Science.gov (United States)

    Jung, Younghun; Decker, Ann M; Wang, Jingcheng; Lee, Eunsohl; Kana, Lulia A; Yumoto, Kenji; Cackowski, Frank C; Rhee, James; Carmeliet, Peter; Buttitta, Laura; Morgan, Todd M; Taichman, Russell S

    2016-05-03

    GAS6 and its receptors (Tryo 3, Axl, Mer or "TAM") are known to play a role in regulating tumor progression in a number of settings. Previously we have demonstrated that GAS6 signaling regulates invasion, proliferation, chemotherapy-induced apoptosis of prostate cancer (PCa) cells. We have also demonstrated that GAS6 secreted from osteoblasts in the bone marrow environment plays a critical role in establishing prostate tumor cell dormancy. Here we investigated the role that endogenous GAS6 and Mer receptor signaling plays in establishing prostate cancer stem cells in the bone marrow microenvironment.We first observed that high levels of endogenous GAS6 are expressed by disseminated tumor cells (DTCs) in the bone marrow, whereas relatively low levels of endogenous GAS6 are expressed in PCa tumors grown in a s.c. Interestingly, elevated levels of endogenous GAS6 were identified in putative cancer stem cells (CSCs, CD133+/CD44+) compared to non-CSCs (CD133-/CD44-) isolated from PCa/osteoblast cocultures in vitro and in DTCs isolated from the bone marrow 24 hours after intracardiac injection. Moreover, we found that endogenous GAS6 expression is associated with Mer receptor expression in growth arrested (G1) PCa cells, which correlates with the increase of the CSC populations. Importantly, we found that overexpression of GAS6 activates phosphorylation of Mer receptor signaling and subsequent induction of the CSC phenotype in vitro and in vivo.Together these data suggest that endogenous GAS6 and Mer receptor signaling contribute to the establishment of PCa CSCs in the bone marrow microenvironment, which may have important implications for targeting metastatic disease.

  14. Estrogen receptor signaling in prostate cancer: Implications for carcinogenesis and tumor progression.

    Science.gov (United States)

    Bonkhoff, Helmut

    2018-01-01

    The androgen receptor (AR) is the classical target for prostate cancer prevention and treatment, but more recently estrogens and their receptors have also been implicated in prostate cancer development and tumor progression. Recent experimental and clinical data were reviewed to elucidate pathogenetic mechanisms how estrogens and their receptors may affect prostate carcinogenesis and tumor progression. The estrogen receptor beta (ERβ) is the most prevalent ER in the human prostate, while the estrogen receptor alpha (ERα) is restricted to basal cells of the prostatic epithelium and stromal cells. In high grade prostatic intraepithelial neoplasia (HGPIN), the ERα is up-regulated and most likely mediates carcinogenic effects of estradiol as demonstrated in animal models. The partial loss of the ERβ in HGPIN indicates that the ERβ acts as a tumor suppressor. The tumor promoting function of the TMPRSS2-ERG fusion, a major driver of prostate carcinogenesis, is triggered by the ERα and repressed by the ERβ. The ERβ is generally retained in hormone naïve and metastatic prostate cancer, but is partially lost in castration resistant disease. The progressive emergence of the ERα and ERα-regulated genes (eg, progesterone receptor (PR), PS2, TMPRSS2-ERG fusion, and NEAT1) during prostate cancer progression and hormone refractory disease suggests that these tumors can bypass the AR by using estrogens and progestins for their growth. In addition, nongenomic estrogen signaling pathways mediated by orphan receptors (eg, GPR30 and ERRα) has also been implicated in prostate cancer progression. Increasing evidences demonstrate that local estrogen signaling mechanisms are required for prostate carcinogenesis and tumor progression. Despite the recent progress in this research topic, the translation of the current information into potential therapeutic applications remains highly challenging and clearly warrants further investigation. © 2017 Wiley Periodicals, Inc.

  15. Wnt signalling via the epidermal growth factor receptor: a role in breast cancer?

    International Nuclear Information System (INIS)

    Musgrove, Elizabeth A

    2004-01-01

    Recent data have suggested the epidermal-growth-factor receptor (EGFR) as a point of convergence for several different classes of receptor. Civenni and colleagues have now demonstrated crosstalk between Wnt signalling and the EGFR, showing that in breast epithelial cells Wnts activate downstream targets of the EGFR, including cyclin D1. Given the role of members of these pathways in the aetiology of breast cancer and as markers of outcome and potential therapeutic targets in breast cancer, this observation has a number of potential implications important for both the basic biology of breast cancer and the clinical management of the disease

  16. Inter-domain tagging implicates caveolin-1 in insulin receptor trafficking and Erk signaling bias in pancreatic beta-cells

    Directory of Open Access Journals (Sweden)

    Tobias Boothe

    2016-05-01

    Full Text Available Objective: The role and mechanisms of insulin receptor internalization remain incompletely understood. Previous trafficking studies of insulin receptors involved fluorescent protein tagging at their termini, manipulations that may be expected to result in dysfunctional receptors. Our objective was to determine the trafficking route and molecular mechanisms of functional tagged insulin receptors and endogenous insulin receptors in pancreatic beta-cells. Methods: We generated functional insulin receptors tagged with pH-resistant fluorescent proteins between domains. Confocal, TIRF and STED imaging revealed a trafficking pattern of inter-domain tagged insulin receptors and endogenous insulin receptors detected with antibodies. Results: Surprisingly, interdomain-tagged and endogenous insulin receptors in beta-cells bypassed classical Rab5a- or Rab7-mediated endocytic routes. Instead, we found that removal of insulin receptors from the plasma membrane involved tyrosine-phosphorylated caveolin-1, prior to trafficking within flotillin-1-positive structures to lysosomes. Multiple methods of inhibiting caveolin-1 significantly reduced Erk activation in vitro or in vivo, while leaving Akt signaling mostly intact. Conclusions: We conclude that phosphorylated caveolin-1 plays a role in insulin receptor internalization towards lysosomes through flotillin-1-positive structures and that caveolin-1 helps bias physiological beta-cell insulin signaling towards Erk activation. Author Video: Author Video Watch what authors say about their articles Keywords: Insulin receptor internalization, Insulin resistance, Pancreatic islet beta-cells, Autocrine insulin signaling

  17. Modulating Neurotrophin Receptor Signaling as a Therapeutic Strategy for Huntington’s Disease

    Science.gov (United States)

    Simmons, Danielle A.

    2017-01-01

    Huntington’s disease (HD) is an autosomal dominant neurodegenerative disorder caused by CAG repeat expansions in the IT15 gene which encodes the huntingtin (HTT) protein. Currently, no treatments capable of preventing or slowing disease progression exist. Disease modifying therapeutics for HD would be expected to target a comprehensive set of degenerative processes given the diverse mechanisms contributing to HD pathogenesis including neuroinflammation, excitotoxicity, and transcription dysregulation. A major contributor to HD-related degeneration is mutant HTT-induced loss of neurotrophic support. Thus, neurotrophin (NT) receptors have emerged as therapeutic targets in HD. The considerable overlap between NT signaling networks and those dysregulated by mutant HTT provides strong theoretical support for this approach. This review will focus on the contributions of disrupted NT signaling in HD-related neurodegeneration and how targeting NT receptors to augment pro-survival signaling and/or to inhibit degenerative signaling may combat HD pathologies. Therapeutic strategies involving NT delivery, peptidomimetics, and the targeting of specific NT receptors (e.g., Trks or p75NTR), particularly with small molecule ligands, are discussed. PMID:29254102

  18. Two signaling molecules share a phosphotyrosine-containing binding site in the platelet-derived growth factor receptor.

    OpenAIRE

    Nishimura, R; Li, W; Kashishian, A; Mondino, A; Zhou, M; Cooper, J; Schlessinger, J

    1993-01-01

    Autophosphorylation sites of growth factor receptors with tyrosine kinase activity function as specific binding sites for Src homology 2 (SH2) domains of signaling molecules. This interaction appears to be a crucial step in a mechanism by which receptor tyrosine kinases relay signals to downstream signaling pathways. Nck is a widely expressed protein consisting exclusively of SH2 and SH3 domains, the overexpression of which causes cell transformation. It has been shown that various growth fac...

  19. Autoinactivation of the stargazin-AMPA receptor complex: subunit-dependency and independence from physical dissociation.

    Directory of Open Access Journals (Sweden)

    Artur Semenov

    Full Text Available Agonist responses and channel kinetics of native α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA receptors are modulated by transmembrane accessory proteins. Stargazin, the prototypical accessory protein, decreases desensitization and increases agonist potency at AMPA receptors. Furthermore, in the presence of stargazin, the steady-state responses of AMPA receptors show a gradual decline at higher glutamate concentrations. This "autoinactivation" has been assigned to physical dissociation of the stargazin-AMPA receptor complex and suggested to serve as a protective mechanism against overactivation. Here, we analyzed autoinactivation of GluA1-A4 AMPA receptors (all flip isoform expressed in the presence of stargazin. Homomeric GluA1, GluA3, and GluA4 channels showed pronounced autoinactivation indicated by the bell-shaped steady-state dose response curves for glutamate. In contrast, homomeric GluA2i channels did not show significant autoinactivation. The resistance of GluA2 to autoinactivation showed striking dependence on the splice form as GluA2-flop receptors displayed clear autoinactivation. Interestingly, the resistance of GluA2-flip containing receptors to autoinactivation was transferred onto heteromeric receptors in a dominant fashion. To examine the relationship of autoinactivation to physical separation of stargazin from the AMPA receptor, we analyzed a GluA4-stargazin fusion protein. Notably, the covalently linked complex and separately expressed proteins expressed a similar level of autoinactivation. We conclude that autoinactivation is a subunit and splice form dependent property of AMPA receptor-stargazin complexes, which involves structural rearrangements within the complex rather than any physical dissociation.

  20. TRPM5, a taste-signaling transient receptor potential ion-channel, is a ubiquitous signaling component in chemosensory cells

    Directory of Open Access Journals (Sweden)

    Hofmann Thomas

    2007-07-01

    Full Text Available Abstract Background A growing number of TRP channels have been identified as key players in the sensation of smell, temperature, mechanical forces and taste. TRPM5 is known to be abundantly expressed in taste receptor cells where it participates in sweet, amino acid and bitter perception. A role of TRPM5 in other sensory systems, however, has not been studied so far. Results Here, we systematically investigated the expression of TRPM5 in rat and mouse tissues. Apart from taste buds, where we found TRPM5 to be predominantly localized on the basolateral surface of taste receptor cells, TRPM5 immunoreactivity was seen in other chemosensory organs – the main olfactory epithelium and the vomeronasal organ. Most strikingly, we found solitary TRPM5-enriched epithelial cells in all parts of the respiratory and gastrointestinal tract. Based on their tissue distribution, the low cell density, morphological features and co-immunostaining with different epithelial markers, we identified these cells as brush cells (also known as tuft, fibrillovesicular, multivesicular or caveolated cells. In terms of morphological characteristics, brush cells resemble taste receptor cells, while their origin and biological role are still under intensive debate. Conclusion We consider TRPM5 to be an intrinsic signaling component of mammalian chemosensory organs, and provide evidence for brush cells being an important cellular correlate in the periphery.

  1. Effects of gamma irradiation on the DNA-protein complex between the estrogen response element and the estrogen receptor

    Energy Technology Data Exchange (ETDEWEB)

    Stisova, Viktorie [Department of Radiation Dosimetry, Nuclear Physics Institute AS CR, Na Truhlarce 39/64, 18086 Praha 8 (Czech Republic); Goffinont, Stephane; Spotheim-Maurizot, Melanie [Centre de Biophysique Moleculaire CNRS, rue Charles Sadron, 45071 Orleans Cedex 2 (France); Davidkova, Marie, E-mail: davidkova@ujf.cas.c [Department of Radiation Dosimetry, Nuclear Physics Institute AS CR, Na Truhlarce 39/64, 18086 Praha 8 (Czech Republic)

    2010-08-15

    Signaling by estrogens, risk factors in breast cancer, is mediated through their binding to the estrogen receptor protein (ER), followed by the formation of a complex between ER and a DNA sequence, called estrogen response element (ERE). Anti-estrogens act as competitive inhibitors by blocking the signal transduction. We have studied in vitro the radiosensitivity of the complex between ERalpha, a subtype of this receptor, and a DNA fragment bearing ERE, as well as the influence of an estrogen (estradiol) or an anti-estrogen (tamoxifen) on this radiosensitivity. We observe that the complex is destabilized upon irradiation with gamma rays in aerated aqueous solution. The analysis of the decrease of binding abilities of the two partners shows that destabilization is mainly due to the damage to the protein. The destabilization is reduced when irradiating in presence of tamoxifen and is increased in presence of estradiol. These effects are due to opposite influences of the ligands on the loss of binding ability of ER. The mechanism that can account for our results is: binding of estradiol or tamoxifen induces distinct structural changes of the ER ligand-binding domain that can trigger (by allostery) distinct structural changes of the ER DNA-binding domains and thus, can differently affect ER-ERE interaction.

  2. Elevated insulin-like growth factor 1 receptor signaling induces antiestrogen resistance through the MAPK/ERK and PI3K/Akt signaling routes

    NARCIS (Netherlands)

    Zhang, Y.; Moerkens, M.; Ramaiahgari, S.; Bont, de H.J.G.M.; Price, L.; Meerman, J.H.N.; Water, van de B.

    2011-01-01

    INTRODUCTION: Insulin-like growth factor 1 (IGF-1) receptor (IGF-1R) is phosphorylated in all breast cancer subtypes. Past findings have shown that IGF-1R mediates antiestrogen resistance through cross-talk with estrogen receptor (ER) signaling and via its action upstream of the epidermal growth

  3. Two signaling molecules share a phosphotyrosine-containing binding site in the platelet-derived growth factor receptor.

    Science.gov (United States)

    Nishimura, R; Li, W; Kashishian, A; Mondino, A; Zhou, M; Cooper, J; Schlessinger, J

    1993-11-01

    Autophosphorylation sites of growth factor receptors with tyrosine kinase activity function as specific binding sites for Src homology 2 (SH2) domains of signaling molecules. This interaction appears to be a crucial step in a mechanism by which receptor tyrosine kinases relay signals to downstream signaling pathways. Nck is a widely expressed protein consisting exclusively of SH2 and SH3 domains, the overexpression of which causes cell transformation. It has been shown that various growth factors stimulate the phosphorylation of Nck and its association with autophosphorylated growth factor receptors. A panel of platelet-derived growth factor (PDGF) receptor mutations at tyrosine residues has been used to identify the Nck binding site. Here we show that mutation at Tyr-751 of the PDGF beta-receptor eliminates Nck binding both in vitro and in living cells. Moreover, the Y751F PDGF receptor mutant failed to mediate PDGF-stimulated phosphorylation of Nck in intact cells. A phosphorylated Tyr-751 is also required for binding of phosphatidylinositol-3 kinase to the PDGF receptor. Hence, the SH2 domains of p85 and Nck share a binding site in the PDGF receptor. Competition experiments with different phosphopeptides derived from the PDGF receptor suggest that binding of Nck and p85 is influenced by different residues around Tyr-751. Thus, a single tyrosine autophosphorylation site is able to link the PDGF receptor to two distinct SH2 domain-containing signaling molecules.

  4. Cell–cell signaling drives the evolution of complex traits: introduction—lung evo-devo

    Science.gov (United States)

    Torday, John S.; Rehan, V. K.

    2009-01-01

    Physiology integrates biology with the environment through cell–cell interactions at multiple levels. The evolution of the respiratory system has been “deconvoluted” (Torday and Rehan in Am J Respir Cell Mol Biol 31:8–12, 2004) through Gene Regulatory Networks (GRNs) applied to cell–cell communication for all aspects of lung biology development, homeostasis, regeneration, and aging. Using this approach, we have predicted the phenotypic consequences of failed signaling for lung development, homeostasis, and regeneration based on evolutionary principles. This cell–cell communication model predicts other aspects of vertebrate physiology as adaptational responses. For example, the oxygen-induced differentiation of alveolar myocytes into alveolar adipocytes was critical for the evolution of the lung in land dwelling animals adapting to fluctuating Phanarezoic oxygen levels over the past 500 million years. Adipocytes prevent lung injury due to oxygen radicals and facilitate the rise of endothermy. In addition, they produce the class I cytokine leptin, which augments pulmonary surfactant activity and alveolar surface area, increasing selection pressure for both respiratory oxygenation and metabolic demand initially constrained by high-systemic vascular pressure, but subsequently compensated by the evolution of the adrenomedullary beta-adrenergic receptor mechanism. Conserted positive selection for the lung and adrenals created further selection pressure for the heart, which becomes progressively more complex phylogenetically in tandem with the lung. Developmentally, increasing heart complexity and size impinges precociously on the gut mesoderm to induce the liver. That evolutionary-developmental interaction is significant because the liver provides regulated sources of glucose and glycogen to the evolving physiologic system, which is necessary for the evolution of the neocortex. Evolution of neocortical control furthers integration of physiologic systems. Such

  5. The Adaptor Protein SAP Directly Associates with CD3ζ Chain and Regulates T Cell Receptor Signaling

    Science.gov (United States)

    Proust, Richard; Bertoglio, Jacques; Gesbert, Franck

    2012-01-01

    Mutations altering the gene encoding the SLAM associated protein (SAP) are responsible for the X-linked lymphoproliferative disease or XLP1. Its absence is correlated with a defective NKT cells development, a decrease in B cell functions and a reduced T cells and NK cells cytotoxic activities, thus leading to an immunodeficiency syndrome. SAP is a small 128 amino-acid long protein that is almost exclusively composed of an SH2 domain. It has been shown to interact with the CD150/SLAM family of receptors, and in a non-canonical manner with SH3 containing proteins such as Fyn, βPIX, PKCθ and Nck1. It would thus play the role of a minimal adaptor protein. It has been shown that SAP plays an important function in the activation of T cells through its interaction with the SLAM family of receptors. Therefore SAP defective T cells display a reduced activation of signaling events downstream of the TCR-CD3 complex triggering. In the present work, we evidence that SAP is a direct interactor of the CD3ζ chain. This direct interaction occurs through the first ITAM of CD3ζ, proximal to the membrane. Additionally, we show that, in the context of the TCR-CD3 signaling, an Sh-RNA mediated silencing of SAP is responsible for a decrease of several canonical T cell signaling pathways including Erk, Akt and PLCγ1 and to a reduced induction of IL-2 and IL-4 mRNA. Altogether, we show that SAP plays a central function in the T cell activation processes through a direct association with the CD3 complex. PMID:22912825

  6. PTP1B-dependent regulation of receptor tyrosine kinase signaling by the actin-binding protein Mena.

    Science.gov (United States)

    Hughes, Shannon K; Oudin, Madeleine J; Tadros, Jenny; Neil, Jason; Del Rosario, Amanda; Joughin, Brian A; Ritsma, Laila; Wyckoff, Jeff; Vasile, Eliza; Eddy, Robert; Philippar, Ulrike; Lussiez, Alisha; Condeelis, John S; van Rheenen, Jacco; White, Forest; Lauffenburger, Douglas A; Gertler, Frank B

    2015-11-01

    During breast cancer progression, alternative mRNA splicing produces functionally distinct isoforms of Mena, an actin regulator with roles in cell migration and metastasis. Aggressive tumor cell subpopulations express Mena(INV), which promotes tumor cell invasion by potentiating EGF responses. However, the mechanism by which this occurs is unknown. Here we report that Mena associates constitutively with the tyrosine phosphatase PTP1B and mediates a novel negative feedback mechanism that attenuates receptor tyrosine kinase signaling. On EGF stimulation, complexes containing Mena and PTP1B are recruited to the EGFR, causing receptor dephosphorylation and leading to decreased motility responses. Mena also interacts with the 5' inositol phosphatase SHIP2, which is important for the recruitment of the Mena-PTP1B complex to the EGFR. When Mena(INV) is expressed, PTP1B recruitment to the EGFR is impaired, providing a mechanism for growth factor sensitization to EGF, as well as HGF and IGF, and increased resistance to EGFR and Met inhibitors in signaling and motility assays. In sum, we demonstrate that Mena plays an important role in regulating growth factor-induced signaling. Disruption of this attenuation by Mena(INV) sensitizes tumor cells to low-growth factor concentrations, thereby increasing the migration and invasion responses that contribute to aggressive, malignant cell phenotypes. © 2015 Hughes, Oudin, et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

  7. Lenalidomide induces lipid raft assembly to enhance erythropoietin receptor signaling in myelodysplastic syndrome progenitors.

    Science.gov (United States)

    McGraw, Kathy L; Basiorka, Ashley A; Johnson, Joseph O; Clark, Justine; Caceres, Gisela; Padron, Eric; Heaton, Ruth; Ozawa, Yukiyasu; Wei, Sheng; Sokol, Lubomir; List, Alan F

    2014-01-01

    Anemia remains the principal management challenge for patients with lower risk Myelodysplastic Syndromes (MDS). Despite appropriate cytokine production and cellular receptor display, erythropoietin receptor (EpoR) signaling is impaired. We reported that EpoR signaling is dependent upon receptor localization within lipid raft microdomains, and that disruption of raft integrity abolishes signaling capacity. Here, we show that MDS erythroid progenitors display markedly diminished raft assembly and smaller raft aggregates compared to normal controls (p = 0.005, raft number; p = 0.023, raft size). Because lenalidomide triggers raft coalescence in T-lymphocytes promoting immune synapse formation, we assessed effects of lenalidomide on raft assembly in MDS erythroid precursors and UT7 cells. Lenalidomide treatment rapidly induced lipid raft formation accompanied by EpoR recruitment into raft fractions together with STAT5, JAK2, and Lyn kinase. The JAK2 phosphatase, CD45, a key negative regulator of EpoR signaling, was displaced from raft fractions. Lenalidomide treatment prior to Epo stimulation enhanced both JAK2 and STAT5 phosphorylation in UT7 and primary MDS erythroid progenitors, accompanied by increased STAT5 DNA binding in UT7 cells, and increased erythroid colony forming capacity in both UT7 and primary cells. Raft induction was associated with F-actin polymerization, which was blocked by Rho kinase inhibition. These data indicate that deficient raft integrity impairs EpoR signaling, and provides a novel strategy to enhance EpoR signal fidelity in non-del(5q) MDS.

  8. Correlated cone noise decreases rod signal contributions to the post-receptoral pathways.

    Science.gov (United States)

    Hathibelagal, Amithavikram R; Feigl, Beatrix; Zele, Andrew J

    2018-04-01

    This study investigated how invisible extrinsic temporal white noise that correlates with the activity of one of the three [magnocellular (MC), parvocellular (PC), or koniocellular (KC)] post-receptoral pathways alters mesopic rod signaling. A four-primary photostimulator provided independent control of the rod and three cone photoreceptor excitations. The rod contributions to the three post-receptoral pathways were estimated by perceptually matching a 20% contrast rod pulse by independently varying the LMS (MC pathway), +L-M (PC pathway), and S-cone (KC pathway) excitations. We show that extrinsic cone noise caused a predominant decrease in the overall magnitude and ratio of the rod contributions to each pathway. Thus, the relative cone activity in the post-receptoral pathways determines the relative mesopic rod inputs to each pathway.

  9. A2A adenosine receptor ligand binding and signalling is allosterically modulated by adenosine deaminase.

    Science.gov (United States)

    Gracia, Eduard; Pérez-Capote, Kamil; Moreno, Estefanía; Barkešová, Jana; Mallol, Josefa; Lluís, Carme; Franco, Rafael; Cortés, Antoni; Casadó, Vicent; Canela, Enric I

    2011-05-01

    A2ARs (adenosine A2A receptors) are highly enriched in the striatum, which is the main motor control CNS (central nervous system) area. BRET (bioluminescence resonance energy transfer) assays showed that A2AR homomers may act as cell-surface ADA (adenosine deaminase; EC 3.5.4.4)-binding proteins. ADA binding affected the quaternary structure of A2ARs present on the cell surface. ADA binding to adenosine A2ARs increased both agonist and antagonist affinity on ligand binding to striatal membranes where these proteins are co-expressed. ADA also increased receptor-mediated ERK1/2 (extracellular-signal-regulated kinase 1/2) phosphorylation. Collectively, the results of the present study show that ADA, apart from regulating the concentration of extracellular adenosine, may behave as an allosteric modulator that markedly enhances ligand affinity and receptor function. This powerful regulation may have implications for the physiology and pharmacology of neuronal A2ARs.

  10. Building tolerance by dismantling synapses: inhibitory receptor signaling in natural killer cells.

    Science.gov (United States)

    Huse, Morgan; Catherine Milanoski, S; Abeyweera, Thushara P

    2013-01-01

    Cell surface receptors bearing immunotyrosine-based inhibitory motifs (ITIMs) maintain natural killer (NK) cell tolerance to normal host tissues. These receptors are difficult to analyze mechanistically because they block activating responses in a rapid and comprehensive manner. The advent of high-resolution single cell imaging techniques has enabled investigators to explore the cell biological basis of the inhibitory response. Recent studies using these approaches indicate that ITIM-containing receptors function at least in part by structurally undermining the immunological synapse between the NK cell and its target. In this review, we discuss these new advances and how they might relate to what is known about the biochemistry of inhibitory signaling in NK cells and other cell types. © 2012 John Wiley & Sons A/S. Published by Blackwell Publishing Ltd.

  11. Functional requirements for inhibitory signal transmission by the immunomodulatory receptor CD300a.

    Science.gov (United States)

    DeBell, Karen E; Simhadri, Venkateswara R; Mariano, John L; Borrego, Francisco

    2012-04-26

    Activation signals can be negatively regulated by cell surface receptors bearing immunoreceptor tyrosine-based inhibitory motifs (ITIMs). CD300a, an ITIM bearing type I transmembrane protein, is expressed on many hematopoietic cells, including subsets of lymphocytes. We have taken two approaches to further define the mechanism by which CD300a acts as an inhibitor of immune cell receptor signaling. First, we have expressed in Jurkat T cells a chimeric receptor consisting of the extracellular domains of killer-cell immunoglobulin-like receptor (KIR)2DL2 fused to the transmembrane and cytoplasmic segments of CD300a (KIR-CD300a) to explore surrogate ligand-stimulated inhibition of superantigen stimulated T cell receptor (TCR) mediated cell signaling. We found that intact CD300a ITIMs were essential for inhibition and that the tyrosine phosphorylation of these ITIMs required the src tyrosine kinase Lck. Tyrosine phosphorylation of the CD300a ITIMs created docking sites for both src homology 2 domain containing protein tyrosine phosphatase (SHP)-1 and SHP-2. Suppression of SHP-1 and SHP-2 expression in KIR-CD300a Jurkat T cells with siRNA and the use of DT40 chicken B cell lines expressing CD300a and deficient in several phosphatases revealed that SHP-1, but not SHP-2 or the src homology 2 domain containing inositol 5' phosphatase SHIP, was utilized by CD300a for its inhibitory activity. These studies provide new insights into the function of CD300a in tuning T and B cell responses.

  12. Apo-ghrelin receptor (apo-GHSR1a Regulates Dopamine Signaling in the Brain

    Directory of Open Access Journals (Sweden)

    Andras eKern

    2014-08-01

    Full Text Available The orexigenic peptide hormone ghrelin is synthesized in the stomach and its receptor growth hormone secretagogue receptor (GHSR1a is expressed mainly in the central nervous system (CNS. In this review we confine our discussion to the physiological role of GHSR1a in the brain. Paradoxically, despite broad expression of GHSR1a in the CNS, other than trace amounts in the hypothalamus, ghrelin is undetectable in the brain. In our efforts to elucidate the function of the ligand-free ghrelin receptor (apo-GHSR1a we identified subsets of neurons that co-express GHSR1a and dopamine receptors. In this review we focus on interactions between apo-GHSR1a and dopamine-2 receptor (DRD2 and formation of GHSR1a:DRD2 heteromers in hypothalamic neurons that regulate appetite, and discuss implications for the treatment of Prader-Willi syndrome. GHSR1a antagonists of distinct chemical structures, a quinazolinone and a triazole, respectively enhance and inhibit dopamine signaling through GHSR1a:DRD2 heteromers by an allosteric mechanism. This finding illustrates a potential strategy for designing the next generation of drugs for treating eating disorders as well as psychiatric disorders caused by abnormal dopamine signaling. Treatment with a GHSR1a antagonist that enhances dopamine/DRD2 activity in GHSR1a:DRD2 expressing hypothalamic neurons has the potential to inhibit the uncontrollable hyperphagia associated with Prader-Willi syndrome. DRD2 antagonists are prescribed for treating schizophrenia, but these block dopamine signaling in all DRD2 expressing neurons and are associated with adverse side effects, including enhanced appetite and excessive weight gain. A GHSR1a antagonist of structural class that allosterically blocks dopamine/DRD2 action in GHSR1a:DRD2 expressing neurons would have no effect on neurons expressing DRD2 alone; therefore, the side effects of DRD2 antagonists would potentially be reduced thereby enhancing patient compliance.

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

    Science.gov (United States)

    Lynch, Jennifer R; Wang, Jenny Yingzi

    2016-05-11

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

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

    Directory of Open Access Journals (Sweden)

    Jennifer R. Lynch

    2016-05-01

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

  15. Peroxisome proliferator-activated receptor gamma signaling in human sperm physiology.

    Science.gov (United States)

    Liu, Li-Li; Xian, Hua; Cao, Jing-Chen; Zhang, Chong; Zhang, Yong-Hui; Chen, Miao-Miao; Qian, Yi; Jiang, Ming

    2015-01-01

    Peroxisome proliferator-activated receptor gamma (PPARγ) is a member of the PPARs, which are transcription factors of the steroid receptor superfamily. PPARγ acts as an important molecule for regulating energy homeostasis, modulates the hypothalamic-pituitary-gonadal (HPG) axis, and is reciprocally regulated by HPG. In the human, PPARγ protein is highly expressed in ejaculated spermatozoa, implying a possible role of PPARγ signaling in regulating sperm energy dissipation. PPARγ protein is also expressed in Sertoli cells and germ cells (spermatocytes). Its activation can be induced during capacitation and the acrosome reaction. This mini-review will focus on how PPARγ signaling may affect fertility and sperm quality and the potential reversibility of these adverse effects.

  16. Role of type I interferon receptor signaling on NK cell development and functions.

    Directory of Open Access Journals (Sweden)

    Jean Guan

    Full Text Available Type I interferons (IFN are unique cytokines transcribed from intronless genes. They have been extensively studied because of their anti-viral functions. The anti-viral effects of type I IFN are mediated in part by natural killer (NK cells. However, the exact contribution of type I IFN on NK cell development, maturation and activation has been somewhat difficult to assess. In this study, we used a variety of approaches to define the consequences of the lack of type I interferon receptor (IFNAR signaling on NK cells. Using IFNAR deficient mice, we found that type I IFN affect NK cell development at the pre-pro NK stage. We also found that systemic absence of IFNAR signaling impacts NK cell maturation with a significant increase in the CD27+CD11b+ double positive (DP compartment in all organs. However, there is tissue specificity, and only in liver and bone marrow is the maturation defect strictly dependent on cell intrinsic IFNAR signaling. Finally, using adoptive transfer and mixed bone marrow approaches, we also show that cell intrinsic IFNAR signaling is not required for NK cell IFN-γ production in the context of MCMV infection. Taken together, our studies provide novel insights on how type I IFN receptor signaling regulates NK cell development and functions.

  17. Identification of potential pathway mediation targets in Toll-like receptor signaling.

    Directory of Open Access Journals (Sweden)

    Fan Li

    2009-02-01

    Full Text Available Recent advances in reconstruction and analytical methods for signaling networks have spurred the development of large-scale models that incorporate fully functional and biologically relevant features. An extended reconstruction of the human Toll-like receptor signaling network is presented herein. This reconstruction contains an extensive complement of kinases, phosphatases, and other associated proteins that mediate the signaling cascade along with a delineation of their associated chemical reactions. A computational framework based on the methods of large-scale convex analysis was developed and applied to this network to characterize input-output relationships. The input-output relationships enabled significant modularization of the network into ten pathways. The analysis identified potential candidates for inhibitory mediation of TLR signaling with respect to their specificity and potency. Subsequently, we were able to identify eight novel inhibition targets through constraint-based modeling methods. The results of this study are expected to yield meaningful avenues for further research in the task of mediating the Toll-like receptor signaling network and its effects.

  18. Caenorhabditis elegans fibroblast growth factor receptor signaling can occur independently of the multi-substrate adaptor FRS2.

    Science.gov (United States)

    Lo, Te-Wen; Bennett, Daniel C; Goodman, S Jay; Stern, Michael J

    2010-06-01

    The components of receptor tyrosine kinase signaling complexes help to define the specificity of the effects of their activation. The Caenorhabditis elegans fibroblast growth factor receptor (FGFR), EGL-15, regulates a number of processes, including sex myoblast (SM) migration guidance and fluid homeostasis, both of which require a Grb2/Sos/Ras cassette of signaling components. Here we show that SEM-5/Grb2 can bind directly to EGL-15 to mediate SM chemoattraction. A yeast two-hybrid screen identified SEM-5 as able to interact with the carboxy-terminal domain (CTD) of EGL-15, a domain that is specifically required for SM chemoattraction. This interaction requires the SEM-5 SH2-binding motifs present in the CTD (Y(1009) and Y(1087)), and these sites are required for the CTD role of EGL-15 in SM chemoattraction. SEM-5, but not the SEM-5 binding sites located in the CTD, is required for the fluid homeostasis function of EGL-15, indicating that SEM-5 can link to EGL-15 through an alternative mechanism. The multi-substrate adaptor protein FRS2 serves to link vertebrate FGFRs to Grb2. In C. elegans, an FRS2-like gene, rog-1, functions upstream of a Ras/MAPK pathway for oocyte maturation but is not required for EGL-15 function. Thus, unlike the vertebrate FGFRs, which require the multi-substrate adaptor FRS2 to recruit Grb2, EGL-15 can recruit SEM-5/Grb2 directly.

  19. IRS-1 acts as an endocytic regulator of IGF-I receptor to facilitate sustained IGF signaling.

    Science.gov (United States)

    Yoneyama, Yosuke; Lanzerstorfer, Peter; Niwa, Hideaki; Umehara, Takashi; Shibano, Takashi; Yokoyama, Shigeyuki; Chida, Kazuhiro; Weghuber, Julian; Hakuno, Fumihiko; Takahashi, Shin-Ichiro

    2018-04-11

    Insulin-like growth factor-I receptor (IGF-IR) preferentially regulates the long-term IGF activities including growth and metabolism. Kinetics of ligand-dependent IGF-IR endocytosis determines how IGF induces such downstream signaling outputs. Here, we find that the insulin receptor substrate (IRS)-1 modulates how long ligand-activated IGF-IR remains at the cell surface before undergoing endocytosis in mammalian cells. IRS-1 interacts with the clathrin adaptor complex AP2. IRS-1, but not an AP2-binding-deficient mutant, delays AP2-mediated IGF-IR endocytosis after the ligand stimulation. Mechanistically, IRS-1 inhibits the recruitment of IGF-IR into clathrin-coated structures; for this reason, IGF-IR avoids rapid endocytosis and prolongs its activity on the cell surface. Accelerating IGF-IR endocytosis via IRS-1 depletion induces the shift from sustained to transient Akt activation and augments FoxO-mediated transcription. Our study establishes a new role for IRS-1 as an endocytic regulator of IGF-IR that ensures sustained IGF bioactivity, independent of its classic role as an adaptor in IGF-IR signaling. © 2018, Yoneyama et al.

  20. Enhanced NMDA receptor-mediated intracellular calcium signaling in magnocellular neurosecretory neurons in heart failure rats.

    Science.gov (United States)

    Stern, Javier E; Potapenko, Evgeniy S

    2013-08-15

    An enhanced glutamate excitatory function within the hypothalamic supraoptic and paraventricluar nuclei is known to contribute to increased neurosecretory and presympathetic neuronal activity, and hence, neurohumoral activation, during heart failure (HF). Still, the precise mechanisms underlying enhanced glutamate-driven neuronal activity in HF remain to be elucidated. Here, we performed simultaneous electrophysiology and fast confocal Ca²⁺ imaging to determine whether altered N-methyl-d-aspartate (NMDA) receptor-mediated changes in intracellular Ca²⁺ levels (NMDA-ΔCa²⁺) occurred in hypothalamic magnocellular neurosecretory cells (MNCs) in HF rats. We found that activation of NMDA receptors resulted in a larger ΔCa²⁺ in MNCs from HF when compared with sham rats. The enhanced NMDA-ΔCa²⁺ was neither dependent on the magnitude of the NMDA-mediated current (voltage clamp) nor on the degree of membrane depolarization or firing activity evoked by NMDA (current clamp). Differently from NMDA receptor activation, firing activity evoked by direct membrane depolarization resulted in similar changes in intracellular Ca²⁺ in sham and HF rats. Taken together, our results support a relatively selective alteration of intracellular Ca²⁺ homeostasis and signaling following activation of NMDA receptors in MNCs during HF. The downstream functional consequences of such altered ΔCa²⁺ signaling during HF are discussed.

  1. The LDL Receptor-Related Protein 1: At the Crossroads of Lipoprotein Metabolism and Insulin Signaling

    Directory of Open Access Journals (Sweden)

    Dianaly T. Au

    2017-01-01

    Full Text Available The metabolic syndrome is an escalating worldwide public health concern. Defined by a combination of physiological, metabolic, and biochemical factors, the metabolic syndrome is used as a clinical guideline to identify individuals with a higher risk for type 2 diabetes and cardiovascular disease. Although risk factors for type 2 diabetes and cardiovascular disease have been known for decades, the molecular mechanisms involved in the pathophysiology of these diseases and their interrelationship remain unclear. The LDL receptor-related protein 1 (LRP1 is a large endocytic and signaling receptor that is widely expressed in several tissues. As a member of the LDL receptor family, LRP1 is involved in the clearance of chylomicron remnants from the circulation and has been demonstrated to be atheroprotective. Recently, studies have shown that LRP1 is involved in insulin receptor trafficking and regulation and glucose metabolism. This review summarizes the role of tissue-specific LRP1 in insulin signaling and its potential role as a link between lipoprotein and glucose metabolism in diabetes.

  2. Differential Role of Insulin/IGF-1 Receptor Signaling in Muscle Growth and Glucose Homeostasis

    Directory of Open Access Journals (Sweden)

    Brian T. O’Neill

    2015-05-01

    Full Text Available Insulin and insulin-like growth factor 1 (IGF-1 are major regulators of muscle protein and glucose homeostasis. To determine how these pathways interact, we generated mice with muscle-specific knockout of IGF-1 receptor (IGF1R and insulin receptor (IR. These MIGIRKO mice showed >60% decrease in muscle mass. Despite a complete lack of insulin/IGF-1 signaling in muscle, MIGIRKO mice displayed normal glucose and insulin tolerance. Indeed, MIGIRKO mice showed fasting hypoglycemia and increased basal glucose uptake. This was secondary to decreased TBC1D1 resulting in increased Glut4 and Glut1 membrane localization. Interestingly, overexpression of a dominant-negative IGF1R in muscle induced glucose intolerance in MIGIRKO animals. Thus, loss of insulin/IGF-1 signaling impairs muscle growth, but not whole-body glucose tolerance due to increased membrane localization of glucose transporters. Nonetheless, presence of a dominant-negative receptor, even in the absence of functional IR/IGF1R, induces glucose intolerance, indicating that interactions between these receptors and other proteins in muscle can impair glucose homeostasis.

  3. Roles for NHERF1 and NHERF2 on the regulation of C3a receptor signaling in human mast cells.

    Directory of Open Access Journals (Sweden)

    Hariharan Subramanian

    Full Text Available BACKGROUND: The anaphylatoxin C3a binds to the G protein coupled receptor (GPCR, C3aR and activates divergent signaling pathways to induce degranulation and cytokine production in human mast cells. Adapter proteins such as the Na(+/H(+ exchange regulatory factor (NHERF1 and NHERF2 have been implicated in regulating functions of certain GPCRs by binding to the class I PDZ (PSD-95/Dlg/Zo1 motifs present on their cytoplasmic tails. Although C3aR possesses a class I PDZ motif, the possibility that it interacts with NHERF proteins to modulate signaling in human mast cells has not been determined. METHODOLOGY/PRINCIPAL FINDINGS: Using reverse transcription PCR and Western blotting, we found that NHERF1 and NHERF2 are expressed in human mast cell lines (HMC-1, LAD2 and CD34(+-derived primary human mast cells. Surprisingly, however, C3aR did not associate with these adapter proteins. To assess the roles of NHERFs on signaling downstream of C3aR, we used lentiviral shRNA to stably knockdown the expression of these proteins in human mast cells. Silencing the expression of NHERF1 and NHERF2 had no effect on C3aR desensitization, agonist-induced receptor internalization, ERK/Akt phosphorylation or chemotaxis. However, loss of NHERF1 and NHERF2 resulted in significant inhibition of C3a-induced mast cell degranulation, NF-κB activation and chemokine production. CONCLUSION/SIGNIFICANCE: This study demonstrates that although C3aR possesses a class I PDZ motif, it does not associate with NHERF1 and NHERF2. Surprisingly, these proteins provide stimulatory signals for C3a-induced degranulation, NF-κB activation and chemokine generation in human mast cells. These findings reveal a new level of complexity for the functional regulation of C3aR by NHERFs in human mast cells.

  4. Toll-like receptor 4 signaling in intracerebral hemorrhage-induced inflammation and injury

    OpenAIRE

    Fang, Huang; Wang, Peng-Fei; Zhou, Yu; Wang, Yan-Chun; Yang, Qing-Wu

    2013-01-01

    Intracerebral hemorrhage (ICH) is a common type of fatal stroke, accounting for about 15% to 20% of all strokes. Hemorrhagic strokes are associated with high mortality and morbidity, and increasing evidence shows that innate immune responses and inflammatory injury play a critical role in ICH-induced neurological deficits. However, the signaling pathways involved in ICH-induced inflammatory responses remain elusive. Toll-like receptor 4 (TLR4) belongs to a large family of pattern recognition ...

  5. Steroid receptor coactivators 1 and 2 mediate fetal-to-maternal signaling that initiates parturition

    OpenAIRE

    Gao, Lu; Rabbitt, Elizabeth H.; Condon, Jennifer C.; Renthal, Nora E.; Johnston, John M.; Mitsche, Matthew A.; Chambon, Pierre; Xu, Jianming; O’Malley, Bert W.; Mendelson, Carole R.

    2015-01-01

    The precise mechanisms that lead to parturition are incompletely defined. Surfactant protein-A (SP-A), which is secreted by fetal lungs into amniotic fluid (AF) near term, likely provides a signal for parturition; however, SP-A–deficient mice have only a relatively modest delay (~12 hours) in parturition, suggesting additional factors. Here, we evaluated the contribution of steroid receptor coactivators 1 and 2 (SRC-1 and SRC-2), which upregulate SP-A transcription, to the parturition process...

  6. Nonlinear signal processing for ultrasonic imaging of material complexity

    Czech Academy of Sciences Publication Activity Database

    Dos Santos, S.; Vejvodová, Šárka; Převorovský, Zdeněk

    2010-01-01

    Roč. 59, č. 2 (2010), s. 108-117 ISSN 1736-6046 Institutional research plan: CEZ:AV0Z20760514 Keywords : nonlinear signal processing * TR-NEWS * symmetry analysis * DORT Subject RIV: BI - Acoustics Impact factor: 0.464, year: 2010 www.eap.ee/proceedings

  7. Inhibition of Wnt/β-catenin signaling by a soluble collagen-derived frizzled domain interacting with Wnt3a and the receptors frizzled 1 and 8.

    Directory of Open Access Journals (Sweden)

    Ismaïl Hendaoui

    Full Text Available The Wnt/β-catenin pathway controls cell proliferation, death and differentiation. Several families of extracellular proteins can antagonize Wnt/β-catenin signaling, including the decoy receptors known as secreted frizzled related proteins (SFRPs, which have a cysteine-rich domain (CRD structurally similar to the extracellular Wnt-binding domain of the frizzled receptors. SFRPs inhibit Wnt signaling by sequestering Wnts through the CRD or by forming inactive complexes with the frizzled receptors. Other endogenous molecules carrying frizzled CRDs inhibit Wnt signaling, such as V3Nter, which is proteolytically derived from the cell surface component collagen XVIII and contains a biologically active frizzled domain (FZC18 inhibiting in vivo cell proliferation and tumor growth in mice. We recently showed that FZC18 expressing cells deliver short-range signals to neighboring cells, decreasing their proliferation in vitro and in vivo through the Wnt/β-catenin signaling pathway. Here, using low concentrations of soluble FZC18 and Wnt3a, we show that they physically interact in a cell-free system. In addition, soluble FZC18 binds the frizzled 1 and 8 receptors' CRDs, reducing cell sensitivity to Wnt3a. Conversely, inhibition of Wnt/β-catenin signaling was partially rescued by the expression of full-length frizzled 1 and 8 receptors, but enhanced by the expression of a chimeric cell-membrane-tethered frizzled 8 CRD. Moreover, soluble, partially purified recombinant FZC18_CRD inhibited Wnt3a-induced β-catenin activation. Taken together, the data indicate that collagen XVIII-derived frizzled CRD shifts Wnt sensitivity of normal cells to a lower pitch and controls their growth.

  8. TARM1 Is a Novel Leukocyte Receptor Complex-Encoded ITAM Receptor That Costimulates Proinflammatory Cytokine Secretion by Macrophages and Neutrophils

    DEFF Research Database (Denmark)

    Radjabova, Valeria; Mastroeni, Piero; Skjødt, Karsten

    2015-01-01

    We identified a novel, evolutionarily conserved receptor encoded within the human leukocyte receptor complex and syntenic region of mouse chromosome 7, named T cell-interacting, activating receptor on myeloid cells-1 (TARM1). The transmembrane region of TARM1 contained a conserved arginine residu...

  9. A complex pattern of chemokine receptor expression is seen in osteosarcoma

    International Nuclear Information System (INIS)

    Luettichau, Irene von; Huss, Ralf; Nelson, Peter J; Segerer, Stephan; Wechselberger, Alexandra; Notohamiprodjo, Mike; Nathrath, Michaela; Kremer, Markus; Henger, Anna; Djafarzadeh, Roghieh; Burdach, Stefan

    2008-01-01

    Osteosarcoma is the most frequent bone tumor in childhood and adolescence. Patients with primary metastatic disease have a poor prognosis. It is therefore important to better characterize the biology of this tumor to define new prognostic markers or therapeutic targets for tailored therapy. Chemokines and their receptors have been shown to be involved in the development and progression of malignant tumors. They are thought to be active participants in the biology of osteosarcoma. The function of specific chemokines and their receptors is strongly associated with the biological context and microenvironment of their expression. In this report we characterized the expression of a series of chemokine receptors in the complex environment that defines osteosarcoma. The overall level of chemokine receptor mRNA expression was determined using TaqMan RT-PCR of microdissected archival patient biopsy samples. Expression was then verified at the protein level by immunohistochemistry using a series of receptor specific antibody reagents to elucidate the cellular association of expression. Expression at the RNA level was found for most of the tested receptors. CCR1 expression was found on infiltrating mononuclear and polynuclear giant cells in the tumor. Cells associated with the lining of intratumoral vessels were shown to express CCR4. Infiltrating mononuclear cells and tumor cells both showed expression of the receptor CCR5, while CCR7 was predominantly expressed by the mononuclear infiltrate. CCR10 was only very rarely detected in few scattered infiltrating cells. Our data elucidate for the first time the cellular context of chemokine receptor expression in osteosarcoma. This is an important issue for better understanding potential chemokine/chemokine receptor function in the complex biologic processes that underlie the development and progression of osteosarcoma. Our data support the suggested involvement of chemokines and their receptors in diverse aspects of the biology

  10. Combinatory annotation of cell membrane receptors and signalling pathways of Bombyx mori prothoracic glands

    Science.gov (United States)

    Moulos, Panagiotis; Samiotaki, Martina; Panayotou, George; Dedos, Skarlatos G.

    2016-01-01

    The cells of prothoracic glands (PG) are the main site of synthesis and secretion of ecdysteroids, the biochemical products of cholesterol conversion to steroids that shape the morphogenic development of insects. Despite the availability of genome sequences from several insect species and the extensive knowledge of certain signalling pathways that underpin ecdysteroidogenesis, the spectrum of signalling molecules and ecdysteroidogenic cascades is still not fully comprehensive. To fill this gap and obtain the complete list of cell membrane receptors expressed in PG cells, we used combinatory bioinformatic, proteomic and transcriptomic analysis and quantitative PCR to annotate and determine the expression profiles of genes identified as putative cell membrane receptors of the model insect species, Bombyx mori, and subsequently enrich the repertoire of signalling pathways that are present in its PG cells. The genome annotation dataset we report here highlights modules and pathways that may be directly involved in ecdysteroidogenesis and aims to disseminate data and assist other researchers in the discovery of the role of such receptors and their ligands. PMID:27576083

  11. Elabela-apelin receptor signaling pathway is functional in mammalian systems.

    Science.gov (United States)

    Wang, Zhi; Yu, Daozhan; Wang, Mengqiao; Wang, Qilong; Kouznetsova, Jennifer; Yang, Rongze; Qian, Kun; Wu, Wenjun; Shuldiner, Alan; Sztalryd, Carole; Zou, Minghui; Zheng, Wei; Gong, Da-Wei

    2015-02-02

    Elabela (ELA) or Toddler is a recently discovered hormone which is required for normal development of heart and vasculature through activation of apelin receptor (APJ), a G protein-coupled receptor (GPCR), in zebrafish. The present study explores whether the ELA-APJ signaling pathway is functional in the mammalian system. Using reverse-transcription PCR, we found that ELA is restrictedly expressed in human pluripotent stem cells and adult kidney whereas APJ is more widely expressed. We next studied ELA-APJ signaling pathway in reconstituted mammalian cell systems. Addition of ELA to HEK293 cells over-expressing GFP-AJP fusion protein resulted in rapid internalization of the fusion receptor. In Chinese hamster ovarian (CHO) cells over-expressing human APJ, ELA suppresses cAMP production with EC50 of 11.1 nM, stimulates ERK1/2 phosphorylation with EC50 of 14.3 nM and weakly induces intracellular calcium mobilization. Finally, we tested ELA biological function in human umbilical vascular endothelial cells and showed that ELA induces angiogenesis and relaxes mouse aortic blood vessel in a dose-dependent manner through a mechanism different from apelin. Collectively, we demonstrate that the ELA-AJP signaling pathways are functional in mammalian systems, indicating that ELA likely serves as a hormone regulating the circulation system in adulthood as well as in embryonic development.

  12. Preprotein import into chloroplasts via the Toc and Tic complexes is regulated by redox signals in Pisum sativum.

    Science.gov (United States)

    Stengel, Anna; Benz, J Philipp; Buchanan, Bob B; Soll, Jürgen; Bölter, Bettina

    2009-11-01

    The import of nuclear-encoded preproteins is necessary to maintain chloroplast function. The recognition and transfer of most precursor proteins across the chloroplast envelopes are facilitated by two membrane-inserted protein complexes, the translocons of the chloroplast outer and inner envelope (Toc and Tic complexes, respectively). Several signals have been invoked to regulate the import of preproteins. In our study, we were interested in redox-based import regulation mediated by two signals: regulation based on thiols and on the metabolic NADP+/NADPH ratio. We sought to identify the proteins participating in the regulation of these transport pathways and to characterize the preprotein subgroups whose import is redox-dependent. Our results provide evidence that the formation and reduction of disulfide bridges in the Toc receptors and Toc translocation channel have a strong influence on import yield of all tested preproteins that depend on the Toc complex for translocation. Furthermore, the metabolic NADP+/NADPH ratio influences not only the composition of the Tic complex, but also the import efficiency of most, but not all, preproteins tested. Thus, several Tic subcomplexes appear to participate in the translocation of different preprotein subgroups, and the redox-active components of these complexes likely play a role in regulating transport.

  13. Structure and Dynamics of the Liver Receptor Homolog 1–PGC1 α Complex

    Energy Technology Data Exchange (ETDEWEB)

    Mays, Suzanne G.; Okafor, C. Denise; Tuntland, Micheal L.; Whitby, Richard J.; Dharmarajan, Venkatasubramanian; Stec, Józef; Griffin, Patrick R.; Ortlund, Eric A.

    2017-03-31

    Peroxisome proliferator-activated gamma coactivator 1-α (PGC1α) regulates energy metabolism by directly interacting with transcription factors to modulate gene expression. Among the PGC1α binding partners is liver receptor homolog 1 (LRH-1; NR5A2), an orphan nuclear hormone receptor that controls lipid and glucose homeostasis. Although PGC1α is known to bind and activate LRH-1, mechanisms through which PGC1α changes LRH-1 conformation to drive transcription are unknown. Here, we used biochemical and structural methods to interrogate the LRH-1–PGC1α complex. Purified, full-length LRH-1, as well as isolated ligand binding domain, bound to PGC1α with higher affinity than to the coactivator, nuclear receptor coactivator-2 (Tif2), in coregulator peptide recruitment assays. We present the first crystal structure of the LRH-1–PGC1α complex, which depicts several hydrophobic contacts and a strong charge clamp at the interface between these partners. In molecular dynamics simulations, PGC1α induced correlated atomic motion throughout the entire LRH-1 activation function surface, which was dependent on charge-clamp formation. In contrast, Tif2 induced weaker signaling at the activation function surface than PGC1α but promoted allosteric signaling from the helix 6/β-sheet region of LRH-1 to the activation function surface. These studies are the first to probe mechanisms underlying the LRH-1–PGC1α interaction and may illuminate strategies for selective therapeutic targeting of PGC1α-dependent LRH-1 signaling pathways.

  14. Reverting doxorubicin resistance in colon cancer by targeting a key signaling protein, steroid receptor coactivator.

    Science.gov (United States)

    Xiong, Sang; Xiao, Gong-Wei

    2018-04-01

    Although there have been notable improvements in treatments against cancer, further research is required. In colon cancer, nearly all patients eventually experience drug resistance and stop responding to the approved drugs, making treatment difficult. Steroid receptor coactivator (SRC) is an oncogenic nuclear receptor coactivator that serves an important role in drug resistance. The present study generated a doxorubicin-resistant colon cancer cell line, in which the upregulation/activation of SRC was responsible for drug resistance, which in turn activated AKT. Overexpression of receptor tyrosine kinase-like epidermal growth factor receptor and insulin-like growth factor 1 receptor also induced SRC expression. It was observed that doxorubicin resistance in colon cancer also induced epithelial to mesenchymal transition, a decrease in expression of epithelial marker E-cadherin and an increase in the expression of mesenchymal markers, including N-cadherin and vimentin. Additionally, the present study indicated that SRC acts as a common signaling node, and inhibiting SRC in combination with doxorubicin treatment in doxorubicin-resistant cells aids in reversing the resistance. Thus, the present study suggests that activation of SRC is responsible for doxorubicin resistance in colon cancer. However, further research is required to understand the complete mechanism of how drug resistance occurs and how it may be tackled to treat patients.

  15. Purification of the active C5a receptor from human polymorphonuclear leukocytes as a receptor - G sub i complex

    Energy Technology Data Exchange (ETDEWEB)

    Rollins, T.E.; Siciliano, S.; Kobayashi, S.; Cianciarulo, D.N.; Bonilla-Argudo, V.; Collier, K.; Springer, M.S. (Merck Sharp and Dohme Research Lab., Rahway, NJ (United States))

    1991-02-01

    The authors have isolated, in an active state, the C5a receptor from human polymorphonuclear leukocytes. The purification was achieved in a single step using a C5a affinity column in which the C5a molecule was coupled to the resin through its N terminus. The purified receptor, like the crude solubilized molecule, exhibited a single class of high-affinity binding sites with a K{sub d} of 30 pM. Further, the binding of C5a retained its sensitivity to guanine nucleotides, implying that the purified receptor contained a guanine nucleotide-binding protein (G protein). SDS/PAGE revealed the presence of three polypeptides with molecular masses of 42, 40, and 36 kDa, which were determined to be the C5a-binding subunit and the {alpha} and {beta} subunits of G{sub i}, respectively. The 36- and 40-kDa polypeptides were identified by immunoblotting and by the ability of pertussis toxin to ADP-ribosylate the 40-kDa molecule. These results confirm their earlier hypothesis that the receptor exists as a complex with a G protein in the presence or absence of C5a. The tight coupling between the receptor and G protein should make possible the identification of the G protein(s) involved in the transduction pathways used by C5a to produce its many biological effects.

  16. Purification of the active C5a receptor from human polymorphonuclear leukocytes as a receptor - Gi complex

    International Nuclear Information System (INIS)

    Rollins, T.E.; Siciliano, S.; Kobayashi, S.; Cianciarulo, D.N.; Bonilla-Argudo, V.; Collier, K.; Springer, M.S.

    1991-01-01

    The authors have isolated, in an active state, the C5a receptor from human polymorphonuclear leukocytes. The purification was achieved in a single step using a C5a affinity column in which the C5a molecule was coupled to the resin through its N terminus. The purified receptor, like the crude solubilized molecule, exhibited a single class of high-affinity binding sites with a K d of 30 pM. Further, the binding of C5a retained its sensitivity to guanine nucleotides, implying that the purified receptor contained a guanine nucleotide-binding protein (G protein). SDS/PAGE revealed the presence of three polypeptides with molecular masses of 42, 40, and 36 kDa, which were determined to be the C5a-binding subunit and the α and β subunits of G i , respectively. The 36- and 40-kDa polypeptides were identified by immunoblotting and by the ability of pertussis toxin to ADP-ribosylate the 40-kDa molecule. These results confirm their earlier hypothesis that the receptor exists as a complex with a G protein in the presence or absence of C5a. The tight coupling between the receptor and G protein should make possible the identification of the G protein(s) involved in the transduction pathways used by C5a to produce its many biological effects

  17. Characterization of Notch1 antibodies that inhibit signaling of both normal and mutated Notch1 receptors.

    Directory of Open Access Journals (Sweden)

    Miguel Aste-Amézaga

    2010-02-01

    Full Text Available Notch receptors normally play a key role in guiding a variety of cell fate decisions during development and differentiation of metazoan organisms. On the other hand, dysregulation of Notch1 signaling is associated with many different types of cancer as well as tumor angiogenesis, making Notch1 a potential therapeutic target.Here we report the in vitro activities of inhibitory Notch1 monoclonal antibodies derived from cell-based and solid-phase screening of a phage display library. Two classes of antibodies were found, one directed against the EGF-repeat region that encompasses the ligand-binding domain (LBD, and the second directed against the activation switch of the receptor, the Notch negative regulatory region (NRR. The antibodies are selective for Notch1, inhibiting Jag2-dependent signaling by Notch1 but not by Notch 2 and 3 in reporter gene assays, with EC(50 values as low as 5+/-3 nM and 0.13+/-0.09 nM for the LBD and NRR antibodies, respectively, and fail to recognize Notch4. While more potent, NRR antibodies are incomplete antagonists of Notch1 signaling. The antagonistic activity of LBD, but not NRR, antibodies is strongly dependent on the activating ligand. Both LBD and NRR antibodies bind to Notch1 on human tumor cell lines and inhibit the expression of sentinel Notch target genes, including HES1, HES5, and DTX1. NRR antibodies also strongly inhibit ligand-independent signaling in heterologous cells transiently expressing Notch1 receptors with diverse NRR "class I" point mutations, the most common type of mutation found in human T-cell acute lymphoblastic leukemia (T-ALL. In contrast, NRR antibodies failed to antagonize Notch1 receptors bearing rare "class II" or "class III" mutations, in which amino acid insertions generate a duplicated or constitutively sensitive metalloprotease cleavage site. Signaling in T-ALL cell lines bearing class I mutations is partially refractory to inhibitory antibodies as compared to cell

  18. Sphingosine Kinases and Sphingosine 1-Phosphate Receptors: Signaling and Actions in the Cardiovascular System

    Directory of Open Access Journals (Sweden)

    Alessandro Cannavo

    2017-08-01

    Full Text Available The sphingosine kinases 1 and 2 (SphK1 and 2 catalyze the phosphorylation of the lipid, sphingosine, generating the signal transmitter, sphingosine 1-phosphate (S1P. The activation of such kinases and the subsequent S1P generation and secretion in the blood serum of mammals represent a major checkpoint in many cellular signaling cascades. In fact, activating the SphK/S1P system is critical for cell motility and proliferation, cytoskeletal organization, cell growth, survival, and response to stress. In the cardiovascular system, the physiological effects of S1P intervene through the binding and activation of a family of five highly selective G protein-coupled receptors, called S1PR1-5. Importantly, SphK/S1P signal is present on both vascular and myocardial cells. S1P is a well-recognized survival factor in many tissues. Therefore, it is not surprising that the last two decades have seen a flourishing of interest and investigative efforts directed to obtain additional mechanistic insights into the signaling, as well as the biological activity of this phospholipid, and of its receptors, especially in the cardiovascular system. Here, we will provide an up-to-date account on the structure and function of sphingosine kinases, discussing the generation, release, and function of S1P. Keeping the bull’s eye on the cardiovascular system, we will review the structure and signaling cascades and biological actions emanating from the stimulation of different S1P receptors. We will end this article with a summary of the most recent, experimental and clinical observations targeting S1PRs and SphKs as possible new therapeutic avenues for cardiovascular disorders, such as heart failure.

  19. The effect of CD4 receptor downregulation and its downstream signaling molecules on HIV-1 latency

    International Nuclear Information System (INIS)

    Kim, Kyung-Chang; Kim, Hyeon Guk; Roh, Tae-Young; Park, Jihwan; Jung, Kyung-Min; Lee, Joo-Shil; Choi, Sang-Yun; Kim, Sung Soon; Choi, Byeong-Sun

    2011-01-01

    Research highlights: → CD4 receptors were downregulated on the surface of HIV-1 latently infected cells. → CD4 downstream signaling molecules were suppressed in HIV-1 latently infected cells. → HIV-1 progeny can be reactivated by induction of T-cell activation signal molecules. → H3K4me3 and H3K9ac were highly enriched in CD4 downstream signaling molecules. → HIV-1 latency can be maintained by the reduction of downstream signaling molecules. -- Abstract: HIV-1 can establish a latent infection in memory CD4 + T cells to evade the host immune response. CD4 molecules can act not only as the HIV-1 receptor for entry but also as the trigger in an intracellular signaling cascade for T-cell activation and proliferation via protein tyrosine kinases. Novel chronic HIV-1-infected A3.01-derived (NCHA) cells were used to examine the involvement of CD4 downstream signaling in HIV-1 latency. CD4 receptors in NCHA cells were dramatically downregulated on its surface but were slightly decreased in whole-cell lysates. The expression levels of CD4 downstream signaling molecules, including P56 Lck , ZAP-70, LAT, and c-Jun, were sharply decreased in NCHA cells. The lowered histone modifications of H3K4me3 and H3K9ac correlated with the downregulation of P56 Lck , ZAP-70, and LAT in NCHA cells. AP-1 binding activity was also reduced in NCHA cells. LAT and c-Jun suppressed in NCHA cells were highly induced after PMA treatment. In epigenetic analysis, other signal transduction molecules which are associated with active and/or latent HIV-1 infection showed normal states in HIV-1 latently infected cells compared to A3.01 cells. In conclusion, we demonstrated that the HIV-1 latent state is sustained by the reduction of downstream signaling molecules via the downregulation of CD4 and the attenuated activity of transcription factor as AP-1. The HIV-1 latency model via T-cell deactivation may provide some clues for the development of the new antireservoir therapy.

  20. The effect of CD4 receptor downregulation and its downstream signaling molecules on HIV-1 latency

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Kyung-Chang [National Institute of Health, Chungbuk (Korea, Republic of); School of Life Science and Biotechnology, Korea University, Seoul (Korea, Republic of); Kim, Hyeon Guk [National Institute of Health, Chungbuk (Korea, Republic of); Roh, Tae-Young [Division of Molecular and Life Science, Pohang University of Science and Technology, Pohang, Gyeongbuk (Korea, Republic of); Division of Integrative Biosciences and Biotechnology, Pohang University of Science and Technology, Pohang, Gyeongbuk (Korea, Republic of); Park, Jihwan [Division of Molecular and Life Science, Pohang University of Science and Technology, Pohang, Gyeongbuk (Korea, Republic of); Jung, Kyung-Min; Lee, Joo-Shil [National Institute of Health, Chungbuk (Korea, Republic of); Choi, Sang-Yun [School of Life Science and Biotechnology, Korea University, Seoul (Korea, Republic of); Kim, Sung Soon [National Institute of Health, Chungbuk (Korea, Republic of); Choi, Byeong-Sun, E-mail: byeongsun@korea.kr [National Institute of Health, Chungbuk (Korea, Republic of)

    2011-01-14

    Research highlights: {yields} CD4 receptors were downregulated on the surface of HIV-1 latently infected cells. {yields} CD4 downstream signaling molecules were suppressed in HIV-1 latently infected cells. {yields} HIV-1 progeny can be reactivated by induction of T-cell activation signal molecules. {yields} H3K4me3 and H3K9ac were highly enriched in CD4 downstream signaling molecules. {yields} HIV-1 latency can be maintained by the reduction of downstream signaling molecules. -- Abstract: HIV-1 can establish a latent infection in memory CD4 + T cells to evade the host immune response. CD4 molecules can act not only as the HIV-1 receptor for entry but also as the trigger in an intracellular signaling cascade for T-cell activation and proliferation via protein tyrosine kinases. Novel chronic HIV-1-infected A3.01-derived (NCHA) cells were used to examine the involvement of CD4 downstream signaling in HIV-1 latency. CD4 receptors in NCHA cells were dramatically downregulated on its surface but were slightly decreased in whole-cell lysates. The expression levels of CD4 downstream signaling molecules, including P56{sup Lck}, ZAP-70, LAT, and c-Jun, were sharply decreased in NCHA cells. The lowered histone modifications of H3K4me3 and H3K9ac correlated with the downregulation of P56{sup Lck}, ZAP-70, and LAT in NCHA cells. AP-1 binding activity was also reduced in NCHA cells. LAT and c-Jun suppressed in NCHA cells were highly induced after PMA treatment. In epigenetic analysis, other signal transduction molecules which are associated with active and/or latent HIV-1 infection showed normal states in HIV-1 latently infected cells compared to A3.01 cells. In conclusion, we demonstrated that the HIV-1 latent state is sustained by the reduction of downstream signaling molecules via the downregulation of CD4 and the attenuated activity of transcription factor as AP-1. The HIV-1 latency model via T-cell deactivation may provide some clues for the development of the new

  1. Volume transmission and receptor-receptor interactions in heteroreceptor complexes: understanding the role of new concepts for brain communication

    Directory of Open Access Journals (Sweden)

    Kjell Fuxe

    2016-01-01

    Full Text Available The discovery of the central monoamine neurons not only demonstrated novel types of brain stem neurons forming global terminal networks all over the brain and the spinal cord, but also to a novel type of communication called volume transmission. It is a major mode of communication in the central nervous system that takes places in the extracellular fluid and the cerebral spinal fluid through diffusion and flow of molecules, like neurotransmitters and extracellular vesicles. The integration of synaptic and volume transmission takes place through allosteric receptor-receptor interactions in heteroreceptor complexes. These heterocomplexes represent major integrator centres in the plasma membrane and their protomers act as moonlighting proteins undergoing dynamic changes and their structure and function. In fact, we propose that the molecular bases of learning and memory can be based on the reorganization of multiples homo and heteroreceptor complexes into novel assembles in the post-junctional membranes of synapses.

  2. Canonical TGF-β Signaling Negatively Regulates Neuronal Morphogenesis through TGIF/Smad Complex-Mediated CRMP2 Suppression.

    Science.gov (United States)

    Nakashima, Hideyuki; Tsujimura, Keita; Irie, Koichiro; Ishizu, Masataka; Pan, Miao; Kameda, Tomonori; Nakashima, Kinichi

    2018-05-16

    Functional neuronal connectivity requires proper neuronal morphogenesis and its dysregulation causes neurodevelopmental diseases. Transforming growth factor-β (TGF-β) family cytokines play pivotal roles in development, but little is known about their contribution to morphological development of neurons. Here we show that the Smad-dependent canonical signaling of TGF-β family cytokines negatively regulates neuronal morphogenesis during brain development. Mechanistically, activated Smads form a complex with transcriptional repressor TG-interacting factor (TGIF), and downregulate the expression of a neuronal polarity regulator, collapsin response mediator protein 2. We also demonstrate that TGF-β family signaling inhibits neurite elongation of human induced pluripotent stem cell-derived neurons. Furthermore, the expression of TGF-β receptor 1, Smad4, or TGIF, which have mutations found in patients with neurodevelopmental disorders, disrupted neuronal morphogenesis in both mouse (male and female) and human (female) neurons. Together, these findings suggest that the regulation of neuronal morphogenesis by an evolutionarily conserved function of TGF-β signaling is involved in the pathogenesis of neurodevelopmental diseases. SIGNIFICANCE STATEMENT Canonical transforming growth factor-β (TGF-β) signaling plays a crucial role in multiple organ development, including brain, and mutations in components of the signaling pathway associated with several human developmental disorders. In this study, we found that Smads/TG-interacting factor-dependent canonical TGF-β signaling regulates neuronal morphogenesis through the suppression of collapsin response mediator protein-2 (CRMP2) expression during brain development, and that function of this signaling is evolutionarily conserved in the mammalian brain. Mutations in canonical TGF-β signaling factors identified in patients with neurodevelopmental disorders disrupt the morphological development of neurons. Thus, our

  3. Selective ligand activity at Nur/retinoid X receptor complexes revealed by dimer-specific bioluminescence resonance energy transfer-based sensors

    Science.gov (United States)

    Giner, Xavier C; Cotnoir-White, David; Mader, Sylvie; Lévesque, Daniel

    2017-01-01

    Retinoid X receptors (RXR) play a role as master regulators due to their capacity to form heterodimers with other nuclear receptors. Accordingly, retinoid signaling is involved in multiple biological processes, including development, cell differentiation, metabolism and cell death. However, the role and functions of RXR in different heterodimer complexes remain unsolved, mainly because most RXR drugs (called rexinoids) are not selective to specific heterodimer complexes. This also strongly limits the use of rexinoids for specific therapeutic approaches. In order to better characterize rexinoids at specific nuclear receptor complexes, we have developed and optimized luciferase protein complementation-based Bioluminescence Resonance Energy Transfer (BRET) assays, which can directly measure recruitment of a co-activator motif fused to yellow fluorescent protein (YFP) by specific nuclear receptor dimers. To validate the assays, we compared rexinoid modulation of co-activator recruitment by RXR homodimer, and heterodimers Nur77/RXR and Nurr1/RXR. Results reveal that some rexinoids display selective co-activator recruitment activities with homo- or hetero-dimer complexes. In particular, SR11237 (BMS649) has increased potency for recruitment of co-activator motif and transcriptional activity with the Nur77/RXR heterodimer compared to other complexes. This technology should prove useful to identify new compounds with specificity for individual dimeric species formed by nuclear receptors. PMID:26148973

  4. Distinct signalling properties of insulin receptor substrate (IRS)-1 and IRS-2 in mediating insulin/IGF-1 action.

    Science.gov (United States)

    Rabiee, Atefeh; Krüger, Marcus; Ardenkjær-Larsen, Jacob; Kahn, C Ronald; Emanuelli, Brice

    2018-07-01

    Insulin/IGF-1 action is driven by a complex and highly integrated signalling network. Loss-of-function studies indicate that the major insulin/IGF-1 receptor substrate (IRS) proteins, IRS-1 and IRS-2, mediate different biological functions in vitro and in vivo, suggesting specific signalling properties despite their high degree of homology. To identify mechanisms contributing to the differential signalling properties of IRS-1 and IRS-2 in the mediation of insulin/IGF-1 action, we performed comprehensive mass spectrometry (MS)-based phosphoproteomic profiling of brown preadipocytes from wild type, IRS-1 -/- and IRS-2 -/- mice in the basal and IGF-1-stimulated states. We applied stable isotope labeling by amino acids in cell culture (SILAC) for the accurate quantitation of changes in protein phosphorylation. We found ~10% of the 6262 unique phosphorylation sites detected to be regulated by IGF-1. These regulated sites included previously reported substrates of the insulin/IGF-1 signalling pathway, as well as novel substrates including Nuclear Factor I X and Semaphorin-4B. In silico prediction suggests the protein kinase B (PKB), protein kinase C (PKC), and cyclin-dependent kinase (CDK) as the main mediators of these phosphorylation events. Importantly, we found preferential phosphorylation patterns depending on the presence of either IRS-1 or IRS-2, which was associated with specific sets of kinases involved in signal transduction downstream of these substrates such as PDHK1, MAPK3, and PKD1 for IRS-1, and PIN1 and PKC beta for IRS-2. Overall, by generating a comprehensive phosphoproteomic profile from brown preadipocyte cells in response to IGF-1 stimulation, we reveal both common and distinct insulin/IGF-1 signalling events mediated by specific IRS proteins. Copyright © 2018 Elsevier Inc. All rights reserved.

  5. Comparative proteomic analysis to dissect differences in signal transduction in activating TSH receptor mutations in the thyroid.

    Science.gov (United States)

    Krause, Kerstin; Boisnard, Alexandra; Ihling, Christian; Ludgate, Marian; Eszlinger, Markus; Krohn, Knut; Sinz, Andrea; Fuhrer, Dagmar

    2012-02-01

    In the thyroid, cAMP controls both thyroid growth and function. Gain-of-function mutations in the thyroid-stimulating hormone receptor (TSHR) lead to constitutive cAMP formation and are a major cause of autonomous thyroid adenomas. The impact of activating TSHR mutations on the signal transduction network of the thyrocyte is not fully understood. To gain more insights into constitutive TSHR signaling, rat thyrocytes (FRTL-5 cells) with stable expression of three activating TSHR mutants (mutTSHR: A623I, L629F and Del613-621), which differ in their functional characteristics in vitro, were analyzed by a quantitative proteomic approach and compared to the wild-type TSHR (WT-TSHR). This study revealed (1) differences in the expression of Rab proteins suggesting an increased TSHR internalization in mutTSHR but not in the WT-TSHR; (2) differential stimulation of PI3K/Akt signaling in mutTSHR vs. WT-TSHR cells, (3) activation of Epac, impairing short-time Akt phosphorylation in both, mutTSHR and WT-TSHR cells. Based on the analysis of global changes in protein expression patterns, our findings underline the complexity of gain-of-function TSHR signaling in thyrocytes, which extends beyond pure cAMP and/or IP formation. Moreover, evidence for augmented endocytosis in the mutTSHR, adds to a new concept of TSHR signaling in thyroid autonomy. Further studies are required to clarify whether the observed differences in Rab, PI3K and Epac signaling may contribute to differences in the phenotypic presentation, i.e. stimulation of function and growth of thyroid autonomy in vivo. Copyright © 2011 Elsevier Ltd. All rights reserved.

  6. Dithiothreitol activation of the insulin receptor/kinase does not involve subunit dissociation of the native α2β2 insulin receptor subunit complex

    International Nuclear Information System (INIS)

    Sweet, L.J.; Wilden, P.A.; Pessin, J.E.

    1986-01-01

    The subunit composition of the dithiothreitol- (DTT) activated insulin receptor/kinase was examined by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis and gel filtration chromatography under denaturing or nondenaturing conditions. Pretreatment of 32 P-labeled insulin receptors with 50 mM DTT followed by gel filtration chromatography in 0.1% SDS demonstrated the dissociation of the α 2 β 2 insulin receptor complex (M/sub r/ 400,000) into the monomeric 95,000 β subunit. In contrast, pretreatment of the insulin receptors with 1-50 mM DTT followed by gel filtration chromatography in 0.1% Triton X-100 resulted in no apparent alteration in mobility compared to the untreated insulin receptors. Resolution of this complex by nonreducing SDS-polyacrylamide gel electrophoresis and autoradiography demonstrated the existence of the α 2 β 2 heterotetrameric complex with essentially no αβ heterodimeric or free monomeric β subunit species present. This suggests that the insulin receptor can reoxidize into the M/sub r/ 400,000 complex after the removal of DTT by gel filtration chromatography. To prevent reoxidation, the insulin receptors were pretreated with 50 mM DTT. Under the conditions the insulin receptors migrated as the M/sub r/ 400,000 α 2 β 2 complex. These results demonstrate that treatment of the insulin receptors with high concentrations of DTT, followed by removal of DTT by gel filtration, results in reoxidation of the reduced α 2 β 2 insulin receptor complex. Further, these results document that although the DTT stimulation of the insulin receptor/kinase does involve reduction of the insulin receptor subunits, it does not result in dissociation of the native α 2 β 2 insulin receptor subunit complex

  7. Signaling cross-talk between peroxisome proliferator-activated receptor/retinoid X receptor and estrogen receptor through estrogen response elements.

    Science.gov (United States)

    Keller, H; Givel, F; Perroud, M; Wahli, W

    1995-07-01

    Peroxisome proliferator-activated receptors (PPARs) and retinoid X receptors (RXRs) are nuclear hormone receptors that are activated by fatty acids and 9-cis-retinoic acid, respectively. PPARs and RXRs form heterodimers that activate transcription by binding to PPAR response elements (PPREs) in the promoter of target genes. The PPREs described thus far consist of a direct tandem repeat of the AGGTCA core element with one intervening nucleotide. We show here that the vitellogenin A2 estrogen response element (ERE) can also function as a PPRE and is bound by a PPAR/RXR heterodimer. Although this heterodimer can bind to several other ERE-related palindromic response elements containing AGGTCA half-sites, only the ERE is able to confer transactivation of test reporter plasmids, when the ERE is placed either close to or at a distance from the transcription initiation site. Examination of natural ERE-containing promoters, including the pS2, very-low-density apolipoprotein II and vitellogenin A2 genes, revealed considerable differences in the binding of PPAR/RXR heterodimers to these EREs. In their natural promoter context, these EREs did not allow transcriptional activation by PPARs/RXRs. Analysis of this lack of stimulation of the vitellogenin A2 promoter demonstrated that PPARs/RXRs bind to the ERE but cannot transactivate due to a nonpermissive promoter structure. As a consequence, PPARs/RXRs inhibit transactivation by the estrogen receptor through competition for ERE binding. This is the first example of signaling cross-talk between PPAR/RXR and estrogen receptor.

  8. Kidins220/ARMS as a functional mediator of multiple receptor signalling pathways.

    Science.gov (United States)

    Neubrand, Veronika E; Cesca, Fabrizia; Benfenati, Fabio; Schiavo, Giampietro

    2012-04-15

    An increasing body of evidence suggests that several membrane receptors--in addition to activating distinct signalling cascades--also engage in substantial crosstalk with each other, thereby adjusting their signalling outcome as a function of specific input information. However, little is known about the molecular mechanisms that control their coordination and integration of downstream signalling. A protein that is likely to have a role in this process is kinase-D-interacting substrate of 220 kDa [Kidins220, also known as ankyrin repeat-rich membrane spanning (ARMS), hereafter referred to as Kidins220/ARMS]. Kidins220/ARMS is a conserved membrane protein that is preferentially expressed in the nervous system and interacts with the microtubule and actin cytoskeleton. It interacts with neurotrophin, ephrin, vascular endothelial growth factor (VEGF) and glutamate receptors, and is a common downstream target of several trophic stimuli. Kidins220/ARMS is required for neuronal differentiation and survival, and its expression levels modulate synaptic plasticity. Kidins220/ARMS knockout mice show developmental defects mainly in the nervous and cardiovascular systems, suggesting a crucial role for this protein in modulating the cross talk between different signalling pathways. In this Commentary, we summarise existing knowledge regarding the physiological functions of Kidins220/ARMS, and highlight some interesting directions for future studies on the role of this protein in health and disease.

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

    Science.gov (United States)

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

    2010-04-01

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

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

    Directory of Open Access Journals (Sweden)

    Kelly J Culhane

    2015-11-01

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

  11. Bile salt receptor complex activates a pathogenic type III secretion system

    Science.gov (United States)

    Li, Peng; Rivera-Cancel, Giomar; Kinch, Lisa N; Salomon, Dor; Tomchick, Diana R; Grishin, Nick V; Orth, Kim

    2016-01-01

    Bile is an important component of the human gastrointestinal tract with an essential role in food absorption and antimicrobial activities. Enteric bacterial pathogens have developed strategies to sense bile as an environmental cue to regulate virulence genes during infection. We discovered that Vibrio parahaemolyticus VtrC, along with VtrA and VtrB, are required for activating the virulence type III secretion system 2 in response to bile salts. The VtrA/VtrC complex activates VtrB in the presence of bile salts. The crystal structure of the periplasmic domains of the VtrA/VtrC heterodimer reveals a β-barrel with a hydrophobic inner chamber. A co-crystal structure of VtrA/VtrC with bile salt, along with biophysical and mutational analysis, demonstrates that the hydrophobic chamber binds bile salts and activates the virulence network. As part of a family of conserved signaling receptors, VtrA/VtrC provides structural and functional insights into the evolutionarily conserved mechanism used by bacteria to sense their environment. DOI: http://dx.doi.org/10.7554/eLife.15718.001 PMID:27377244

  12. Bile salt receptor complex activates a pathogenic type III secretion system

    Energy Technology Data Exchange (ETDEWEB)

    Li, Peng; Rivera-Cancel, Giomar; Kinch, Lisa N.; Salomon, Dor; Tomchick, Diana R.; Grishin, Nick V.; Orth, Kim

    2016-07-05

    Bile is an important component of the human gastrointestinal tract with an essential role in food absorption and antimicrobial activities. Enteric bacterial pathogens have developed strategies to sense bile as an environmental cue to regulate virulence genes during infection. We discovered thatVibrio parahaemolyticusVtrC, along with VtrA and VtrB, are required for activating the virulence type III secretion system 2 in response to bile salts. The VtrA/VtrC complex activates VtrB in the presence of bile salts. The crystal structure of the periplasmic domains of the VtrA/VtrC heterodimer reveals a β-barrel with a hydrophobic inner chamber. A co-crystal structure of VtrA/VtrC with bile salt, along with biophysical and mutational analysis, demonstrates that the hydrophobic chamber binds bile salts and activates the virulence network. As part of a family of conserved signaling receptors, VtrA/VtrC provides structural and functional insights into the evolutionarily conserved mechanism used by bacteria to sense their environment.

  13. Mediator and p300/CBP-Steroid Receptor Coactivator Complexes Have Distinct Roles, but Function Synergistically, during Estrogen Receptor α-Dependent Transcription with Chromatin Templates

    OpenAIRE

    Acevedo, Mari Luz; Kraus, W. Lee

    2003-01-01

    Ligand-dependent transcriptional activation by nuclear receptors involves the recruitment of various coactivators to the promoters of hormone-regulated genes assembled into chromatin. Nuclear receptor coactivators include histone acetyltransferase complexes, such as p300/CBP-steroid receptor coactivator (SRC), as well as the multisubunit mediator complexes (“Mediator”), which may help recruit RNA polymerase II to the promoter. We have used a biochemical approach, including an in vitro chromat...

  14. Mu and delta opioid receptors oppositely regulate motor impulsivity in the signaled nose poke task.

    Directory of Open Access Journals (Sweden)

    Mary C Olmstead

    Full Text Available Impulsivity is a primary feature of many psychiatric disorders, most notably attention deficit hyperactivity disorder and drug addiction. Impulsivity includes a number of processes such as the inability to delay gratification, the inability to withhold a motor response, or acting before all of the relevant information is available. These processes are mediated by neural systems that include dopamine, serotonin, norepinephrine, glutamate and cannabinoids. We examine, for the first time, the role of opioid systems in impulsivity by testing whether inactivation of the mu- (Oprm1 or delta- (Oprd1 opioid receptor gene alters motor impulsivity in mice. Wild-type and knockout mice were examined on either a pure C57BL6/J (BL6 or a hybrid 50% C57Bl/6J-50% 129Sv/pas (HYB background. Mice were trained to respond for sucrose in a signaled nose poke task that provides independent measures of associative learning (responses to the reward-paired cue and motor impulsivity (premature responses. Oprm1 knockout mice displayed a remarkable decrease in motor impulsivity. This was observed on the two genetic backgrounds and did not result from impaired associative learning, as responses to the cue signaling reward did not differ across genotypes. Furthermore, mutant mice were insensitive to the effects of ethanol, which increased disinhibition and decreased conditioned responding in wild-type mice. In sharp contrast, mice lacking the Oprd1 gene were more impulsive than controls. Again, mutant animals showed no deficit in associative learning. Ethanol completely disrupted performance in these animals. Together, our results suggest that mu-opioid receptors enhance, whereas delta-opioid receptors inhibit, motor impulsivity. This reveals an unanticipated contribution of endogenous opioid receptor activity to disinhibition. In a broader context, these data suggest that alterations in mu- or delta-opioid receptor function may contribute to impulse control disorders.

  15. Astrocyte reactivity to unconjugated bilirubin requires TNF-α and IL-1β receptor signaling pathways.

    Science.gov (United States)

    Fernandes, Adelaide; Barateiro, Andreia; Falcão, Ana Sofia; Silva, Sandra Leit-Ao; Vaz, Ana Rita; Brito, Maria Alexandra; Silva, Rui Fernando Marques; Brites, Dora

    2011-01-01

    Jaundice and sepsis are common neonatal conditions that can lead to neurodevelopment sequelae, namely if present at the same time. We have reported that tumor necrosis factor (TNF)-α and interleukin (IL)-1β are produced by cultured neurons and mainly by glial cells exposed to unconjugated bilirubin (UCB). The effects of these cytokines are mediated by cell surface receptors through a nuclear factor (NF)-κB-dependent pathway that we have showed to be activated by UCB. The present study was designed to evaluate the role of TNF-α and IL-1β signaling on astrocyte reactivity to UCB in rat cortical astrocytes. Exposure of astrocytes to UCB increased the expression of both TNF-α receptor (TNFR)1 and IL-1β receptor (IL-1R)1, but not TNFR2, as well as their activation, observed by augmented binding of receptors' molecular adaptors, TRAF2 and TRAF6, respectively. Silencing of TNFR1, using siRNA technology, or blockade of IL-1β cascade, using its endogenous antagonist, IL-1 receptor antagonist (IL-1ra), prevented UCB-induced cytokine release and NF-κB activation. Interestingly, lack of TNF-α signal transduction reduced UCB-induced cell death for short periods of incubation, although an increase was observed after extended exposure; in contrast, inhibition of IL-1β cascade produced a sustained blockade of astrocyte injury by UCB. Together, our data show that inflammatory pathways are activated during in vitro exposure of rat cortical astrocytes to UCB and that this activation is prolonged in time. This supports the concept that inflammatory pathways play a role in brain damage by UCB, and that they may represent important pharmacological targets. Copyright © 2010 Wiley-Liss, Inc.

  16. Distinct Signaling Cascades Elicited by Different Formyl Peptide Receptor 2 (FPR2 Agonists

    Directory of Open Access Journals (Sweden)

    Fabio Cattaneo

    2013-04-01

    Full Text Available The formyl peptide receptor 2 (FPR2 is a remarkably versatile transmembrane protein belonging to the G-protein coupled receptor (GPCR family. FPR2 is activated by an array of ligands, which include structurally unrelated lipids and peptide/proteins agonists, resulting in different intracellular responses in a ligand-specific fashion. In addition to the anti-inflammatory lipid, lipoxin A4, several other endogenous agonists also bind FPR2, including serum amyloid A, glucocorticoid-induced annexin 1, urokinase and its receptor, suggesting that the activation of FPR2 may result in potent pro- or anti-inflammatory responses. Other endogenous ligands, also present in biological samples, include resolvins, amyloidogenic proteins, such as beta amyloid (Aβ-42 and prion protein (Prp106–126, the neuroprotective peptide, humanin, antibacterial peptides, annexin 1-derived peptides, chemokine variants, the neuropeptides, vasoactive intestinal peptide (VIP and pituitary adenylate cyclase activating polypeptide (PACAP-27, and mitochondrial peptides. Upon activation, intracellular domains of FPR2 mediate signaling to G-proteins, which trigger several agonist-dependent signal transduction pathways, including activation of phospholipase C (PLC, protein kinase C (PKC isoforms, the phosphoinositide 3-kinase (PI3K/protein kinase B (Akt pathway, the mitogen-activated protein kinase (MAPK pathway, p38MAPK, as well as the phosphorylation of cytosolic tyrosine kinases, tyrosine kinase receptor transactivation, phosphorylation and nuclear translocation of regulatory transcriptional factors, release of calcium and production of oxidants. FPR2 is an attractive therapeutic target, because of its involvement in a range of normal physiological processes and pathological diseases. Here, we review and discuss the most significant findings on the intracellular pathways and on the cross-communication between FPR2 and tyrosine kinase receptors triggered by different FPR2

  17. Signaling by Kit protein-tyrosine kinase--the stem cell factor receptor.

    Science.gov (United States)

    Roskoski, Robert

    2005-11-11

    Signaling by stem cell factor and Kit, its receptor, plays important roles in gametogenesis, hematopoiesis, mast cell development and function, and melanogenesis. Moreover, human and mouse embryonic stem cells express Kit transcripts. Stem cell factor exists as both a soluble and a membrane-bound glycoprotein while Kit is a receptor protein-tyrosine kinase. The complete absence of stem cell factor or Kit is lethal. Deficiencies of either produce defects in red and white blood cell production, hypopigmentation, and sterility. Gain-of-function mutations of Kit are associated with several human neoplasms including acute myelogenous leukemia, gastrointestinal stromal tumors, and mastocytomas. Kit consists of an extracellular domain, a transmembrane segment, a juxtamembrane segment, and a protein kinase domain that contains an insert of about 80 amino acid residues. Binding of stem cell factor to Kit results in receptor dimerization and activation of protein kinase activity. The activated receptor becomes autophosphorylated at tyrosine residues that serve as docking sites for signal transduction molecules containing SH2 domains. The adaptor protein APS, Src family kinases, and Shp2 tyrosyl phosphatase bind to phosphotyrosine 568. Shp1 tyrosyl phosphatase and the adaptor protein Shc bind to phosphotyrosine 570. C-terminal Src kinase homologous kinase and the adaptor Shc bind to both phosphotyrosines 568 and 570. These residues occur in the juxtamembrane segment of Kit. Three residues in the kinase insert domain are phosphorylated and attract the adaptor protein Grb2 (Tyr703), phosphatidylinositol 3-kinase (Tyr721), and phospholipase Cgamma (Tyr730). Phosphotyrosine 900 in the distal kinase domain binds phosphatidylinositol 3-kinase which in turn binds the adaptor protein Crk. Phosphotyrosine 936, also in the distal kinase domain, binds the adaptor proteins APS, Grb2, and Grb7. Kit has the potential to participate in multiple signal transduction pathways as a result of

  18. Role of Neurokinin 3 Receptor Signaling in Oral Squamous Cell Carcinoma.

    Science.gov (United States)

    Obata, Kyoichi; Shimo, Tsuyoshi; Okui, Tatsuo; Matsumoto, Kenichi; Takada, Hiroyuki; Takabatake, Kiyofumi; Kunisada, Yuki; Ibaragi, Soichiro; Yoshioka, Norie; Kishimoto, Koji; Nagatsuka, Hitoshi; Sasaki, Akira

    2017-11-01

    The neurokinin 3 receptor (NK-3R) is differentially expressed in the central nervous system including cases of human oral squamous cell carcinoma. However, the role of NK-3R signaling in oral squamous cell carcinoma is not well known. NK-3R expression in surgically resected oral squamous cell carcinoma was examined immunohistochemically and the strength of the expression was quantified. We evaluated the function of NK-3R signaling using NK-3R antagonist in human oral squamous cell carcinoma bone invasion mouse model. NK-3R was significantly expressed in tumor cells that had invaded the bone matrix compared to the oral side tumor cells. SB222200, a selective antagonist of NK-3R, significantly suppressed the radiographic osteolytic lesion and tumorigenesis. NK-3R signaling is a potential target for the treatment of oral squamous cell carcinoma in cases of bone destruction. Copyright© 2017, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.

  19. Disabled is a putative adaptor protein that functions during signaling by the sevenless receptor tyrosine kinase.

    Science.gov (United States)

    Le, N; Simon, M A

    1998-08-01

    DRK, the Drosophila homolog of the SH2-SH3 domain adaptor protein Grb2, is required during signaling by the sevenless receptor tyrosine kinase (SEV). One role of DRK is to provide a link between activated SEV and the Ras1 activator SOS. We have investigated the possibility that DRK performs other functions by identifying additional DRK-binding proteins. We show that the phosphotyrosine-binding (PTB) domain-containing protein Disabled (DAB) binds to the DRK SH3 domains. DAB is expressed in the ommatidial clusters, and loss of DAB function disrupts ommatidial development. Moreover, reduction of DAB function attenuates signaling by a constitutively activated SEV. Our biochemical analysis suggests that DAB binds SEV directly via its PTB domain, becomes tyrosine phosphorylated upon SEV activation, and then serves as an adaptor protein for SH2 domain-containing proteins. Taken together, these results indicate that DAB is a novel component of the SEV signaling pathway.

  20. Epigenetics of Estrogen Receptor Signaling: Role in Hormonal Cancer Progression and Therapy

    International Nuclear Information System (INIS)

    Mann, Monica; Cortez, Valerie; Vadlamudi, Ratna K.

    2011-01-01

    Estrogen receptor (ERα) signaling plays a key role in hormonal cancer progression. ERα is a ligand-dependent transcription factor that modulates gene transcription via recruitment to the target gene chromatin. Emerging evidence suggests that ERα signaling has the potential to contribute to epigenetic changes. Estrogen stimulation is shown to induce several histone modifications at the ERα target gene promoters including acetylation, phosphorylation and methylation via dynamic interactions with histone modifying enzymes. Deregulation of enzymes involved in the ERα -mediated epigenetic pathway could play a vital role in ERα driven neoplastic processes. Unlike genetic alterations, epigenetic changes are reversible, and hence offer novel therapeutic opportunities to reverse ERα driven epigenetic changes. In this review, we summarize current knowledge on mechanisms by which ERα signaling potentiates epigenetic changes in cancer cells via histone modifications

  1. Epigenetics of Estrogen Receptor Signaling: Role in Hormonal Cancer Progression and Therapy

    Energy Technology Data Exchange (ETDEWEB)

    Mann, Monica; Cortez, Valerie [Department of Cellular and Structural Biology, UTHSCSA, 7703 Floyd Curl Drive, San Antonio, TX 78229 (United States); Vadlamudi, Ratna K., E-mail: vadlamudi@uthscsa.edu [Department of Obstetrics and Gynecology, UTHSCSA, 7703 Floyd Curl Drive, San Antonio, TX 78229 (United States)

    2011-03-29

    Estrogen receptor (ERα) signaling plays a key role in hormonal cancer progression. ERα is a ligand-dependent transcription factor that modulates gene transcription via recruitment to the target gene chromatin. Emerging evidence suggests that ERα signaling has the potential to contribute to epigenetic changes. Estrogen stimulation is shown to induce several histone modifications at the ERα target gene promoters including acetylation, phosphorylation and methylation via dynamic interactions with histone modifying enzymes. Deregulation of enzymes involved in the ERα -mediated epigenetic pathway could play a vital role in ERα driven neoplastic processes. Unlike genetic alterations, epigenetic changes are reversible, and hence offer novel therapeutic opportunities to reverse ERα driven epigenetic changes. In this review, we summarize current knowledge on mechanisms by which ERα signaling potentiates epigenetic changes in cancer cells via histone modifications.

  2. Signal Peptide and Denaturing Temperature are Critical Factors for Efficient Mammalian Expression and Immunoblotting of Cannabinoid Receptors*

    Science.gov (United States)

    WANG, Chenyun; WANG, Yingying; WANG, Miao; CHEN, Jiankui; YU, Nong; SONG, Shiping; KAMINSKI, Norbert E.; ZHANG, Wei

    2013-01-01

    Summary Many researchers employed mammalian expression system to artificially express cannabinoid receptors, but immunoblot data that directly prove efficient protein expression can hardly be seen in related research reports. In present study, we demonstrated cannabinoid receptor protein was not able to be properly expressed with routine mammalian expression system. This inefficient expression was rescued by endowing an exogenous signal peptide ahead of cannabinoid receptor peptide. In addition, the artificially synthesized cannabinoid receptor was found to aggregate under routine sample denaturing temperatures (i.e., ≥95°C), forming a large molecular weight band when analyzed by immunoblotting. Only denaturing temperatures ≤75°C yielded a clear band at the predicted molecular weight. Collectively, we showed that efficient mammalian expression of cannabinoid receptors need a signal peptide sequence, and described the requirement for a low sample denaturing temperature in immunoblot analysis. These findings provide very useful information for efficient mammalian expression and immunoblotting of membrane receptors. PMID:22528237

  3. Receptor⁻Receptor Interactions in Multiple 5-HT1A Heteroreceptor Complexes in Raphe-Hippocampal 5-HT Transmission and Their Relevance for Depression and Its Treatment.

    Science.gov (United States)

    Borroto-Escuela, Dasiel O; Narváez, Manuel; Ambrogini, Patrizia; Ferraro, Luca; Brito, Ismel; Romero-Fernandez, Wilber; Andrade-Talavera, Yuniesky; Flores-Burgess, Antonio; Millon, Carmelo; Gago, Belen; Narvaez, Jose Angel; Odagaki, Yuji; Palkovits, Miklos; Diaz-Cabiale, Zaida; Fuxe, Kjell

    2018-06-03

    Due to the binding to a number of proteins to the receptor protomers in receptor heteromers in the brain, the term "heteroreceptor complexes" was introduced. A number of serotonin 5-HT1A heteroreceptor complexes were recently found to be linked to the ascending 5-HT pathways known to have a significant role in depression. The 5-HT1A⁻FGFR1 heteroreceptor complexes were involved in synergistically enhancing neuroplasticity in the hippocampus and in the dorsal raphe 5-HT nerve cells. The 5-HT1A protomer significantly increased FGFR1 protomer signaling in wild-type rats. Disturbances in the 5-HT1A⁻FGFR1 heteroreceptor complexes in the raphe-hippocampal 5-HT system were found in a genetic rat model of depression (Flinders sensitive line (FSL) rats). Deficits in FSL rats were observed in the ability of combined FGFR1 and 5-HT1A agonist cotreatment to produce antidepressant-like effects. It may in part reflect a failure of FGFR1 treatment to uncouple the 5-HT1A postjunctional receptors and autoreceptors from the hippocampal and dorsal raphe GIRK channels, respectively. This may result in maintained inhibition of hippocampal pyramidal nerve cell and dorsal raphe 5-HT nerve cell firing. Also, 5-HT1A⁻5-HT2A isoreceptor complexes were recently demonstrated to exist in the hippocampus and limbic cortex. They may play a role in depression through an ability of 5-HT2A protomer signaling to inhibit the 5-HT1A protomer recognition and signaling. Finally, galanin (1⁻15) was reported to enhance the antidepressant effects of fluoxetine through the putative formation of GalR1⁻GalR2⁻5-HT1A heteroreceptor complexes. Taken together, these novel 5-HT1A receptor complexes offer new targets for treatment of depression.

  4. Novel TPR-containing subunit of TOM complex functions as cytosolic receptor for Entamoeba mitosomal transport.

    Science.gov (United States)

    Makiuchi, Takashi; Mi-ichi, Fumika; Nakada-Tsukui, Kumiko; Nozaki, Tomoyoshi

    2013-01-01

    Under anaerobic environments, the mitochondria have undergone remarkable reduction and transformation into highly reduced structures, referred as mitochondrion-related organelles (MROs), which include mitosomes and hydrogenosomes. In agreement with the concept of reductive evolution, mitosomes of Entamoeba histolytica lack most of the components of the TOM (translocase of the outer mitochondrial membrane) complex, which is required for the targeting and membrane translocation of preproteins into the canonical aerobic mitochondria. Here we showed, in E. histolytica mitosomes, the presence of a 600-kDa TOM complex composed of Tom40, a conserved pore-forming subunit, and Tom60, a novel lineage-specific receptor protein. Tom60, containing multiple tetratricopeptide repeats, is localized to the mitosomal outer membrane and the cytosol, and serves as a receptor of both mitosomal matrix and membrane preproteins. Our data indicate that Entamoeba has invented a novel lineage-specific shuttle receptor of the TOM complex as a consequence of adaptation to an anaerobic environment.

  5. Generating "fragment-based virtual library" using pocket similarity search of ligand-receptor complexes.

    Science.gov (United States)

    Khashan, Raed S

    2015-01-01

    As the number of available ligand-receptor complexes is increasing, researchers are becoming more dedicated to mine these complexes to aid in the drug design and development process. We present free software which is developed as a tool for performing similarity search across ligand-receptor complexes for identifying binding pockets which are similar to that of a target receptor. The search is based on 3D-geometric and chemical similarity of the atoms forming the binding pocket. For each match identified, the ligand's fragment(s) corresponding to that binding pocket are extracted, thus forming a virtual library of fragments (FragVLib) that is useful for structure-based drug design. The program provides a very useful tool to explore available databases.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2010-02-12

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

  7. Enhanced Androgen Signaling With Androgen Receptor Overexpression in the Osteoblast Lineage Controls Skeletal Turnover, Matrix Quality and Bone Architecture

    National Research Council Canada - National Science Library

    Wiren, Kristine M; Jepsen, Karl

    2006-01-01

    .... We genetically engineered transgenic mice in which androgen receptor (AR) overexpression is skeletally targeted in two separate models to better understand the role of androgen signaling directly in bone...

  8. Epidermal growth factor receptor signalling in human breast cancer cells operates parallel to estrogen receptor α signalling and results in tamoxifen insensitive proliferation

    International Nuclear Information System (INIS)

    Moerkens, Marja; Zhang, Yinghui; Wester, Lynn; Water, Bob van de; Meerman, John HN

    2014-01-01

    Tamoxifen resistance is a major problem in the treatment of estrogen receptor (ER) α -positive breast cancer patients. Although the mechanisms behind tamoxifen resistance are still not completely understood, clinical data suggests that increased expression of receptor tyrosine kinases is involved. Here, we studied the estrogen and anti-estrogen sensitivity of human breast cancer MCF7 cells that have a moderate, retroviral-mediated, ectopic expression of epidermal growth factor receptor (MCF7-EGFR). Proliferation of MCF7-EGFR and parental cells was induced by 17β-estradiol (E2), epidermal growth factor (EGF) or a combination of these. Inhibition of proliferation under these conditions was investigated with 4-hydroxy-tamoxifen (TAM) or fulvestrant at 10 -12 to 10 -6 M. Cells were lysed at different time points to determine the phosphorylation status of EGFR, MAPK 1/3 , AKT and the expression of ERα. Knockdown of target genes was established using smartpool siRNAs. Transcriptomics analysis was done 6 hr after stimulation with growth factors using Affymetrix HG-U133 PM array plates. While proliferation of parental MCF7 cells could only be induced by E2, proliferation of MCF7-EGFR cells could be induced by either E2 or EGF. Treatment with TAM or fulvestrant did significantly inhibit proliferation of MCF7-EGFR cells stimulated with E2 alone. EGF treatment of E2/TAM treated cells led to a marked cell proliferation thereby overruling the anti-estrogen-mediated inhibition of cell proliferation. Under these conditions, TAM however did still inhibit ERα- mediated transcription. While siRNA-mediated knock-down of EGFR inhibited the EGF- driven proliferation under TAM/E2/EGF condition, knock down of ERα did not. The TAM resistant cell proliferation mediated by the conditional EGFR-signaling may be dependent on the PI3K/Akt pathway but not the MEK/MAPK pathway, since a MEK inhibitor (U0126), did not block the proliferation. Transcriptomic analysis under the various E2/TAM

  9. A new inhibitor of the ?-arrestin/AP2 endocytic complex reveals interplay between GPCR internalization and signalling

    OpenAIRE

    Beautrait, Alexandre; Paradis, Justine S.; Zimmerman, Brandon; Giubilaro, Jenna; Nikolajev, Ljiljana; Armando, Sylvain; Kobayashi, Hiroyuki; Yamani, Lama; Namkung, Yoon; Heydenreich, Franziska M.; Khoury, Etienne; Audet, Martin; Roux, Philippe P.; Veprintsev, Dmitry B.; Laporte, St?phane A.

    2017-01-01

    In addition to G protein-coupled receptor (GPCR) desensitization and endocytosis, ?-arrestin recruitment to ligand-stimulated GPCRs promotes non-canonical signalling cascades. Distinguishing the respective contributions of ?-arrestin recruitment to the receptor and ?-arrestin-promoted endocytosis in propagating receptor signalling has been limited by the lack of selective analytical tools. Here, using a combination of virtual screening and cell-based assays, we have identified a small molecul...

  10. The biology of skin wetness perception and its implications in manual function and for reproducing complex somatosensory signals in neuroprosthetics

    Science.gov (United States)

    Ackerley, Rochelle

    2017-01-01

    Our perception of skin wetness is generated readily, yet humans have no known receptor (hygroreceptor) to signal this directly. It is easy to imagine the sensation of water running over our hands or the feel of rain on our skin. The synthetic sensation of wetness is thought to be produced from a combination of specific skin thermal and tactile inputs, registered through thermoreceptors and mechanoreceptors, respectively. The present review explores how thermal and tactile afference from the periphery can generate the percept of wetness centrally. We propose that the main signals include information about skin cooling, signaled primarily by thinly myelinated thermoreceptors, and rapid changes in touch, through fast-conducting, myelinated mechanoreceptors. Potential central sites for integration of these signals, and thus the perception of skin wetness, include the primary and secondary somatosensory cortices and the insula cortex. The interactions underlying these processes can also be modeled to aid in understanding and engineering the mechanisms. Furthermore, we discuss the role that sensing wetness could play in precision grip and the dexterous manipulation of objects. We expand on these lines of inquiry to the application of the knowledge in designing and creating skin sensory feedback in prosthetics. The addition of real-time, complex sensory signals would mark a significant advance in the use and incorporation of prosthetic body parts for amputees in everyday life. PMID:28123008

  11. Machines vs. ensembles: effective MAPK signaling through heterogeneous sets of protein complexes.

    Directory of Open Access Journals (Sweden)

    Ryan Suderman

    Full Text Available Despite the importance of intracellular signaling networks, there is currently no consensus regarding the fundamental nature of the protein complexes such networks employ. One prominent view involves stable signaling machines with well-defined quaternary structures. The combinatorial complexity of signaling networks has led to an opposing perspective, namely that signaling proceeds via heterogeneous pleiomorphic ensembles of transient complexes. Since many hypotheses regarding network function rely on how we conceptualize signaling complexes, resolving this issue is a central problem in systems biology. Unfortunately, direct experimental characterization of these complexes has proven technologically difficult, while combinatorial complexity has prevented traditional modeling methods from approaching this question. Here we employ rule-based modeling, a technique that overcomes these limitations, to construct a model of the yeast pheromone signaling network. We found that this model exhibits significant ensemble character while generating reliable responses that match experimental observations. To contrast the ensemble behavior, we constructed a model that employs hierarchical assembly pathways to produce scaffold-based signaling machines. We found that this machine model could not replicate the experimentally observed combinatorial inhibition that arises when the scaffold is overexpressed. This finding provides evidence against the hierarchical assembly of machines in the pheromone signaling network and suggests that machines and ensembles may serve distinct purposes in vivo. In some cases, e.g. core enzymatic activities like protein synthesis and degradation, machines assembled via hierarchical energy landscapes may provide functional stability for the cell. In other cases, such as signaling, ensembles may represent a form of weak linkage, facilitating variation and plasticity in network evolution. The capacity of ensembles to signal effectively

  12. Monitoring ligand-dependent assembly of receptor ternary complexes in live cells by BRETFect.

    Science.gov (United States)

    Cotnoir-White, David; El Ezzy, Mohamed; Boulay, Pierre-Luc; Rozendaal, Marieke; Bouvier, Michel; Gagnon, Etienne; Mader, Sylvie

    2018-03-13

    There is currently an unmet need for versatile techniques to monitor the assembly and dynamics of ternary complexes in live cells. Here we describe bioluminescence resonance energy transfer with fluorescence enhancement by combined transfer (BRETFect), a high-throughput technique that enables robust spectrometric detection of ternary protein complexes based on increased energy transfer from a luciferase to a fluorescent acceptor in the presence of a fluorescent intermediate. Its unique donor-intermediate-acceptor relay system is designed so that the acceptor can receive energy either directly from the donor or indirectly via the intermediate in a combined transfer, taking advantage of the entire luciferase emission spectrum. BRETFect was used to study the ligand-dependent cofactor interaction properties of the estrogen receptors ERα and ERβ, which form homo- or heterodimers whose distinctive regulatory properties are difficult to dissect using traditional methods. BRETFect uncovered the relative capacities of hetero- vs. homodimers to recruit receptor-specific cofactors and regulatory proteins, and to interact with common cofactors in the presence of receptor-specific ligands. BRETFect was also used to follow the assembly of ternary complexes between the V2R vasopressin receptor and two different intracellular effectors, illustrating its use for dissection of ternary protein-protein interactions engaged by G protein-coupled receptors. Our results indicate that BRETFect represents a powerful and versatile technique to monitor the dynamics of ternary interactions within multimeric complexes in